CN114058699A - Application of PPDPF in pancreatic cancer diagnosis and medicine preparation - Google Patents

Abstract

The invention provides the application of pancreatic progenitor cell differentiation and proliferation factor (PPDPF) in pancreatic cancer diagnosis and drug preparation. The present invention reveals that PPDPF is highly expressed in pancreatic cancer and promotes the progression of pancreatic cancer, which can be used as a novel target for diagnosis or treatment. Using it as a target, it is possible to develop drugs that inhibit pancreatic cancer, diagnose pancreatic cancer or symptoms, evaluate prognosis, and screen therapeutic drugs and diagnostic reagents.

Description

Application of PPDPF in pancreatic cancer diagnosis and medicine preparation

Technical Field

The invention belongs to the field of biotechnology and pharmacology, and particularly relates to application of PPDPF in pancreatic cancer diagnosis and medicine preparation.

Background

Pancreatic cancer is one of the most common cancers in the world, known as "the king of cancer". The tumor is a tumor with extremely high malignancy degree, the morbidity of the tumor is high in invisibility, the median survival time is only 3-6 months, the 5-year survival rate is lower than 5%, and the tumor is one of the tumors with the worst prognosis. Pancreatic Ductal Adenocarcinoma (PDAC) is the most common pathological type of Pancreatic cancer, accounting for approximately 90% of Pancreatic cancers. Surgical resection is the only possible cure for pancreatic adenocarcinoma. Diagnosis is a significant challenge for pancreatic cancer, however, and most patients are diagnosed with pancreatic cancer that is already advanced, i.e., locally advanced or metastatic tumor, meaning that 80% -85% of patients are diagnosed with pancreatic cancer that cannot be surgically resected. If new targets for diagnosing and treating pancreatic cancer can be found, the method has very important significance for improving the prognosis of pancreatic cancer.

Traditional therapeutic agents for pancreatic cancer include the nucleoside analog gemcitabine. Treatment with erlotinib or albumin paclitaxel in combination with gemcitabine also showed some therapeutic benefit. Despite aggressive treatment regimens, the median survival for pancreatic cancer is still only around 6 months.

At present, the molecular mechanisms and cellular biological properties involved in regulating pancreatic cancer cell infiltration and metastasis are not clear. In recent years, research on the pathogenesis, particularly the metastatic mechanism, of pancreatic cancer has been a focus.

Pancreatic cancer-associated targets that are being investigated in the art include: PI3 kinase/Akt/mTOR pathway, HER2 signaling pathway, RAF/MEK/ERK pathway, KRAS, Epidermal Growth Factor Receptor (EGFR), JAK/ATAT signaling pathway, insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor receptor pathway (VEGF), transforming growth factor-Beta, platelet-derived growth factors (PDGFs), hyaluronic acid, Matrix Metalloproteinases (MMPs), Hh inhibitors, albumin-binding paclitaxel, PEP02(MM-398), Endo TAG-1, and the like.

Although the biological characteristics of pancreatic cancer have been well studied, the progress of the identification of new molecular targets remains extremely slow and there are very few potential drug candidates, probably due to the genetic complexity of pancreatic cancer. In addition, due to the short survival time of the disease, it is difficult to have the opportunity to test the effectiveness of any possible immunotherapy or other therapy.

Therefore, there is an urgent need in the art to develop new diagnostic or therapeutic targets for pancreatic cancer, thereby providing new approaches for the disease research and clinical treatment of pancreatic cancer.

Disclosure of Invention

The invention aims to provide application of PPDPF in pancreatic cancer diagnosis and medicine preparation.

In a first aspect of the invention, there is provided the use of pancreatic progenitor differentiation and proliferation factors for: as a target for inhibiting pancreatic cancer; as a marker for diagnosis or prognosis of pancreatic cancer; preparing a diagnostic reagent for diagnosing or prognosing pancreatic cancer; or as a target for screening drugs for inhibiting pancreatic cancer.

In another aspect of the present invention, there is provided the use of a down-regulator of pancreatic progenitor differentiation and proliferation factors for the preparation of a composition for inhibiting pancreatic cancer.

In a preferred embodiment, said inhibiting pancreatic cancer comprises: preventing, alleviating or treating pancreatic cancer.

In another preferred embodiment, the composition is further used for: inhibiting anchorage-independent growth of pancreatic cancer cells; inhibiting the tumorigenic capacity of pancreatic cancer cells; inhibit phosphorylation of Erk and/or AKT 308; and/or inhibiting activation of the RAS.

In another preferred embodiment, the pancreatic progenitor differentiation and proliferation factor down-regulator includes (but is not limited to): substances which down-regulate the activity of pancreatic progenitor cells or substances which down-regulate the expression, stability or reduce the effective duration of pancreatic progenitor cells.

In another preferred embodiment, the down-regulator includes a down-regulator selected from (but not limited to): agents that knock-out or silence pancreatic progenitor differentiation and proliferation factors; binding molecules (e.g., antibodies or ligands) that specifically bind to pancreatic progenitor cell differentiation and proliferation factors; chemical small molecule antagonists or inhibitors against pancreatic progenitor differentiation and proliferation factors; an agent that inhibits anchorage-independent growth or tumorigenesis of pancreatic cancer cells mediated by pancreatic progenitor cell differentiation and proliferation factors; an agent that inhibits Erk and/or AKT308 phosphorylation mediated by pancreatic progenitor cell differentiation and proliferation factors; an agent that inhibits RAS activation mediated by pancreatic progenitor cell differentiation and proliferation factors; or agents that interfere with the interaction of pancreatic progenitor cell differentiation with proliferation factors and effector molecules.

In another preferred embodiment, the agent for knocking out or silencing pancreatic progenitor differentiation and proliferation factors includes (but is not limited to): the kit comprises CRISPR gene editing reagents aiming at pancreatic progenitor cell differentiation and proliferation factors, interference molecules specifically interfering with the expression of coding genes of the pancreatic progenitor cell differentiation and proliferation factors, homologous recombination reagents or site-directed mutation reagents aiming at the pancreatic progenitor cell differentiation and proliferation factors, and the homologous recombination reagents or the site-directed mutation reagents are used for carrying out loss-of-function mutation on the pancreatic progenitor cell differentiation and proliferation factors.

In another preferred embodiment, the interfering molecule comprises an siRNA, shRNA, miRNA, antisense nucleic acid, or the like, or a construct capable of forming the siRNA, shRNA, miRNA, antisense nucleic acid, or the like.

In another preferred example, the CRISPR gene editing reagent for pancreatic progenitor cell differentiation and proliferation factor is sgRNA, and the nucleotide sequence thereof is shown in SEQ ID No. 19 and/or SEQ ID No. 20.

In another preferred embodiment, said PPDPF is selected from the group consisting of: (a) polypeptide with amino acid sequence as shown in SEQ ID No. 1; (b) a PPDPF derivative which is formed by substituting, deleting or adding one or more (such as 1-20, 1-10, 1-5, 1-3 or 1-2) amino acid residues in the amino acid sequence shown in the (a) and has the polypeptide function of the (a) or (b), or an active fragment thereof; (c) PPDPF derivatives with homology of more than or equal to 90 percent (such as homology of more than or equal to 92 percent, more than or equal to 94 percent, more than or equal to 96 percent, more than or equal to 98 percent or more than or equal to 99 percent) or active fragments thereof compared with the amino acid sequence shown in SEQ ID NO. 1.

In another preferred mode, an expression construct (expression vector) for introducing a down-regulator such as a sgRNA or an interfering molecule into a cell includes: viral vectors, non-viral vectors; preferably, the expression vector comprises: adeno-associated virus vectors, lentiviral vectors, and adenoviral vectors.

In another preferred embodiment, the pancreatic cancer includes (but is not limited to): pancreatic ductal adenocarcinoma, pancreatic intraepithelial neoplasia.

In another aspect of the present invention, there is provided a use of a reagent specifically recognizing or amplifying pancreatic progenitor cell differentiation and proliferation factors for preparing a diagnostic reagent or kit for diagnosis or prognosis of pancreatic cancer.

In a preferred embodiment, the agents include (but are not limited to): a binding molecule (e.g., an antibody or ligand) that specifically binds to a pancreatic progenitor differentiation and proliferation factor protein; primers for specifically amplifying pancreatic progenitor cell differentiation and proliferation factor genes; a probe for specifically recognizing pancreatic progenitor cell differentiation and proliferation factor genes; or a chip for specifically recognizing the pancreatic progenitor cell differentiation and proliferation factor genes.

In another aspect of the present invention, there is provided a pharmaceutical composition or kit for inhibiting tumor, comprising: down-regulators of pancreatic progenitor differentiation and proliferation factors; preferably, the down regulator is sgRNA for gene editing of pancreatic progenitor cell differentiation and proliferation factors, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 19 and/or SEQ ID NO. 20.

In another aspect of the present invention, there is provided a method of screening for a potential substance inhibiting pancreatic cancer, the method comprising: (1) treating an expression system expressing pancreatic progenitor differentiation and proliferation factors with a candidate substance; and, (2) detecting the expression or activity of pancreatic progenitor differentiation and proliferation factors in said system; a candidate substance is a potential substance for reducing pancreatic cancer if the candidate substance statistically downregulates (significantly downregulates, e.g., by more than 10%, more than 20%, more than 50%, more than 80%, etc., or renders it non-expressed or inactive) the expression or activity of pancreatic progenitor differentiation and proliferation factors.

In a preferred mode, the system of step (1) is a pancreatic cancer cell (culture) system; the step (2) further comprises the following steps: detecting the non-anchorage dependent growth ability or clonogenic ability of pancreatic cancer cells in said system; if the anchorage-independent growth ability or the clonogenic ability of the candidate substance is reduced (significantly reduced, such as reduced by more than 10%, more than 20%, more than 50%, more than 80%, etc.), the candidate substance is a potential substance for reducing pancreatic cancer.

In another preferred mode, the system of step (1) further expresses RAS and its downstream signaling pathway molecules Erk and AKT; the step (2) further comprises the following steps: and detecting the activated GTP-Ras level and/or detecting the phosphorylation levels of Erk and AKT in the system, wherein if the activated GTP-Ras level is obviously reduced and/or the phosphorylation levels of Erk and AKT are obviously reduced (such as reduction by more than 10%, more than 20%, more than 50%, more than 80% and the like), the candidate substance is a potential substance for reducing pancreatic cancer.

In another preferred mode, the step (1) includes: in the test group, adding a candidate substance to the expression system; and/or, the step (2) comprises: detecting the expression or activity of pancreatic progenitor cell differentiation and proliferation factors in the system, or detecting anchorage-independent growth or clonogenic capacity of pancreatic cancer cells, or detecting the phosphorylation levels of Erk and AKT in the system; and comparing the expression system with a control group, wherein the control group is an expression system without the candidate substance; a candidate substance is a potential substance for reducing pancreatic cancer if it statistically down-regulates the expression or activity of pancreatic progenitor cell differentiation and proliferation factors, or statistically decreases the anchorage-independent growth or clonogenic capacity of pancreatic cancer cells, or decreases the phosphorylation levels of Erk and AKT.

In another preferred embodiment, the candidate substance includes (but is not limited to): regulatory molecules or constructs thereof (such as shRNA, siRNA, gene editing entity, expression vector, recombinant viral or non-viral construct, etc.), chemical small molecules (such as specific inhibitor or antagonist), interactive molecules, etc. designed for pancreatic progenitor cell development and growth factor, fragments or variants thereof, coding genes thereof or upstream and downstream molecules thereof or signaling pathways thereof.

In another preferred embodiment, the system is selected from: cell systems (e.g., cells or cell cultures expressing pancreatic progenitor differentiation and proliferation factors), subcellular (culture) systems, solution systems, tissue systems, organ systems, or animal systems.

In another preferred example, the method further comprises: the obtained potential substance is subjected to further cell experiments and/or animal experiments to further select and determine a substance useful for inhibiting pancreatic cancer from the candidate substances.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

Figure 1, mRNA level expression of PPDPF in pancreatic cancer tissues was significantly upregulated; the RT-PCR method is applied to detect 39 the expression level of PPDPPF mRNA in pancreatic cancer specimens (T) and matched paracancer normal tissues (N).

Figure 2, protein expression level of PPDPF in pancreatic cancer tissues was significantly upregulated; the PPDPPF protein level in paired N (paracancer normal), T (tumor) tissues of pancreatic cancer patients was measured by immunohistochemistry.

FIG. 3, chip analysis of PPDPPF expression level in pancreatic cancer tissues; h-score (p ═ 0.0003) of PPDPF protein in N (N ═ 90) and T (N ═ 90) in paired pancreatic cancer tissues.

FIG. 4, PPDPPF expression correlates with poor prognosis in pancreatic cancer patients. Total survival of patients with high and low PPDPF expression (PPDPF high, n-47; PPDPF low, n-43; p-0.03).

FIG. 5 PPDPF overexpression promotes anchorage-independent growth of pancreatic cancer cells in vitro. (A) Protein overexpression efficiency of PPDPPF in stably transfected pancreatic cancer cells was examined by Western blotting. (B) The effect of over-expressing PPDPF on the non-anchored growth of pancreatic cancer cells was examined by the soft agar method. The above statistics were all triplicated and expressed as mean ± sd. Represents P < 0.05; represents P < 0.01; represents P < 0.001.

Figure 6, endogenous PPDPF knock-out significantly inhibited the in vitro anchorage-independent growth capacity of pancreatic cancer cells. (A) The PPDPPF protein expression level in hepatocellular carcinoma cells was detected by Western blotting. The effect of endogenous PPDPPF gene knockout on the anchorage-independent growth capacity of pancreatic cancer cells HPAC (B) and Miapaca2(C) was examined by soft agarose method. The statistical results are all three times of repeated results and are expressed by mean value plus or minus standard deviation, and represents that P is less than 0.05; represents P < 0.01; represents P < 0.001.

Figure 7, endogenous PPDPF knockout inhibits the tumorigenic capacity of pancreatic cancer cells in nude mice. In the experiment of the tumor formation of the nude mice,nude mice injected with HPAC control cells or endogenous PPDPF knock-out cells thereof (A) nude mice photographs of tumors, (B) tumor photographs, (C) tumor weights (g) and (D) tumor growth curves (mm)³) (ii) a The above statistics are expressed as mean ± standard deviation. P<0.05；**P<0.01；***，P<0.001。

FIG. 8, the ability of over-expressing PPDPF to significantly promote the tumorigenesis of pancreatic cancer cells in nude mice. In nude mouse tumorigenicity experiment, nude mice injected with Miapaca2 cell control cell or PPDPF-overexpressing cell (A) tumorigenic nude mouse photo, (B) tumor photo, (C) tumor weight (g) and (D) tumor growth curve (mm)³). The above statistics are expressed as mean ± standard deviation. P<0.05；**P<0.01；***，P<0.001。

FIG. 9, KRAS-G12D-driven PDAC model, PPDPF expression in PanIN was significantly elevated. Control mice (LSL-KRAS-G12D) and PDX-Cre, 10 months old, respectively; pancreatic tissue from LSL-KRAS-G12D mice was immunohistochemically stained for PPDPF. 1,2,3 represent three different mice per group.

Figure 10, pancreatic ductal adenocarcinoma mouse pancreatic tissue HE staining results. (A) Pancreatic ductal adenocarcinoma model mice 8 months old and (B)10 months old (PDX-CRE; LSL-KRAS-G12D) and PPDPPF knockout mice (PDX-CRE; LSL-KRAS-G12D; PPDPF loxP/loxP) were stained for pancreatic tissue HE.

FIG. 11, PPDPDFF overexpression promotes phosphorylation activation of Erk and AKT in different pancreatic cancer cells, at resting and EGF treatment. (A) HPAC, (B) Miapaca2 and (C) Capan pancreatic cancer control cells and PPDPPF overexpressing cells the phosphorylation levels of Erk and AKT were measured by Western Blotting at rest and EGF (10ng/ml) treatment.

Figure 12, knockout PPDPF inhibits activation of phosphorylation levels of Erk and AKT in pancreatic cancer cells, both at rest and EGF treatment. HPAC pancreatic cancer control cells and PPDPPF KO cells were tested for Erk and AKT phosphorylation by Western Blotting at rest and EGF (10ng/ml) treatment.

Figure 13, overexpression of PPDPF promotes RAS activation in pancreatic cancer cells, at rest and EGF treatment. The amount of RAS-GTP active form was measured by GST-pulldown and Western Blotting in the presence of the Cap pancreatic cancer cells at rest and EGF (5ng/ml) treatment.

Figure 14, in Miapaca2 pancreatic cells, at rest, the knockout PPDPF significantly inhibited RAS activation. Miapaca2 pancreatic cancer cells were assayed for the amount of RAS-GTP active form in the resting state by GST-pulldown and Western Blotting. T-Ras, total Ras.

Detailed Description

The inventor discloses that the pancreatic progenitor cell differentiation and proliferation factor (PPDPF) is highly expressed in pancreatic cancer and promotes the development of pancreatic cancer by analyzing and detecting the protein expression between normal pancreatic tissues and pancreatic cancer tissues and clinically analyzing the correlation between the protein and pancreatic cancer prognosis and other information, and the PPDPF can be used as a novel target point for diagnosis or treatment. By taking the compound as a target, medicaments for inhibiting (including relieving or treating) pancreatic cancer can be developed, and diagnosis and prognosis evaluation can be carried out on pancreatic cancer or symptoms.

The inventors have also explored the effect of PPDPF on pancreatic cancer cell growth and tumorigenicity in vitro and in vivo through in vivo and in vitro experiments. In a KRAS-G12D-driven PDAC mouse model, the influence of a pancreas-specific PPDPF knockout on the occurrence and development of pancreatic ductal adenocarcinoma and related molecular mechanisms are explored, so that important insights are provided for potential therapeutic targets and biomarkers of the pancreatic ductal adenocarcinoma.

PPDPF

PPDPF is located on human chromosome 20 and encodes a protein of about 12kD, and the function of this gene has been poorly studied and is unknown in mammals. The amino acid sequence of human PPDPF can be as shown in SEQ ID NO. 1 or as shown in Gene ID NO. 79144; NP-077275.1; the amino acid sequence of the murine PPDPF can be shown as SEQ ID NO. 2 or as Gene ID No. 66496; NP _ 079874.1. PPDPF homologues from other species and uses thereof are also encompassed by the present invention.

The PPDPF of the present invention may be naturally occurring, e.g., it may be isolated or purified from a mammal. In addition, the PPDPF can also be artificially prepared, for example, recombinant PPDPF can be produced according to the conventional genetic engineering recombination technology for experimental or clinical application. In use, recombinant PPDPF may be used. The PPDPF comprises full-length PPDPF or a biologically active fragment thereof. Preferably, the amino acid sequence of PPDPF may be substantially identical to the sequence shown in SEQ ID NO. 1. The corresponding nucleotide coding sequence is conveniently derived from the amino acid sequence of PPDPF.

The amino acid sequence of PPDPF formed by substitution, deletion, or addition of one or more amino acid residues is also included in the present invention. PPDPF or a biologically active fragment thereof includes a partial substitution sequence of conserved amino acids, which does not affect its activity or retains some of its activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and ensure that the biological activity of the resulting molecule is not altered. These techniques allow one of skill in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al, Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummings Pub. Co. P224.

Any biologically active fragment of PPDPF can be used in the present invention. As used herein, a biologically active fragment of PPDPF is meant to be a polypeptide that still retains all or part of the function of full-length PPDPF. Typically, the biologically active fragment retains at least 50% of the activity of full-length PPDPF. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length PPDPF.

After intensive study on clinical data, the inventor determines PPDPF as a research target of pancreatic cancer. Around this target, the present inventors have conducted extensive experimental demonstrations, including demonstrations at the cellular level as well as at the animal level, including in particular:

(1) the expression of PPDPF was significantly increased in pancreatic cancer tissues;

(2) PPDPF expression is negatively correlated with overall survival in pancreatic cancer patients;

(3) overexpression of PPDPF in pancreatic cancer cells promotes anchorage-independent growth capacity of pancreatic cancer cells;

(4) the expression of endogenous PPDPF knockout significantly inhibits the anchorage-independent growth capacity of pancreatic cancer cells;

(5) PPDPF promotes the tumorigenic capacity of pancreatic cancer cells in vivo;

(6) in a transgenic mouse model, PPDPF knockout inhibits the development of pancreatic cancer;

(7) PPDPF overexpression in pancreatic cancer cells promotes p-Erk and p-AKT₃₀₈Activation of (2);

(8) PPDPF knockout in pancreatic cancer cells significantly inhibits p-Erk and p-AKT₃₀₈Activation of (2);

(9) overexpression of PPDPF in pancreatic cancer cells promotes RAS activation;

(10) pancreatic cancer cells Miapaca2 knockout PPDPF significantly inhibited RAS activation.

PPDPF downregulators and uses thereof

Based on the above new findings of the present inventors, the present invention provides a use of PPDPF or a down-regulator of a gene encoding PPDPF for preparing a pharmaceutical composition for inhibiting pancreatic cancer.

As used herein, the PPDPF or gene encoding thereof down-regulator includes inhibitors, antagonists, blockers, and the like, which terms are used interchangeably.

The PPDPF or the gene encoding the PPDPF is any substance which can reduce the activity of the PPDPF, reduce the stability of the PPDPF or the gene encoding the PPDPF, reduce the expression of the PPDPF, reduce the effective action time of the PPDPF, or inhibit the transcription and translation of the PPDPF gene, and the substances can be used for the invention, can be used as substances which are useful for reducing the PPDPF, and can be used for inhibiting pancreatic cancer. For example, the down-regulating agent is: an interfering RNA molecule or antisense nucleotide that specifically interferes with PPDPF gene expression; is an antibody or ligand that specifically binds to a protein encoded by the PPDPF gene; an agent that inhibits anchorage-independent growth or tumorigenesis of pancreatic cancer cells mediated by pancreatic progenitor cell differentiation and proliferation factors; an agent that inhibits Erk and/or AKT308 phosphorylation mediated by pancreatic progenitor cell differentiation and proliferation factors; or an agent that inhibits RAS activation mediated by pancreatic progenitor cell differentiation and proliferation factors. And so on.

As an alternative of the invention, the down-regulator may be a small molecule compound directed against PPDPF. Screening of such small molecule compounds can be performed by one skilled in the art using routine screening methods in the art. For example, in the examples of the present invention, several alternative screening methods are provided in conjunction with the regulatory mechanisms disclosed herein.

As an alternative of the present invention, the down-regulator may be a PPDPF-specific interfering RNA molecule (e.g., siRNA, shRNA, miRNA, etc.), and the present inventors can understand that such an interfering RNA molecule can be prepared according to the PPDPF sequence information provided in the present invention. The method for preparing the interfering RNA molecule is not particularly limited, and includes, but is not limited to: chemical synthesis, in vitro transcription, and the like. The interfering RNA may be delivered into the cell by using an appropriate transfection reagent, or may also be delivered into the cell using a variety of techniques known in the art.

As a preferred mode of the present invention, targeted gene editing can be performed using CRISPR/Cas (e.g., Cas9) system to knock out PPDPF gene in the region targeted to disease. A common method for knocking out PPDPF gene includes: co-transferring the sgRNA or a nucleic acid capable of forming the sgRNA, Cas9mRNA or a nucleic acid capable of forming the Cas9mRNA into a targeted region or targeted cell. After the target site is determined, known methods can be employed to cause the sgRNA and Cas9 to be introduced into the cell. The nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector, or the nucleic acid capable of forming the Cas9mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into cells, so that active sgrnas and Cas9 mrnas are formed in the cells. In a particularly preferred mode of the invention, the CRISPR gene editing reagent aiming at PPDPF is sgRNA, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 19 and/or SEQ ID NO. 20.

As an alternative of the present invention, PPDPF can be specifically targeted by homologous recombination to be defective or absent in expression. The Cre and loxp methods can also be used to selectively knock-out, reduce expression, or inactivate a gene of interest in the genome of an animal or cell.

The inventor finds that PPDPF promotes the clonogenic capacity of pancreatic cancer cells, and the expression of knockout endogenous PPDPF in the pancreatic cancer cells obviously inhibits the in vitro clonogenic capacity of the pancreatic cancer cells. Based on this, as a preferred mode of the present invention, some agents inhibiting non-anchorage dependent growth or tumorigenesis of pancreatic cancer cells mediated by PPDPF are available as down-regulators having an effect on inhibiting pancreatic cancer.

The present inventors found that PPDPPF promotes p-Erk and p-AKT₃₀₈Activation of PPDPPF knock-out in pancreatic cancer cells significantly inhibited p-Erk and p-AKT₃₀₈Activation of (2). Based on this, as a preferred mode of the present invention, some inhibition of p-Erk and p-AKT mediated by PPDPF₃₀₈Activating agents are useful downregulators that have an effect on inhibiting pancreatic cancer.

The inventors found that PPDPF promotes activation of RAS, and that knockout of PPDPF in pancreatic cancer cells significantly inhibits activation of RAS. Based on this, as a preferred mode of the present invention, some agents inhibiting the activation of RAS mediated by PPDPF are available as down-regulators having an effect on inhibiting pancreatic cancer.

The above are some representative ways to down-regulate PPDPF, and it is understood that other methods known in the art to regulate PPDPF can be adopted after the general scheme of the present invention is known to those skilled in the art, and these methods are also included in the present invention.

Applications related to diagnosis and prognosis evaluation

In the present invention, targets that have an important regulatory effect on the development and progression of pancreatic cancer are disclosed. Based on this new discovery by the present inventors, PPDPF can be used as a target (or marker) for inhibiting pancreatic cancer, or screening drugs for inhibiting pancreatic cancer: (i) performing a tumor typing, differential diagnosis, and/or susceptibility analysis; (ii) evaluating the treatment medicine, the curative effect and the prognosis of the relevant pancreatic cancer population, and selecting a proper treatment method. For example, individuals with abnormal gene expression of PPDPF can be isolated and more targeted therapies can be performed.

The prognosis of pancreatic cancer in a subject who provides a sample to be evaluated can be predicted by determining the expression or activity of PPDPF in the sample to be evaluated, and selecting an appropriate drug for treatment. Typically, a threshold value for PPDPF may be specified, and treatment with a regimen that inhibits PPDPF is considered when PPDPF expression is above the specified threshold. The threshold value can be easily determined by those skilled in the art, and for example, a threshold value of PPDPF expression abnormality can be obtained by comparing the expression of PPDPF in normal human cells or tissues with the expression of PPDPF in patient cells or tissues. The specific value of the threshold may be different according to the measurement parameters, measurement instruments, and the like.

The presence or absence of the gene of PPDPF and the expression thereof can be detected by various techniques known in the art, and such techniques are encompassed by the present invention. For example, the conventional techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR and the like can be used, and these methods can be used in combination.

The invention also provides reagents for detecting the presence or absence and expression of PPDPF or a gene encoding PPDPF in an analyte. Preferably, when the detection at the gene level is performed, a primer specifically amplifying PPDPF may be used; or a probe that specifically recognizes PPDPF to determine the presence or absence of the PPDPF gene; when the protein level is detected, an antibody or ligand that specifically binds to a protein encoded by PPDPF may be used to determine PPDPF expression.

The design of a specific probe for the PPDPF gene is well known to those skilled in the art, for example, a probe is prepared which specifically binds to a specific site on the PPDPF gene, but not to genes other than the PPDPF gene, and which carries a detectable signal.

Methods for detecting PPDPF expression in an analyte using an antibody that specifically binds PPDPF are also well known to those skilled in the art.

The present invention also provides a kit for detecting the presence or absence and expression of the PPDPF gene in an analyte, the kit comprising: primers for specifically amplifying PPDPF gene; a probe that specifically recognizes the PPDPF gene; or an antibody or ligand that specifically binds to a protein encoded by the PPDPPF gene.

In addition, the kit may further include various reagents required for DNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like.

In addition, the kit may further comprise instructions for use and/or nucleic acid sequence analysis software, and the like.

Drug screening

After close correlation between high expression or high activity of PPDPF and pancreatic cancer is known, substances inhibiting expression or activity of PPDPF or a gene encoding PPDPF can be screened based on the characteristics. From these substances, a drug which is truly useful for inhibiting pancreatic cancer can be found.

Accordingly, the present invention provides a method of screening for a potential substance (candidate substance or drug candidate) that inhibits pancreatic cancer, the method comprising: treating a PPDPF-expressing system with a candidate substance; and detecting the expression or activity of PPDPF in said system; if the candidate substance can inhibit the expression or activity of PPDPF, the candidate substance is a potential substance for inhibiting pancreatic cancer. The PPDPF-expressing system is preferably a cell (or cell culture) system, and the cell may be a cell endogenously expressing PPDPF; or may be a cell that recombinantly expresses PPDPF. In addition, it is also possible to assess whether the potential substance is useful by observing the interaction of PPDPF with its upstream and downstream proteins.

In combination with the results of the studies of the present inventors, as a preferable mode of the screening means of the present invention, the effectiveness of the potential substance (candidate substance or drug candidate) can be further determined by analyzing the anchorage-independent growth ability or clonogenic ability of pancreatic cancer cells. This can be assayed by culturing pancreatic cancer cells expressing PPDPF. The observable decrease in non-anchorage-dependent growth or clonogenic capacity is predictive of the effectiveness of this potential agent.

In combination with the results of the studies of the present inventors, as a preferred mode of the screening mode of the present invention, the effectiveness of the potential substance (candidate substance or drug candidate) can be further determined by detecting the phosphorylation levels of Erk and AKT (p-Erk and p-AKT) in the system. This can be assayed by culturing pancreatic cancer cells expressing PPDPF and containing the RAS signaling pathway. A decrease in phosphorylation levels of Erk and AKT downstream RAS signaling pathway molecules is indicative of a decrease in RAS signaling pathway activity, indicative of the potential agent's effectiveness. The determination of the phosphorylation level of a protein can be performed by techniques that are routine in the art.

In a preferred embodiment of the present invention, a Control group (Control) may be provided in order to make it easier to observe the change in the expression or activity of PPDPF during screening, and the Control group may be a system expressing PPDPF without adding the candidate substance. The control group includes but is not limited to: blank control without candidate substance, blank plasmid control, etc.

As a preferred embodiment of the present invention, the method further comprises: the obtained potential substances are subjected to further cell experiments and/or animal experiments to further select and identify substances which are truly useful for inhibiting pancreatic cancer.

In another aspect, the invention also provides a potential substance for inhibiting pancreatic cancer, which is obtained by the screening method. These preliminarily selected substances may constitute a screening library so that one may finally select therefrom substances useful for inhibiting the expression and activity of PPDPF and, in turn, pancreatic cancer.

Pharmaceutical composition

The invention also provides a pharmaceutical composition, which contains an effective amount (such as 0.000001-50 wt%, preferably 0.00001-20 wt%, more preferably 0.0001-10 wt%) of the PPDPF or the down-regulator of the coding gene thereof, and a pharmaceutically acceptable carrier.

As a preferred embodiment of the present invention, there is provided a composition for inhibiting pancreatic cancer, comprising an effective amount of PPDPF or a down-regulator of a gene encoding thereof, and a pharmaceutically acceptable carrier.

In a preferred form of the invention, the down-regulating agents include, but are not limited to: an agent that knocks out or silences PPDPF, a binding molecule (e.g., an antibody or ligand) that specifically binds PPDPF, a chemical small molecule antagonist or inhibitor directed against PPDPF, and the like. In a more specific manner, the down-regulating agents include, but are not limited to: a CRISPR gene editing agent directed to PPDPF, an interfering molecule that specifically interferes with the expression of the gene encoding PPDPF, a homologous recombination or site-directed mutation agent directed to PPDPF, which subjects PPDPF to loss-of-function mutation.

As used herein, the "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for administration of the therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell transfection reagent.

Once the use of the PPDPF or gene encoding a down-regulator is known, the down-regulator or gene encoding it, or a pharmaceutical composition thereof, may be administered to a mammal or human using a variety of methods well known in the art.

Preferably, it can be carried out by means of gene therapy. For example, a down-regulator of PPDPF may be administered directly to a subject by a method such as injection; alternatively, an expression unit (e.g., an expression vector or virus, etc., or siRNA) carrying a downregulator for PPDPF can be delivered to a target site and allowed to express an active PPDPF downregulator, depending on the type of downregulator, in a manner known to those skilled in the art.

The effective amount of the PPDPF or gene encoding thereof of the present invention to be administered may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the PPDPF or the down-regulation of the coding gene thereof, such as bioavailability, metabolism, half-life and the like; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

In the specific examples of the present invention, some dosing regimens for animals such as mice are given. The conversion from the administered dose in animals such as mice to the administered dose suitable for humans is easily done by the person skilled in the art, and can be calculated, for example, according to the Meeh-Rubner formula: Meeh-Rubner formula: a ═ kx (W)^2/3)/10,000. Wherein A is the body surface area in m²Calculating; w is body weight, calculated as g; k is constant and varies with species of animal, in general, mouse and rat 9.1, guinea pig 9.8, rabbit 10.1, cat 9.9, dog 11.2, monkey 11.8, human 10.6. It will be appreciated that the conversion to a given dose may vary depending on the drug and clinical situation, as assessed by an experienced pharmacist.

The invention also provides a kit containing the pharmaceutical composition or directly containing the PPDPF or the downward regulator of the coding gene thereof. In addition, the kit can also comprise instructions for the use of the drugs in the kit.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

1. Pancreatic cancer sample collection, animal source

Clinical samples of pancreatic cancer used in all examples were obtained from the university of Compound denier, Zhongshan Hospital, with patient consent and approval of the institutional ethics committee for use. Fresh pancreatic cancer samples and paired normal pancreatic tissues were obtained from Zhongshan Hospital surgery and rapidly placed in liquid nitrogen and subsequently stored in an ultra-low temperature freezer at-80 ℃. One part of the sample is used for extracting mRNA of the tissue, and reverse transcription is carried out to form cDNA, and the cDNA is used for detecting fluorescence quantitative PCR; the other part is used for manufacturing a pancreatic cancer tissue chip, and is subjected to Immunohistochemical (IHC) staining and pathological information analysis. When the statistical result is P <0.05, the result is considered to have a significant difference; when P <0.01, the results are considered to be very different.

Mice (LSL-KRAS-G12D) were obtained from Shanghai, south China, Square Biotech, Inc.

PDX-Cre is obtained from Shanghai's Square model Biotech, Inc., and then mated with LSL-KRAS-G12D mouse to obtain PDX-CRE; LSL-KRAS-G12D mice.

KRAS-G12D-driven Pancreatic Ductal Adenocarcinoma (PDAC) animal model establishment method: spontaneous PDAC will occur as long as this mutation is present. Animals with mutations develop significant panIN (pancreatic intraepithelial neoplasma) at 8-10 months and progress to PDAC at a later date.

PDX-CRE; LSL-KRAS-G12D; PPDPF loxP/loxP mouse establishment method: PPDPPFloxP/LoxP mice and PDX-CRE; LSL-KRAS-G12D.

2. Construction of plasmids and selection of Stable cell lines

(1) Construction of plasmids

The PPDPDPPF gene was cloned from cDNA of pancreatic cancer cells, and the enzyme-cleaved cloned gene fragment was embedded into the P23 lentiviral core vector (purchased from Addgene) at KpnI and ClaI cleavage sites. The designed Cas9 sequence was synthesized, the synthesized primers were diluted to 100uM, followed by 5min at 95 ℃ and slow annealing for ligation of lentiCRISPRV2 lentiviral core vector. Primers for the cloned gene of PPDPF and its Cas9 sequence are shown in table 1.

TABLE 1

(2) Virus package

The core plasmid with the target fragment and the packaging vector were transferred into 293T cells 60-70% in length (core vector: PAX: PMD ratio 4:3:1) by calcium phosphate transfection, and virus supernatants were collected at 24 hours and 48 hours, respectively. The cell debris in the virus supernatant was then removed with a 0.45 μm filter, the filtered virus solution was transferred to a 50ml centrifuge tube, 5ml of sterile sucrose solution (20% m/v) was carefully added to the bottom of the tube (no air bubbles were generated) and the sample was weight-balanced and placed in a high speed centrifuge at 4 ℃, 20000rpm, for 2 h. And (3) discarding the supernatant in a biological safety cabinet, adding 2ml of serum-free DMEM into each tube, standing at 4 ℃ overnight for precipitation and dissolution, subpackaging in the biological safety cabinet the next day, and freezing in a refrigerator at-80 ℃ for later use.

(3) Screening for Stable cell lines

The cells are paved in a six-well plate one day in advance, the cells in the six-well plate grow to 20-30% in the next day, then 300 mu l of concentrated virus liquid is added, a certain amount of DMEM culture medium is added, the mixture is mixed evenly, and the mixture is incubated at 37 ℃. The day night virus solution was removed and fresh DMEM growth medium was added. When the core plasmid is a P23 vector, since the vector contains green fluorescent protein, namely GFP, when the cells are expanded to a 10cm large dish, the cells with GFP can be sorted out by a flow cytometry sorter, then the sorted cells are subjected to amplification culture and finally detected by Western Blotting, and the expression efficiency of the cells is identified. And the core virus plasmid is lenticrisprrv 2 lentivirus core vector, cells are amplified to a large dish, Puromycin (2 mug/ml) is added for screening, and a new culture medium containing Puromycin is replaced every 3 days, and the steps are repeated for three times. Mu.l of the selected cells were seeded into a new 10cm dish (cells were kept as monoclonal as possible). After the monoclonal cells were grown to a certain number, the monoclonal was picked up with a 200. mu.l gun and inoculated into a 48-well plate. And finally, detecting the PPDPF knockout efficiency in the monoclonal by using Western Blotting.

3. Western blotting detection

The supernatant from the dish was discarded, washed three times with pre-cooled low temperature PBS, followed by addition of an appropriate amount of RIPA lysate (0.5% sodium deoxycholate, 1% NP-40, 50mM Tris, 0.1% SDS, pH 7.4; containing protease and phosphatase inhibitors in a ratio of 1: 100) and lysis on ice for 15 minutes. The lysed liquid was then transferred to a centrifuge tube and placed in a 4 ℃ centrifuge for 15 minutes at 13000rpm, and the supernatant was transferred to a new centrifuge tube and placed on ice for future use. Mu.l of sample was taken for determination of protein concentration, and 5. mu.l of sample was typically diluted 10-fold and added to a 96-well plate with standards, three replicates per sample, followed by 200. mu.l/well Bradford. And (4) putting the prepared sample into a microplate reader for protein concentration detection. Then according to the protein concentration measured by the sample, corresponding RIPA lysate is respectively added to adjust the concentration of the sample to the same concentration. Then 6 Xloading buffer was added and the protein was denatured for 5 minutes in a metal bath. After the protein denaturation is complete, the sample is subjected to transient centrifugation, and then an equal amount of protein sample is added to the SDS-PAGE gel well, run for 20 minutes at low pressure (80V) in order to concentrate the sample proteins, and run at high pressure (140V) until complete. After running, the gel containing the split protein samples and the membrane of the methanol treated PVDF were clamped together and the proteins were transferred to the PVDF membrane by wet transfer. The PVDF membrane was then blocked with blocking solution (5% BSA in TBST) for 1 hour at room temperature, the blocking solution was decanted (care was taken not to pour the PVDF membrane), and the diluted primary antibody solution (5% BSA in TBST added with the corresponding concentration of primary antibody) was added. And then putting the mixture into a cold storage room at the temperature of 2-8 ℃ for incubation overnight. The following day, the primary antibody was recovered and washed three times with TBST for 5 minutes each, after which the secondary antibody (dilution ratio 1:2000) was added and allowed to stand on a shaker at room temperature for 1 hour. After completion, the secondary antibody solution was removed, washed three times with TBST for 5 minutes each, finally washed with TBS for 5 minutes, and then developed with addition of substrate in a Western substrate developing apparatus.

4. Soft Agar experiment

The anchorage-independent growth capacity of tumor cells was examined by soft agar cloning experiments. Prepared 1.25% in advanceAnd 60ml of 1% sterilized agar each, sealing with tin foil paper, and placing in a 65 ℃ incubator for later use. Deionized water was used after filter sterilization with a 0.22 μm filter. Rinse the 24-well plate with sterilized deionized water and then suck the deionized water away. Sucking 4ml 2 × DMEM, 4ml 1.25% agarose and 2ml fetal calf serum, mixing, adding the mixed liquid into a 24-well plate (1 ml/well) as a lower layer part, and standing and naturally cooling for later use. Tumor cells were counted and the cells were diluted with 1 × DMEM and quantified to 1 × 10⁴And/ml. The quantified 1ml of cells, 1ml of fetal bovine serum, 1.5ml of 1% agarose and 1.5ml of 2 × DMEM were then mixed well. And the mixed liquid was added to a 24-well plate (500. mu.l/well) as an upper layer portion. The 24-well plate was incubated at 37 ℃ for about 2 weeks in an incubator, and after one week of inoculation, observation was performed every other day. When the clone size and number were seen to meet the experimental requirements, photographs were taken and counted. 4 replicates were made for each sample.

5. Purification of GST-RBD protein

The BL21 strain was transformed with a vector encoding RBD protein (PGEX 4T-1). The next day, the monoclonal strain was added to 250 ml of LB medium, incubated at 37 ℃ and 220rpm/min, and shaken overnight. When the OD of the culture bacterial liquid is 0.6-1, IPTG with proper concentration is added to induce GST-PRC-RBD expression, the temperature is 30 ℃, the rpm is 220/min, and the operation is carried out for 5 hours. And collecting bacterial liquid for centrifugation at 5000rpm/min for 5 min. At the end of centrifugation, the supernatant was removed as much as possible and the pellet was washed 3 times with pre-cooled PBS. The pellet was resuspended in 10ml of PBS containing protease inhibitor and vortexed. Ultrasonication was performed on ice (400W, 3s on, 2s off, total duration 30min) until the lysate was clear. After the ultrasonic treatment, 20% TritonX-100 was added to the lysate to a final concentration of 1%, and the lysate was dissolved in a shaker at room temperature for 30 min. Then 14000rpm/min, 4 ℃, centrifugation for 20 minutes, and collection of supernatant. Mu.l of Glut4B beads were added to the supernatant and incubated at 4 ℃ for 60 minutes. The beads were then washed three times with PBS and finally eluted with glutathione to elute the bound protein of interest from the beads. And finally, detecting the purity of the target protein by using a Coomassie brilliant blue method.

6. GST pull down experiment for detecting RAS-GTP

To examine the RAS-GTP activity, the present inventors constructed a GST pull down experiment. Cells are cultured in a large dish, and after the cells grow to meet the experimental requirements, cell supernatants are discarded. Then washed twice with pre-cooled PBS and 1ml IP lysis buffer was added to the large dish. And the cells were scraped off with a 1ml tip, lysed on ice for 30 minutes, and shaken once every 5 minutes to allow for sufficient lysis. The lysis solution was then transferred to a 2mL centrifuge tube and centrifuged for 30 minutes at 14000rpm, 4 ℃. At the end of centrifugation, the supernatant was transferred to a new 1.5ml centrifuge tube. The solution in the centrifuge tube was added to purified GST protein and placed on a ferris wheel in a freezer and incubated overnight. The following day, glutathione sepharose beads (glutathione sepharose) were washed three times in advance with IP lysis buffer, the ethanol in the beads was removed, then 30 μ l of glutathione sepharose beads was added to each sample, and incubated in a freezer at 4 ℃ for 4 hours. The incubated beads were centrifuged at 500g for 2 min, the supernatant was removed, 1ml of IP lysis buffer was added, the mixture was placed on a shaker and incubated for 5min, the sample was centrifuged again, and the above steps were repeated three times. And 30. mu.l of 2 Xloading buffer was added to the beads. The sample was then placed in a metal bath at 100 ℃ for 5 minutes, repeated twice with shaking of the sample for 1 minute in between. Finally, the sample is detected and analyzed by Western Blotting.

7. Rat tail identification

The obtained rat tails were placed in 1.5ml centrifuge tubes, then 100. mu.l buffer L and 2. mu.l protease were added to each tube for 1 hour at 56 ℃, followed by a metal bath at 95 ℃ for 5 minutes, and finally placed in a refrigerator at 4 ℃ for further use. Mouse PCR primers are shown in Table 2.

TABLE 2

The PCR reaction systems for PPDPF, PDX-CRE and KRAS are shown in Table 3.

TABLE 3

PCR reaction conditions for mouse PPDPF: 95 ℃ for 5 min; at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃ for 30s (second to fourth steps, 40 cycles); 72 ℃ for 5 min; hold at 4 ℃.

And (3) identification result: wild type (Wild type) band size: 335 bp; PPDPF KO band size: 400 bp;

PCR conditions for mouse PDX-Cre: 95 ℃ for 5 min; at 95 ℃ for 30 s; at 62 ℃ for 40 s; 72 ℃ for 40s (second to fourth steps, 35 cycles); 72 ℃ for 5 min; hold at 4 ℃.

And (3) identification result: PDX-Cre band size: 400 bp;

mouse LSL-KRAS PCR reaction conditions: 94 ℃ for 5 min; 94 ℃ for 50 s; 56 ℃ for 45 s; 72 ℃ for 45s (second to fourth steps, 32 cycles); 72 ℃ for 8 min; hold at 4 ℃.

And (3) identification result: LSL-KRAS band size: 350 bp.

8. Preparation of cDNA

(1) RNA extraction

Pancreatic cancer samples from Zhongshan Hospital, university of Compound Dane were ground to powder using a tissue grinder, and 500ul of TRIzol (Invitrogen) solution was added to the samples, and dissolved on ice for 2h, and mixed well with shaking. The solution was transferred to a 1.5ml centrifuge tube and 200. mu.l of chloroform was added, mixed by inversion, and centrifuged on ice for 5min followed by 13000rpm for 15 min. The supernatant was pipetted into another fresh tube, an equal volume of isopropanol was added, followed by shaking, standing on ice for 10min, and centrifugation at 13000rpm for 15 min. The supernatant was removed and the pellet was washed twice with 75% ethanol in DEPC water (now ready). And finally, performing instantaneous centrifugation, sucking dry ethanol, standing at room temperature, airing until the ethanol is completely volatilized, and adding DEPC (diethyl phthalate) for dissolving. The concentration and quality of the RNA was then determined using a Nano-drop instrument.

(2) Reverse transcription of RNA

The amount of sample (2-3 ug total) to be added was calculated based on the RNA concentration measured, and reverse transcription was performed using a reverse transcription kit from Promega corporation, and the reverse transcription system was 50. mu.l for mRNA extracted from the tissue, and is shown in Table 4.

TABLE 4

9. Fluorescent quantitative PCR

The cDNA obtained from the clinical samples was diluted 10-fold and subjected to fluorescent quantitative PCR using 2X SYBR Mix. Each cDNA sample was plated with three replicate wells and the reaction program was set up as follows: melting at 95 deg.C for 3 min; then annealing at 95 ℃ for 20 seconds and 60 ℃ for 30 seconds, and circulating for 40 times; then 30 seconds at 60 ℃; then 20 ℃ for 10 seconds. The reaction system is shown in Table 5.

TABLE 5

The amplification reaction was then performed using a fluorescent quantitative PCR instrument. And the GAPDH gene was used as an internal control. The primers corresponding to the genes detected in the experiment were designed by Primerbank website and are shown in Table 6.

TABLE 6

10. Embedding of tissue paraffin and sectioning of samples

The fresh tissue obtained was placed in a 7ml centrifuge tube, fixed with 4% paraformaldehyde, and placed in a freezer overnight. The next day, the mixture was dehydrated overnight by changing to 75% ethanol. On the third day, the formaldehyde-fixed tissue is placed in an embedding frame, soaked for 1 hour in 80% ethanol, then soaked for 45 minutes in 95% ethanol, and repeated twice; soaking with anhydrous ethanol for 25 min, and repeating twice; soaking with n-butanol for 1 hr, and repeating twice. The embedding frame containing the tissue sample was placed in melted paraffin for 2 hours (65 ℃), and finally the sample was embedded with a paraffin embedding machine.

The embedded tissue samples were then serially sectioned using a paraffin microtome to a thickness of 5 μm and the cut samples were developed in warm water at 42 ℃. Samples were selected to spread out as much as possible and mounted on slides. Then dried overnight (37 ℃ C.), and stored at room temperature.

11. HE staining

The paraffin-sliced samples were placed in a 65 ℃ oven for 45 minutes, then transferred to xylene, soaked twice for 10 minutes each, and then soaked twice for 10 minutes each in absolute ethanol. Then, the mixture was immersed in 95% ethanol, 75% ethanol and 50% ethanol, respectively, each for 10 minutes/time. Washing with distilled water for 1 minute, then staining with hematoxylin for 5-10 minutes, and quickly washing with tap water for 1-2 seconds. Then added to ethanol containing 1% hydrochloric acid to rapidly wash for 3 seconds, followed by rinsing with tap water for 10 minutes or more. Washing with distilled water for 2 min, dyeing with 0.5% eosin staining solution for 20 s, and soaking in 75% ethanol, 95% ethanol, 100% ethanol and xylene for two minutes. Residual xylene around the sample was wiped off with a glass wiping paper, and then a suitable amount of neutral resin in which 30% xylene was dissolved was dropped to perform mounting.

12. Immunohistochemical staining

Dewaxing the paraffin section sample in xylene, and repeating the dewaxing twice for 10 minutes/time; soaking in anhydrous ethanol twice for 10 min. Soaking in 95% ethanol, 75% ethanol, and 50% ethanol for 10 min/time. The sections were then rinsed three times with 0.01M PBS for 5 min/time. 0.01M sodium citrate buffer was placed in the staining jar in advance, and placed in a water bath to heat together to 98 ℃, and then the sections treated as described above were placed and incubated for 30 minutes. And then taking out the sample, and naturally cooling at room temperature. After the temperature had dropped to room temperature, the sections were rinsed three times for 5 minutes in 0.01M PBS, and then the samples were treated with 0.3% H2O 2/methanol solution for half an hour in order to remove endogenous peroxidase. Followed by three washes with 0.01M PBS for 5 minutes each. Absorbent paper was added in advance to the wet box and 0.01M PBS was added to reduce liquid evaporation, then the treated sample was added and a blocking-resistant solution (100. mu.l/sample) was dropped on, and the box was covered with a paper mirror and closed at room temperature for 2 hours. Removing the blocking solution, adding primary antibody to the sample, covering with a piece of lens wiping paper, and incubating in a cold storage overnight. The next day, the samples were rinsed three times with 0.01M PBS for five minutes each. Then, 100. mu.l of secondary antibody was added thereto, and the mixture was incubated at 37 ℃ for 1 hour. Followed by one 0.01M PBS rinse for 5 minutes, and two 0.05M Tris-HCl rinses for 5 minutes/time. 100. mu.l of a color developing solution was added to each sample, and the mixture was left to develop color at room temperature. And after the color development is stable, washing the sample by using tap water for 2 minutes, and placing the sample into hematoxylin for dyeing for 5-8 minutes. Followed by a 10min rinse with tap water for anti-blue, followed by drying overnight in a fume hood. On the third day, the cells were soaked in 75%, 80% and 95% ethanol for 10 minutes, respectively, and then placed in absolute ethanol for two times of 10 minutes each, and then soaked in xylene for two times of 10 minutes each. The experiment was finally mounted with a neutral resin containing 30% xylene and after the neutral resin had solidified, the sample was ready for taking pictures under a microscope.

13. Subcutaneous tumor formation of nude mice

Male nude mice of 5 weeks size purchased from shanghai seelbika laboratory animals ltd were housed in standard SPF-grade animal houses. Counting the constructed stable HCC cell strain, and quantifying the liver cancer cells to 1 × 10 with serum-free medium (DMEM)⁶Cells/50 ul, then adding thawed Matrigel gel, mixing uniformly according to the volume ratio of 1:1, and injecting the mixture to the subcutaneous side of the nude mouse, wherein the injection volume is 100 ul. Tumor volume was measured starting from one week and then every four days. Finally, mice were treated, tumor weights were measured, and samples were embedded with paraffin for subsequent testing.

14. Statistical analysis

Survival curves were drawn according to the Kaplan-Meier method and analyzed by log rank test. Statistical analysis was performed using GraphPad-Prism 5 and SPSS 22(IBM) software. Results are representative of at least three independent experiments in triplicate (expressed as mean ± standard deviation). The difference between the two groups was analyzed by student's t-test. When the statistical result is p <0.05, the result is a significant difference and is expressed by a; when the result is p <0.01, it indicates an extremely significant difference, and is denoted by x; when the result is p <0.005, it is denoted by x.

Example 1 significantly increased PPDPPF expression in pancreatic cancer tissues

In order to detect the expression level of PPDPF in pancreatic cancer tissues, the present inventors extracted mRNA from 39 pairs of paired pancreatic cancer tissues and paracancerous normal tissues, detected the mRNA expression level of PPDPF in pancreatic cancer tissues and normal pancreatic tissues using the RT-PCR method, and included GAPDH as an internal reference, and according to the detection results, 64.1% (25/39) of pancreatic cancer tissues showed that the transcription level of PPDPF was up-regulated, while the transcription level of PPDPF was down-regulated in pancreatic cancer tissues was only 14 pairs (fig. 1).

To verify the above results from protein levels, PPDPF protein expression was examined by immunohistochemistry. The results showed that the expression level of PPDPF in pancreatic cancer tissues was significantly increased compared to normal tissues (fig. 2).

To further confirm the expression pattern of PPDPF in pancreatic cancer, the present inventors performed Immunohistochemistry (IHC) on tissue chips containing 90 pairs of pancreatic cancer tissue (T) and paracancerous normal tissue (N), and examined the expression of PPDPF in tumor tissue and normal tissue. The results showed that the expression level of PPDPF was significantly increased in pancreatic cancer tissues compared to normal tissues (fig. 3). Staining results of the immuno-chips were scored using the Vectra system. The staining intensity of tumor tissue cells was scored as 0-3, 0 as no staining, 1 as weak staining, 2 as medium staining, and 3 as strong staining. And finally, calculating a comprehensive score according to the proportion of each component, wherein the score value is represented by H-score. According to statistical scores, PPDPF expression was significantly higher in pancreatic cancer tissues than in paracancerous normal tissues (p ═ 0.0003). The expression pattern of PPDPF in pancreatic cancer tissues was similar to that in liver cancer expression patterns.

The above experimental results show that in pancreatic cancer tissues, PPDPPF expression is significantly higher than that of corresponding paracancerous normal tissues.

Example 2 expression of PPDPPF is negatively correlated with Total survival

Pancreatic cancer patients were divided into two groups, low PPDPF (score < 80) and high PPDPF (score > 80) based on PPDPF expression level. The relation between PPDPPF expression and clinical pathological characteristics in 90 pancreatic cancer tissues was further analyzed. Among them, 47 cases were highly expressed and 43 cases were lowly expressed. The PPDPPF expression and clinical information were then analyzed for correlation.

The results showed that highly expressed PPDPF was closely inversely correlated with overall survival (p ═ 0.03) in pancreatic cancer patients (fig. 4). This has not been reported before.

The data indicate that PPDPPF can be used as a prognostic marker of pancreatic cancer and can play a cancer promotion role in the occurrence and development of pancreatic cancer.

Example 3 overexpression of PPDPPF in pancreatic cancer cells promotes Anchor independent growth of pancreatic cancer cells

Recent studies report that Circ-FOXM1 can up-regulate the expression levels of colon cancer-associated transfer factor 1(MACC1) and PPDPF, thereby enhancing proliferation and invasion of non-small cell lung cancer cells, indicating that PPDPF can promote proliferation and invasion of lung cancer cells. Therefore, the present inventors examined the effect of PPDPF on the anchorage-independent growth capacity of pancreatic cancer cells using a soft agarose method. According to the existing pancreatic cancer cell lines in the laboratory, lentiviruses are constructed by a calcium transfer method, then SW1990 pancreatic cancer cell lines are infected by lentiviruses of a P23 empty vector or a PPDPF overexpression vector, and a cell sorter is used for sorting out positive clones with GFP green fluorescence to study the function of PPDPF. Subsequent assessment of the efficiency of PPDPF overexpression by Western Blotting technique revealed that there was no Flag-tagged PPDPF expression in the control P23 empty vector pancreatic cancer cells, whereas PPDPF-overexpressed pancreatic cancer cells had significant Flag-PPDPF expression (fig. 5A). The experimental results showed that overexpression of PPDPF gene promoted anchorage-independent growth of pancreatic cancer cell lines (fig. 5B).

Statistical results show that PPDPF over-expression of SW1990 pancreatic cancer cells significantly improved anchorage-independent growth capacity (p < 0.005) compared to control pancreatic cancer cells, indicating that PPDPF over-expression improves anchorage-independent growth capacity of pancreatic cancer cells.

Example 4 knock-out expression of endogenous PPDPF significantly inhibited the in vitro anchorage-independent growth capacity of pancreatic cancer cells

To further demonstrate the role of PPDPF in pancreatic cancer cells, the present inventors knocked out the expression of PPDPF in the human pancreatic cancer cell lines HPAC and Miapaca2 using CRISPR/Cas9 technology. Firstly, the inventor utilizes CRISPR/Cas9 interference technology to connect two designed interference sequences to CRISPR/Cas9lentiCRIPRV2 vector, then transfers the two interference sequences into 293T as core plasmid and two packaging plasmids, collects supernatant at 48 hours, and carries out ultrafiltration concentration. Finally, the obtained virus was added to pancreatic cancer cells, and positive clones were selected by puro. The PPDPF knockout efficiency is identified by using a Western Blotting method.

The results are shown in fig. 6A, and the inventors have seen that PPDPF expression cannot be detected in two pancreatic cancer cells, indicating that the PPDPF knock-out was successful. The inventors examined the anchorage-independent growth capacity of pancreatic cancer cell lines by soft agarose method, and statistical results showed that clonogenic was significantly reduced (p < 0.005) in PPDPF-knocked-out pancreatic cancer cells compared to HPAC and Miapaca2 pancreatic cancer cells that normally expressed PPDPF (fig. 6B and C).

The results of the in vitro experiments show that the endogenous PPDPF gene knock-out can effectively inhibit the non-anchorage-dependent growth capacity of pancreatic cancer cells.

Example 5 PPDPPF promotes the in vivo tumorigenicity of pancreatic cancer cells

The above results show that PPDPF can promote the growth of pancreatic cancer cells in vitro, and in order to further clarify whether PPDPF can promote the proliferation of pancreatic cancer cells in vivo, the inventors designed a nude mouse subcutaneous tumor formation experiment to detect the effect of PPDPF on the tumor formation ability of pancreatic cancer cells in nude mice.

The inventors injected HPAC control group and PPDPF knockout group cells at the flanks of nude mice respectively, periodically monitored the tumor growth, and finally measured the tumor volume and weight. The results showed that tumors produced by PPDPF knocked-out HPAC cells grew slowly (fig. 7D) and were smaller than those produced by HPAC control cells (fig. 7A and B). Statistical results showed that endogenous PPDPF knocked-out HPAC cells produced significantly reduced tumor weight compared to HPAC control group (fig. 7C, P <0.001), and tumor volume was also significantly less than that produced by HPAC control cells (fig. 7D, P < 0.001). These data indicate that PPDPF knockout significantly inhibits tumorigenic development of pancreatic cancer cells in vivo, which is consistent with the results of in vitro experiments.

To verify the above results, the present inventors injected the constructed cells of Miapaca2 control group and PPDPF overexpression group into the flanks of nude mice, respectively, and monitored the tumor growth, and finally measured the tumor volume and weight. The results showed that none of the mice injected with Miapaca2 control cells had tumor growth, whereas all of the mice overexpressing Miapaca2 cells had significant tumor formation (fig. 8).

The data of the two cell line tumor forming experiments show that the PPDPF knockout remarkably inhibits the tumor forming capability of pancreatic cancer cells, while the PPDPF overexpression remarkably promotes the in vivo tumor forming capability of pancreatic cancer cells, and the cancer promotion effect of PPDPF in pancreatic cancer is revealed.

Example 6 PPDPF expression increases with the progression of PDAC in KRAS-G12D-driven animal models of PDAC

To further illustrate the effect of PPDPF on the development of pancreatic carcinogenesis, the present inventors constructed an animal model of Pancreatic Ductal Adenocarcinoma (PDAC) driven by KRAS-G12D. The present inventors tested 10-month old control mice (LSL-KRAS-G12D) and PDX-CRE; pancreatic tissue from LSL-KRAS-G12D mice, and immunohistochemical staining for PPDPF was performed. The pancreatic tissue of the control mice was found to be normal and essentially no expression of PPDPF was detected. While in KRAS-G12D mice, a large number of panins appeared and PPDPF expression was significantly upregulated (fig. 9), revealing that PPDPF expression increased as PDAC developed in KRAS-G12D driven mouse models of PDAC, consistent with the present inventors' clinical observations.

Example 7 PPDPF knockout inhibits the development of pancreatic cancer in transgenic mouse models

The present inventors specifically knocked out the PPDPF gene expression in pancreatic tissues (PDX-CRE; LSL-KRAS-G12D control mice and PDX-CRE; LSL-KRAS-G12D; PPDPF loxP/loxP mice), and thus observed the effect of PPDPF on pancreatic cancer at the level of transgenic mice. Since PanIN (pancreatic intraepithelial neoplasia) is present in the pancreas of 7-10 month old mice (PDX-CRE; LSL-KRAS-G12D mice), the inventors examined pancreatic tissues of 8 month old control and PPDPF KO mice. The experimental results showed that PanIN was present in the pancreas of 8-month-old control mice (PDX-CRE; LSL-KRAS-G12D mice) for the most part, but was difficult to detect in the pancreas tissue of PPDPPF KO mice (PDX-CRE; LSL-KRAS-G12D; PPDPPF loxP/loxP mice) (FIG. 10A). To further verify the above results, the present inventors performed the same test again when the mice grew to 10 months of age. The results showed that the control mice had a significant increase in PanIN number at 10 months of age compared to 8 months, while PPDPF KO significantly inhibited the formation of PanIN (fig. 10B).

This result indicates that expression of knockout PPDPF significantly reduces the pancreatic ductal adenocarcinoma pre-lesion PanIN in a mouse model of pancreatic ductal adenocarcinoma, further validating the carcinotropic effect of PPDPF in pancreatic cancer.

Example 8 overexpression of PPDPDPF in pancreatic cancer cells promotes activation of p-Erk and p-AKT308

The inventors found that formation of PanIN in mice with a pancreatic knock-out of PPDPF was significantly inhibited in a pancreatic ductal adenocarcinoma model of transgenic mice that was directly driven by KRAS, so the inventors speculated that PPDPF might affect the signaling pathway of KRAS. Therefore, the inventor utilizes the constructed over-expressed PPDPF to stabilize pancreatic cancer cell strains, and studies the influence of PPDPPF over-expression on activation of RAS downstream signal pathway molecules Erk and AKT phosphorylation under the conditions of resting state and EGF treatment. The results show that at rest, the phosphorylation levels of Erk and AKT are significantly increased when PPDPF is overexpressed in pancreatic cancer cells (fig. 11). Under EGF (10ng/mL) treatment, PPDPPF overexpression significantly increased the level of phosphorylation of AKT in HPAC cells. Background Erk phosphorylation in PPDPF-overexpressing cells was more significantly altered than AKT, although there was no significant increase in Erk phosphorylation under EGF stimulation (fig. 11A). Phosphorylation levels of Erk and AKT were activated more rapidly on average in PPDPF-overexpressed Miapaca2(KRAS G12C) and capan (KRAS wt) cells, and phosphorylation levels were higher (fig. 11B and C).

Example 9 knockdown of PPDPPF in pancreatic cancer cells significantly inhibited activation of p-Erk and p-AKT308

To further verify the above results, the inventors constructed a PPDPF knockout monoclonal strain using CRISPR/Cas9 technology, and then examined the influence of PPDPF knockout on activation of KRAS downstream signaling pathway Erk and AKT phosphorylation under resting state and EGF treatment, respectively. The results show that when PPDPF is knocked out in pancreatic cancer cells, the phosphorylation level of AKT is significantly inhibited in the resting state, and there is no significant change in the phosphorylation level of AKT even in the EGF-treated state, whereas the phosphorylation level of AKT in control cells is significantly increased after stimulation with EGF (fig. 12). Also, after PPDPF knock-out, the rise in Erk phosphorylation was also weaker than in control cells when EGF was treated (fig. 12).

In combination with the results of example 8, supra, it is suggested that PPDPF modulates activation of MAPK and PI3K signaling pathways downstream of Kas.

Example 10 overexpression of PPDPDPPF in pancreatic cancer cells promotes activation of RAS-GTP active forms

Based on the above results, the present inventors further explored whether PPDPF could directly affect activation of RAS, thereby affecting the signal path of RAS.

The inventor utilizes the constructed over-expressed PPDPF to stabilize pancreatic cancer cell strains and researches the influence of PPDPPF over-expression on RAS-GTP activity form under the conditions of resting state and EGF treatment. The results show that at resting state, when PPDPF is overexpressed in pancreatic cancer cells, the level of RAS-GTP active form is significantly upregulated. While in the EGF (5ng/mL) treated state, the level of RAS-GTP overexpressed by PPDPF rose faster, indicating that overexpression of PPDPPF promotes activation of RAS in pancreatic cancer cells by EGF (FIG. 13).

This result revealed that overexpression of PPDPF can significantly increase the level of RAS activation in pancreatic cancer cells.

Example 11 knockdown of PPDPPF in pancreatic cancer cells Miapaca2 significantly inhibited RAS activation

In view of the fact that over-expression of PPDPF can significantly promote activation of RAS, to further validate this result, the present inventors constructed a PPDPF-knocked pancreatic cancer Miapaca2 monoclonal strain using CRISPR/Cas9 technology. The inventor utilizes the constructed PPDPF knockout cell strain to detect the influence of PPDPF knockout on RAS-GTP active form in a resting state.

The PPDPPF knockout pancreatic cancer Miapaca2 monoclonal strain uses an interference sequence as follows:

sgRNA1：5’-ATGGCGGCCATCCCCTCCAG-3’(SEQ ID NO:19)；

sgRNA2：5’-CAGCAGTACCGAGTGCCCCG-3’(SEQ ID NO:20)。

the results show that when PPDPF is knocked out in pancreatic cancer cells, the level of RAS-GTP active form is significantly reduced (fig. 14).

The results of examples 10 and 11 suggest that PPDPF modulates RAS activation, thereby promoting the development of pancreatic ductal adenocarcinoma.

Example 12 screening method

(1) PPDPF expression or activity based screens

Cell: pancreatic cancer cells overexpressing PPDPF.

Test group: culturing the PPDPF-overexpressing pancreatic cancer cells and administering a candidate agent;

control group: culturing the PPDPPF overexpressing pancreatic cancer cells without administration of a candidate agent.

The PPDPF expression or activity in the test group and the control group is respectively detected and compared. If the expression or activity of PPDPF in the test group is statistically lower (e.g., 30% or less lower) than that in the control group, it indicates that the candidate is a potential agent for inhibiting pancreatic cancer.

The activity profile can be judged by observing: activated RAS signaling pathway, including RAS-GTP levels, phosphorylation levels of downstream ERK and AKT. If the RAS signaling pathway activity is obviously reduced, including the RAS-GTP level is obviously reduced, and the phosphorylation levels of downstream ERK and AKT are obviously reduced, the candidate is a potential substance for inhibiting pancreatic cancer.

(2) Screening based on pancreatic cancer cell cultures

Cell: pancreatic cancer cells overexpressing PPDPF.

Test group: culturing the PPDPF-overexpressing pancreatic cancer cells and administering a candidate agent;

control group: culturing the PPDPPF overexpressing pancreatic cancer cells without administration of a candidate agent.

The non-anchorage dependent growth capacity of pancreatic cancer cells in the test group and the control group were measured separately and compared. If the pancreatic cancer cells in the test group have a significantly lower (e.g., 30% or less) anchorage-independent growth capacity than the control group, this indicates that the candidate is a potential agent for inhibiting pancreatic cancer.

Further, if the in vivo tumorigenic capacity of the test group is significantly lower (e.g., 30% or less lower) than that of the control group, it may be judged by an in vivo tumorigenic experiment that the candidate is a potential agent for inhibiting pancreatic cancer.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Claims (14)

1. Application of pancreatic progenitor cell differentiation and proliferation factors for: as a target for suppressing pancreatic cancer; as a diagnostic or prognostic marker for pancreatic cancer; Preparation of diagnostic reagents for the diagnosis or prognosis of pancreatic cancer; or As a target for screening drugs that inhibit pancreatic cancer. 2. The application of a down-regulating agent of pancreatic progenitor cell differentiation and proliferation factor for preparing a composition for inhibiting pancreatic cancer. 3. application as claimed in claim 2 is characterized in that, described composition is also used for: Inhibits anchorage-independent growth of pancreatic cancer cells; Inhibit the tumorigenic ability of pancreatic cancer cells; Inhibit phosphorylation of Erk and/or AKT308; and/or Inhibits RAS activation. 4. application as claimed in claim 1 or 2 is characterized in that, the down-regulating agent of described pancreatic progenitor cell differentiation and proliferation factor comprises: the substance that down-regulates pancreatic progenitor cell differentiation and proliferation factor activity or down-regulates pancreatic progenitor cell differentiation and proliferation The expression, stability of a factor, or a substance that reduces the duration of its effective action. 5. application as claimed in claim 4 is characterized in that, described down-regulating agent comprises and is selected from: Reagents for knocking out or silencing pancreatic progenitor cell differentiation and proliferation factors; Binding molecules that specifically bind to pancreatic progenitor cell differentiation and proliferation factors; Chemical small molecule antagonists or inhibitors of pancreatic progenitor cell differentiation and proliferation factors; Agents that inhibit anchorage-independent growth or tumorigenesis of pancreatic cancer cells mediated by pancreatic progenitor cell differentiation and proliferation factors; Agents that inhibit the phosphorylation of Erk and/or AKT308 mediated by pancreatic progenitor cell differentiation and proliferation factors; Agents that inhibit RAS activation mediated by pancreatic progenitor differentiation and proliferation factors; or Agents that interfere with the differentiation of pancreatic progenitor cells interacting with proliferation factors and effector molecules. 6. The application according to claim 5, wherein the reagent for knocking out or silencing pancreatic progenitor cell differentiation and proliferation factor comprises: a CRISPR gene editing reagent for pancreatic progenitor cell differentiation and proliferation factor, which specifically interferes with pancreatic progenitor cell differentiation and proliferation factor. Interfering molecules for gene expression encoding progenitor cell differentiation and proliferation factors, homologous recombination reagents or site-directed mutagenesis reagents for pancreatic progenitor cell differentiation and proliferation factors, said homologous recombination reagents or site-directed mutagenesis reagents Loss-of-function mutation was performed. 7. The application according to claim 6, wherein the CRISPR gene editing reagent for pancreatic progenitor cell differentiation and proliferation factor is sgRNA, and its nucleotide sequence is such as SEQ ID NO: 19 and/or SEQ ID NO :20 shown. 8. Use of a reagent for specifically identifying or amplifying pancreatic progenitor cell differentiation and proliferation factors, for preparing a diagnostic reagent or kit for diagnosing or prognosing pancreatic cancer. 9. The use according to claim 8, wherein the reagent comprises: a binding molecule that specifically binds to the differentiation and proliferation factor protein of pancreatic progenitor cells; a primer that specifically amplifies the differentiation and proliferation factor gene of pancreatic progenitor cells ; a probe that specifically recognizes the differentiation and proliferation factor genes of pancreatic progenitor cells; or, a chip that specifically recognizes the differentiation and proliferation factor genes of pancreatic progenitor cells. 10. A pharmaceutical composition or kit for inhibiting tumors, comprising: a down-regulating agent for pancreatic progenitor cell differentiation and proliferation factor; The nucleotide sequence of the edited sgRNA is shown in SEQ ID NO:19 and/or SEQ ID NO:20. 11. A method of screening for potential substances that inhibit pancreatic cancer, the method comprising: (1) treating an expression system with a candidate substance that expresses pancreatic progenitor cell differentiation and proliferation factors; and (2) Detecting the expression or activity of pancreatic progenitor cell differentiation and proliferation factors in the system; if the candidate substance statistically down-regulates the expression or activity of pancreatic progenitor cell differentiation and proliferation factors, then the candidate substance reduces pancreatic cancer potential substances. 12. The method of claim 11, wherein the system in step (1) is a pancreatic cancer cell system; step (2) further comprises: detecting the anchorage-independent growth ability of pancreatic cancer cells in the system or clonogenicity; if its anchorage-independent growth or clonogenicity decreases, the candidate substance is a potential substance to reduce pancreatic cancer. 13. The method of claim 11, wherein the system of step (1) also expresses RAS and its downstream signaling pathway molecules Erk and AKT; step (2) further comprises: detecting activated GTP in the system -Ras levels, and/or detection of phosphorylation levels of Erk and AKT, if the level of activated GTP-Ras is significantly decreased, and/or the phosphorylation levels of Erk and AKT are significantly decreased, the candidate substance is a potential substance for reducing pancreatic cancer . 14. The method according to any one of claims 11 to 13, wherein step (1) comprises: in the test group, adding candidate substances to the expression system; and/or Step (2) includes: detecting the expression or activity of pancreatic progenitor cell differentiation and proliferation factors in the system, or detecting the anchorage-independent growth ability or cloning ability of pancreatic cancer cells, or detecting the expression of Erk and AKT in the system. Phosphorylation level; and compared with a control group, wherein the control group is an expression system without adding the candidate substance; if the candidate substance statistically down-regulates the expression or activity of pancreatic progenitor cell differentiation and proliferation factors, or Statistically reducing the anchorage-independent growth ability or clonogenic ability of pancreatic cancer cells, or reducing the level of Ras-GTP and/or phosphorylation of Erk and AKT, the candidate substance is a potential substance for reducing pancreatic cancer .

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