CN118141902B

CN118141902B - Application of PPA2 in the treatment and prognosis assessment of colorectal cancer

Info

Publication number: CN118141902B
Application number: CN202410394664.4A
Authority: CN
Inventors: 何伟玲; 王子洋; 邸宇琴; 张翔; 柯尊富; 覃家乐; 王有鹏; 叶绿兰; 温祥琼
Original assignee: First Affiliated Hospital of Sun Yat Sen University
Current assignee: First Affiliated Hospital of Sun Yat Sen University
Priority date: 2024-04-02
Filing date: 2024-04-02
Publication date: 2024-11-12
Anticipated expiration: 2044-04-02
Also published as: CN118141902A

Abstract

The invention relates to application of PPA2 in preparing colorectal cancer therapeutic drugs and/or prognosis judgment. The biomarker PPA2 which is negatively related to colorectal cancer metastasis is discovered for the first time, the metastasis risk and prognosis situation of a patient are judged by detecting the change of PPA2 in tumor tissues of the colorectal cancer patient, the metastasis risk of the patient is reduced by activating PPA2, and the treatment effect is improved. According to the invention, PPA2 is found to be low-expressed in tumor tissues through clinical colorectal patient specimens and queue follow-up data, and is negatively related to poor prognosis of patients; meanwhile, PPA2 is shown to obviously inhibit colorectal cancer metastasis through a series of in vitro function experiments and animal models, and further the function is found to be related to PPA2 negative regulation HIF1α, and PPA2 expression is activated, so that the curative effect can be enhanced, and the prognosis of a patient can be improved. The invention provides a new target spot and a new method for diagnosis, prognosis evaluation and treatment of colorectal cancer.

Description

Use of PPA2 in colorectal cancer treatment and prognosis evaluation

Technical Field

The invention belongs to the field of biological medicine, and relates to application of PPA2 in colorectal cancer treatment and prognosis evaluation.

Background

Colorectal cancer is a common tumor worldwide, with new cases and cancer-related mortality rates third among both men and women in the united states. However, colorectal cancer is hidden, 25% of patients have distant metastasis at the time of treatment, and half of patients finally have distant metastasis in the treatment process, so that the traditional curative effects of operation, radiotherapy and chemotherapy and the like are poor in curative effect on metastatic colorectal cancer, and the survival rate of 5 years is only 6%. Thus, the discovery of key functional targets and effective biomarkers for colorectal cancer metastasis is of great importance for improving colorectal cancer patient prognosis.

Inorganic pyrophosphatase 2 (Inorganic Pyrophosphatase, ppa 2) belongs to the family of inorganic pyrophosphatases and catalyzes the hydrolysis of pyrophosphates to inorganic phosphates, which is critical for cellular phosphate metabolism. Inorganic pyrophosphates are byproducts of several enzymatic reactions in cells, including DNA and RNA synthesis, fatty acid and amino acid activation, etc., which are not efficiently exported across cell membranes. Inorganic pyrophosphatase activity has been reported to be critical for proper regulation of mitochondrial membrane potential and mitochondrial function, and PPA2 mutations may trigger some severe cardiomyopathy. However, the effect of PPA2 in tumors is currently being studied.

Disclosure of Invention

Aiming at the problems of high metastasis tendency but difficult reasonable evaluation in advance in colorectal cancer in the prior art, the invention carries out in-depth research on colorectal cancer metastasis mechanism, makes clear that PPA2 is an important target point related to colorectal cancer metastasis, predicts metastasis risk by detecting PPA2 expression level in tumor tissues of patients, enhances treatment effect by improving PPA2 expression, improves prognosis, provides a new strategy for colorectal cancer treatment scheme and provides scientific basis for prognosis evaluation.

In order to solve the technical problems, the invention is realized by the following technical scheme.

The first aspect of the invention provides the use of PPA2 for the manufacture of a medicament for the treatment of colorectal cancer.

In a second aspect the invention provides the use of PPA2 for the manufacture of a product for use in the assisted diagnosis and/or prognosis evaluation of colorectal cancer.

Preferably, the index of prognosis evaluation is selected from one or more of colorectal cancer cell metastasis risk and survival rate.

In a third aspect, the invention provides the use of an agent for detecting PPA2 expression levels in the manufacture of a composition for use in the assisted diagnosis and/or prognosis evaluation of colorectal cancer.

Preferably, the reagent for detecting the expression level of PPA2 comprises a primer for detecting the expression level of PPA2 gene and/or a reagent for detecting the expression level of PPA2 protein.

Preferably, the primer for detecting the expression level of PPA2 gene is selected from the following primer pairs: the upstream primer sequence of the primer pair is shown as SEQ ID NO:1 (aagaagttcaaac cgggttacc), the sequence of the downstream primer is shown in SEQ ID NO:2 (gcaacggaaagggc tatcaga).

Preferably, the reagent for detecting the expression level of PPA2 protein is selected from PPA2 monoclonal antibodies and/or PPA2 polyclonal antibodies.

Preferably, the anti-PPA2 antibody for detecting the expression level of PPA2 protein can be selected from one or more of ab180859 (abcam), MA5-26973 (Thermo FISHER SCIEN TIFIC) and 16662-1-AP (proteintech).

In a fourth aspect, the invention provides a pharmaceutical composition for the treatment of colorectal cancer comprising one or more of PPA2 nucleotides, PPA2 proteins, and pharmaceutically acceptable excipients.

Preferably, the pharmaceutically acceptable pharmaceutical excipients are selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants and colorants.

In a fifth aspect, the invention provides a kit for use in the prediction and/or prognosis of colorectal cancer efficacy, comprising reagents for detecting PPA2 expression levels; and one or more of PCR enzymes, PCR buffers, dNTPs, fluorogenic substrates.

Preferably, the fluorogenic substrate is selected from Syber Green or a fluorescently labeled probe.

It is to be understood that, unless otherwise specified, in the context of the present invention, PPA2 includes PPA2 nucleotides as well as PPA2 proteins encoded by PPA2 nucleotides. The PPA2 inhibitor is a substance capable of specifically down-regulating the PPA2 expression level and/or the transcription level of mature mRNA thereof and/or the expression level or activity of PPA2 protein, and for example, the PPA2 expression level and/or activity can be down-regulated by antisense oligonucleotide, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponge, MIRNA ERASERS, TARGET MASKING and/or multi-target or the like, so long as the reduction of the PPA2 level and/or activity can be achieved. The primer and/or primer pair refers to a PC R primer for synthesizing a PPA2 gene cDNA strand in PCR, thereby being used for detecting the expression level of PPA2 gene mRNA. In addition to the primers and/or primer pairs listed in the present invention, it is fully within the ability of one skilled in the art to design corresponding primers and/or primer pairs based on the gene sequence of PPA2 by means of conventional methods in the art including, but not limited to, molecular biology, etc., and to screen the designed primers and/or primer pairs by means of conventional experiments, as long as specific detection of PPA2 expression levels can be achieved; the PPA2 protein expression level may also be detected using reagents and methods conventional in the art.

Compared with the prior art, the invention has the following technical effects:

(1) According to the invention, the PP A2 is found to be a gene which is extremely related to colorectal cancer metastasis through clinical colorectal cancer patient specimens and queue follow-up data, and the expression level of the PP A2 in colorectal cancer tissues is obviously reduced. The expression level of PPA2 exhibits a clear negative correlation with the poor prognosis of colorectal patients, and can be used to assess the risk of metastasis and prognosis of colorectal patients to guide the formulation of treatment regimens.

(2) The invention proves that PPA2 can inhibit the transfer capacity of colorectal cancer cells through a series of in-vivo and in-vitro experiments. Can obviously inhibit colorectal metastasis by promoting PPA2 expression and improve the treatment effect. The correlation between PPA2 and colorectal cancer metastasis is revealed, so that the correlation has important practical significance for solving the problems of clinical curative effect difference and blank prognosis evaluation among individuals and better realizing accurate treatment. Provides a new drug treatment target for human to attack colorectal cancer, thereby providing a new direction for subsequent drug research and development, clinical treatment and the like, and having extremely high social value and market application prospect.

Drawings

FIG. 1 is a schematic representation of the results of immunohistochemical detection of cancer and paracancer PPA2 expression in colorectal cancer patients.

FIG. 2 is a schematic diagram showing the results of WB detection in colorectal cancer patients for cancer and paracancer PPA2 expression.

FIG. 3 is a schematic representation of the expression results of PPA2 in cancer and paracancestral normal tissues of colorectal cancer patients in TCGA and GEO colorectal cancer databases.

FIG. 4 is a schematic representation of the results of immunohistochemical detection of PPA2 expression in tissues of patients with metastatic and non-metastatic colorectal cancer.

FIG. 5 is a graph showing the effect of PPA2 expression levels in the TCGA colorectal cancer database on prognosis at early and late stages of tumor, respectively.

FIG. 6 is a graph showing the effect of PPA2 expression in tumor tissue of colorectal cancer patients on patient survival.

FIG. 7 is a schematic diagram showing the detection results of the knockdown efficiency of shRNA on PPA2 and the overexpression efficiency of an overexpression vector on PPA 2.

FIG. 8 is a graph showing the effect of hypoxia treatment on Transwell migration of colorectal cancer stable transgenic cells.

FIG. 9 is a graph showing the effect of hypoxia treatment on scratch migration in stable colorectal cancer transformants.

FIG. 10 is a graph showing the effect of WB assay on HIF1α expression levels in colorectal cancer stable transformants.

FIG. 11 is a graph showing the effect of PPA2 knockdown on a mouse spleen injected colorectal cancer cell liver metastasis model.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention more clear and clear, the present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Without being limited by any particular explanation, the cell lines listed in the context of the present invention, including HEK293T, DLD, SW1116, etc., were all cultured according to the prior art, all identified by short tandem repeat analysis by the chinese collection of typical cultures (wuhan) and verified for the presence of mycoplasma contamination using the PCR detection kit (shanghai Biothrive Sci), while being stored in liquid nitrogen and used in subsequent experiments. All of the reagents used in the present invention are commercially available. For the use of clinical specimens, the patient signs informed consent, and related procedures and methods accord with medical ethics requirements and quality management specifications of clinical tests of medicines. The experimental methods used in the present invention, such as nucleic acid extraction, whole genome sequencing, primer design, immunohistochemistry, western blot, cell experiments, and the like, are all conventional methods and techniques in the art. Representative results of selection from the biological experimental replicates are presented in the context figures, and data are presented as mean±sd and mean±sem as specified in the figures. All in vitro experiments were repeated at least three times and animal experiments were repeated twice. The data were analyzed using GRAPHPAD PRISM 8.0.0 software. And comparing the average value difference of two or more groups by adopting conventional medical statistical methods such as t-test, chi-square test, analysis of variance and the like. * p < 0.05 was considered a significant difference.

Example 1

A biopsy sample of colorectal cancer patients attached to the first hospital at the university of Zhongshan was taken, and the patient inclusion criteria were as follows: (1) age 18 years or older (including 18 years old), both male and female; (2) Histological or pathological evidence of colorectal cancer in patients (all stages I-IV); (3) Collecting paraffin sections prepared from fresh tumor biopsies; (4) expected lifetime of 3 months or more. Firstly, detecting PPA2 expression level in tumor tissues and paracancerous tissues of a patient by adopting immunohistochemistry, wherein the specific steps are as follows:

(1) Embedding the biopsy sample tissue into paraffin for sealing, preserving the paraffin sample at room temperature, and preserving the cut white slices in a refrigerator at 4 ℃.

(2) And during experimental detection, taking out the paraffin section, and placing the paraffin section in a baking oven at 60 ℃ for baking for about 1h, so that the section is attached to a glass slide.

(3) The sections were successively placed in two portions of xylene for dewaxing for 10 minutes each.

(4) The sections were sequentially put into gradient alcohol (100%, 90%, 80%, 70%) for hydration treatment for 5min each time, and then washed three times with PBS for 3min each time.

(5) Immersing the slice in EDTA antigen retrieval liquid, and heating for 15min by using high fire in a microwave oven to carry out antigen retrieval. After heating, the sections were allowed to cool naturally to room temperature in antigen retrieval solution, washed 3 times with PBS for 3min each.

(6) The slices are placed in a light-proof wet box, a small amount of distilled water is added, 3% hydrogen peroxide is used for covering tissues, and the slices are placed for 20min at room temperature, so that endogenous peroxidase is thoroughly removed.

(7) PBS was washed three times, each for 3min. The goat serum blocking solution is dripped to cover the tissues, and the inside of the wet box is blocked for 30min to reduce the nonspecific binding of the primary antibody.

(8) Removing the blocking solution, directly adding primary antibody to cover tissues without cleaning, and placing in a wet box at 4 ℃ for blocking overnight.

(9) The next day was left at room temperature for 30min, then washed with PBS for 3min, and repeated 3 times.

(10) The biotin-labeled secondary antibody was added dropwise, incubated at room temperature for 30min, washed with PBS for 3min, and repeated 3 times.

(11) DAB is dripped for color development, and the reaction is stopped after the color development is completed by putting the mixture into water.

(12) Dripping hematoxylin for dyeing for 3min for counterstaining, and putting into water for stopping coloring; then 1% hydrochloric acid alcohol is added dropwise for differentiation for 3s, and the mixture is washed in running water for 30-45min until the color of the slice is stable for blue-turning treatment.

(13) Gradient alcohol dehydration: soaking in 70%, 80%, 90%, 100% ethanol and xylene sequentially for 5min each time, and sealing.

Clinical pathology immunohistochemical sections of patients were scored by two independent researchers, respectively, on the premise of unknown patient information. Staining scores were determined based on staining intensity and positive cell proportion. The staining intensity was scored as follows: 0 (no staining), 1 (shallow), 2 (medium) and 3 (strong). The PPA2 positive cell proportion was scored as follows: 1 (< 25%), 2 (25-50%), 3 (50-75%) and 4 (75-100%). The final fraction was obtained by multiplying the staining intensity and the proportion of positively stained cells, and the detection results are shown in fig. 1. The results show that PPA2 expression levels in cancer tissues of colorectal cancer patients are significantly lower than normal tissue expression levels (< 0.001).

Subsequently, the expression level of PPA2 in colorectal cancer tissues and paracancerous tissues was further verified by Western Blot, and the specific steps are as follows:

(1) 250mg of colorectal cancer or paracancestral tissue was weighed into an EP tube, washed twice with PBS, and then cell lysates containing 1X protease inhibitor and phosphatase inhibitor were added at 250 mg/mL. The glass homogenizer was homogenized at low speed for 30 seconds each time with an ice bath for 1 minute between each time until the tissue was completely lysed. Placing on ice for cracking for 30min, and shaking every 10 min. After sufficient lysis, the supernatant was immediately transferred to a new centrifuge tube by centrifugation at 14000rpm at 4℃for 15 minutes.

(2) After protein quantification, 5 Xprotein loading buffer is added according to the sample volume, and the mixture is uniformly mixed, and water bath is carried out for 5min at 95 ℃.

(3) And (3) carrying out protein electrophoresis on the protein sample obtained in the step (2), setting the initial voltage to 80V, and adjusting the voltage to 120V when the protein Marker strips are obviously separated.

(4) And (3) transferring the membrane after the protein electrophoresis is finished, firstly, placing the protein gel into a membrane transferring liquid for balancing, then adding a proper amount of membrane transferring liquid into a container, sequentially placing a sponge, filter paper, a PVD F membrane (activated by methanol), the gel, the filter paper and the sponge, removing gas, and placing a membrane transferring groove into ice water, wherein the membrane transferring condition is 300mA constant flow for 1h.

(5) After the film transfer, the film is placed in 5% milk and is sealed in a shaker for 1h at room temperature.

(6) After the blocking, the membrane was washed once with 1 XTBE buffer, the desired band was cut, the corresponding primary antibody was added and placed on a 4℃shaker overnight.

(7) The primary antibody was recovered and the membrane was washed three times with 1 XTBE buffer for 5min each.

(8) The secondary antibody was incubated at room temperature for 1h, then the membrane was washed three times with TBST buffer for 5min each.

(9) After the completion of the film washing, chemiluminescence was performed, ECL luminescence was prepared (a: b=1:1), and luminescence development was performed using a Biorad chemiluminescent apparatus.

The experimental detection results are shown in fig. 2. The results showed that, consistent with the tissue IHC analysis results described above, PPA2 expression levels in colorectal cancer tissues were significantly reduced. Analysis was performed by using the TC GA and GSEA databases, grouped into carcinoma (T) and paracancerous normal tissue (N), and the results are shown in FIG. 3. The results showed that, consistent with the analysis of the clinical samples described above, PPA2 expression levels in colorectal cancer tissues were significantly lower than that in paracancerous normal tissues (< p < 0.001).

Further, samples of patients with metastatic and non-metastatic colorectal cancer were collected, and immunohistochemical staining analysis was performed according to the method described above, and the results are shown in fig. 4. The results show that PPA2 expression levels in tumor tissue of patients in the metastatic group were significantly lower than those in the non-metastatic group (< 0.001). The results are shown in FIG. 5 by analysis using TCGA colon (READ) and rectal Cancer (COAD) databases, and early tumor (I/II) and late tumor (III/IV) groupings. The results show that PPA2 is inversely related to the late poor prognosis of the tumor, while there is no obvious correlation to the early prognosis of the tumor. PPA2 is suggested to potentially affect prognosis by affecting metastasis of advanced colorectal tumors.

In the previous experiments it was clear that PPA2 is low expressed in colorectal cancer tissue and high in normal tissue, and that PPA2 expression levels in colorectal cancer tissue are inversely related to tumor metastasis. In this regard, to clarify the effect of PPA2 on survival of colorectal cancer patients, it was found by analysis of follow-up data from patients with colorectal cancer in the first hospital affiliated with the university of zhong mountain that patients could benefit significantly from high expression of PPA2, and Overall Survival (OS) of patients exhibiting high expression of PPA2 levels in colorectal cancer tissues was significantly higher than those with low expression of PPA2 levels (< 0.05) (see fig. 6).

Example 2

The effect of PPA2 expression levels on colorectal cancer patient survival and therapeutic efficacy was demonstrated in clinical samples in the foregoing examples. To investigate the effect of PPA2 on colorectal cancer in depth, further studies were performed by a series of in vitro experiments.

First, shRNA for reducing expression level of PPA2 in cells and over-expression plasmid for increasing expression level of PPA2 in cells were designed and respective activities were verified, specifically as follows:

(1) Designing shRNAs knocking down sequences in a Sigma website, designing primers according to a shRNA small hairpin structure, synthesizing the primers, diluting the synthesized shRNAs primers to 100 mu M by adding water, taking 10 mu L of the upstream and downstream primers respectively, uniformly mixing, using a PCR instrument to gradient and cool from 100 ℃ to room temperature, annealing to synthesize double chains, and transferring the double chains to a pLKO.1 lentiviral vector; in this example, two shRNAs were taken as examples, shPPA2#1 (SEQ ID NO:3, GATCATTAGTTGAATCGGTAT) and shPPA2#2 (SEQ ID NO:4, GCCTCTTCTTTAAGAATGTAA), respectively; PPA2 (sequence shown as SEQ ID NO: 5) was cloned into pCDH-Flag eukaryotic cells to construct an overexpression vector.

(2) Carrying out double enzyme digestion on pLKO.1 and pCDH-Flag by adopting EcoRI and AgeI endonuclease respectively, carrying out enzyme digestion, and then, running glue to obtain two single strips, and cutting large strips for glue recovery; the annealed duplex is then ligated to the support after gel recovery.

(3) 293T cells in the logarithmic growth phase are inoculated in a 10cm culture dish, and the inoculation density is about 70%; 6000ng of the objective plasmid loaded with shPPA2#1, shPPA2#2 or PPA2, 4500ng of PSPAX2 and 1500ng of PMD2.G were transfected into 293T cells and cultured for 48h.

(4) Collecting culture medium after 48 hr, filtering cell debris with 0.45 μm filter to obtain virus solution, packaging, and preserving at-80deg.C. The day before infection, colorectal cancer cells (DLD 1, SW 1116) to be infected in the logarithmic growth phase are inoculated to a 6cm culture dish, the original culture medium is discarded, 3mL of virus liquid is added, 3uL of polybrene is added to promote virus infection, the virus liquid is discarded after 24 hours, and the fresh culture medium is replaced for continuous culture.

(5) After 48h of infection, the culture medium is discarded, a culture medium with proper concentration of puromycin is added, at the moment, cells which successfully integrate exogenous genes have puromycin resistance and can survive, stable cell strains can be obtained after continuous screening for 7-10 days, and whether the PPA2 expression level is knocked down or over-expressed successfully is determined by qPCR or WB detection.

The method in which the expression level of PPA2 in colorectal cancer cells is detected using WB is the same as in example 1; the method for detecting PPA2 expression level in colorectal cancer cells by qPCR is specifically as follows:

(1) After washing the cells with PBS, 1mL Trizol was added to each well and left at room temperature for 2min to allow the lysate to evenly distribute on the cell surface and lyse the cells. The cells were detached by pipetting gun blow and subsequently transferred to 1mL centrifuge tubes without rnase.

(2) Standing at room temperature for 5min, centrifuging at 4deg.C at 10000 Xg for 15min, absorbing supernatant, adding chloroform, shaking vigorously, and standing at room temperature for 3min.

(3) Centrifuging at 4deg.C and 10000 Xg for 15min, carefully sucking the water phase layer into a new centrifuge tube, adding equal volume of isopropanol, mixing, and standing at room temperature for 10min.

(4) Centrifuge at 4℃for 10min at 10000 Xg, remove supernatant and wash with 1mL of 75% ethanol (DEPC water).

(5) Centrifugation was performed at 7500 Xg at 4℃for 5min, the supernatant was discarded, the pellet was dried by uncapping, 12. Mu.L of DEPC water was added, and RNA was dissolved by incubation at 60℃for 10min and the concentration was measured. Total RNA was reverse transcribed into cDNA using HIS CRIPT II reverse transcriptase (Vazyme #R201-01) while genomic DNA was removed. Based on the measured RNA concentration, the reaction system was calculated, as shown in Table 1 below.

TABLE 1 reverse transcription reaction system

(6) According to 1:5 as a template, qPCR was performed using ChamQ SYBR QPCR MASTER Mix (Vazyme #q712) from nupran; the 96/384 Kong Yingguang quantitative PCR instrument was Biorad. Wherein the reaction system configuration (for example, total volume of 20. Mu.L) is as follows: 2X Taq Pro Universal SYBR QPCR MASTER Mix 10. Mu.L, upstream primer 0.4. Mu.L, downstream primer 0.4. Mu.L, cDNA 2. Mu.L, ddH2O 7.2. Mu.L; the primer sequences and amplification procedures used are shown in tables 2 and 3 below.

TABLE 2 primer sequences

TABLE 3qPCR amplification procedure

The detection results are shown in FIG. 7. The results show that the two shRNAs designed according to the invention can generate obvious inhibition activity on PPA2, reduce the expression level of PPA2 protein and RNA in colorectal cancer cells, and the PPA2 over-expression vector can obviously increase the expression level of PPA2 protein and RNA in colorectal cancer cells.

The effect of differences in PPA2 expression levels on colorectal cancer cell metastasis was then studied using a Transwell experiment and a scratch experiment, wherein the Transwell experiment was performed as follows:

(1) shNT, shPPA2, vector and PPA2 OE colorectal cancer cells constructed in the above method were digested, and then the cells were resuspended in serum-free medium, counted and the cell density was adjusted to 100 ten thousand/mL.

(2) A Transwell culture cell with a pore size of 8.0 μm was placed in a 24-well plate, 600. Mu.L of a culture solution containing 10% fetal bovine serum was added to the lower layer of the cell, 100. Mu.L of the serum-free cell suspension counted in step (1) was added to the cell, one set was cultured under normal conditions (37℃5% CO2 incubator), and the other set was cultured in an anaerobic incubator for 48 hours.

(3) The Transwell chamber was removed, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, and the microporous membrane was carefully wiped off with a cotton swab to remove the upper cells, washed twice with PBS, photographed under a microscope, and the lower cells were counted.

The detection results are shown in FIG. 8. The results show that shPPA a2 slightly promotes tumor cell migration under normal oxygen conditions, and the effect is more remarkable under anoxic conditions, whereas PPA 2OE can remarkably inhibit tumor cell migration under anoxic conditions.

The scratch test comprises the following specific steps:

(1) The marker pen is adopted to draw a transverse line on the back of the 6-hole plate by comparison with a ruler, and the transverse line is crossed at intervals of about 0.5 cm to 1 cm. Each hole passes through at least 5 lines.

(2) About 200 ten thousand cells were added to the wells, and the specific number was varied from cell to cell, and the wells were kept overnight to allow for well bottom filling.

(3) The gun head is vertical and can not incline compared with the ruler in the next day, and the gun head is vertical to the transverse line scratch on the back as much as possible.

(4) The cells were washed 3 times with PBS, the streaked cells were removed, and serum-free medium was added.

(5) One group was cultured under conventional conditions (37 ℃, 5% co ₂ incubator) and the other group was cultured in anaerobic incubator. Samples were taken at 0, 12, 24 hours, observed under a microscope and photographed.

The detection results are shown in FIG. 9. The results show that shPPA a2 slightly promotes tumor cell migration under normoxic conditions, and this effect is more pronounced in hypoxic conditions (p < 0.001), whereas PPA2 OE inhibits tumor cell migration more pronounced in hypoxic conditions (p < 0.001).

Hif1α is a key molecule of cell response to hypoxia, and is subject to continuous ubiquitination degradation under normoxic conditions, and is stable under hypoxic conditions, and is transcribed to activate a plurality of downstream target genes, thereby playing important roles in promoting angiogenesis, cell proliferation, metastasis, metabolic reprogramming and the like. Hif1α has been reported to be abnormally upregulated in a variety of tumors and is closely related to various malignant behaviors of tumor cells. Therefore, shNT, shPPA2 (shPPA. Sup. 2# 1), vector, PPA2 OE colorectal cancer intracellular HIF1α expression levels were examined under normoxic and anoxic conditions according to the WB detection method described in example 1, and the results are shown in FIG. 10. The results show that under hypoxic conditions, hif1α expression levels were significantly increased in shPPA cells compared to shNT cells, and significantly decreased in PPA2 OE cells compared to Vector cells.

Further, the effect of PPA2 on tumor metastasis in vivo was studied as follows:

(1) Female C57BL/6J mice of 6 weeks of age were housed in an SPF environment.

(2) After digestion and PBS resuspension, colorectal cancer cells of shNT and shPPA2 (shPPA # 2#1 and shPPA2# 2) were counted and adjusted to 2500 ten thousand/mL. A transverse incision of about 1cm is made at the skin position about 1-2cm below the left chest rib, and the skin layer and the muscle layer are sheared.

(3) The pancreas tail adhered with the spleen is clamped and pulled out of the body together with the spleen, the insulin needle sucks the uniformly mixed cell suspension, and the cell suspension is slowly injected into 100 mu L, so that the color of the spleen is lightened, the needle head is slowly withdrawn, and the spleen and fat are slightly pushed back to the abdominal cavity. The muscle layer and the skin layer were sutured separately, and after about two months, the mice were anesthetized, sacrificed and dissected for liver observation and detection.

The detection results are shown in FIG. 11. The results show that shPPA2 can significantly promote colorectal cancer cell liver metastasis.

As is clear from the above, PPA2 is a gene related to colorectal cancer metastasis, and the lower expression level of PPA2 has obvious positive correlation with poor prognosis, so that the PPA2 can be used for predicting colorectal cancer metastasis risk and prognosis, and provides a reference for the patient to specify a treatment scheme. Meanwhile, the PPA2 expression is promoted to have an important function of inhibiting colorectal cancer metastasis, and accordingly, the metastasis risk of a patient can be reduced by improving the expression level of PPA2 in vivo, and the prognosis of the patient can be improved; and further finds that the function is related to PPA2 negative regulation of HIF1a, and the invention also provides a new thought for research of targeted HIF1a treatment. The correlation between PPA2 genes and colorectal cancer metastasis is revealed, so that the correlation has important significance for solving the problem of blank metastasis risk assessment among individuals, and is beneficial to better realizing accurate treatment. Provides a new drug treatment target for human to attack colorectal cancer, thereby providing a new direction for subsequent drug research and development, clinical treatment and the like, and having extremely high social value and market application prospect.

The above detailed description describes the analysis method according to the present invention. It should be noted that the above description is only intended to help those skilled in the art to better understand the method and idea of the present invention, and is not intended to limit the related content. Those skilled in the art may make appropriate adjustments or modifications to the present invention without departing from the principle of the present invention, and such adjustments and modifications should also fall within the scope of the present invention.

Claims

1. Use of PPA2 gene and/or protein in the preparation of drugs for treating colorectal cancer.

2. Use of the PPA2 gene and/or protein in the preparation of products for auxiliary diagnosis and/or prognosis assessment of colorectal cancer.

3. The use according to claim 2, characterized in that the prognostic evaluation indicator is selected from one or more of the risk of colorectal cancer cell metastasis and survival rate.

4. Use of a reagent for detecting PPA2 expression level in the preparation of a composition for auxiliary diagnosis and/or prognosis evaluation of colorectal cancer, wherein the reagent for detecting PPA2 expression level comprises primers for detecting PPA2 gene expression level and/or a reagent for detecting PPA2 protein expression level.

5. The use according to claim 4, characterized in that the primers for detecting the expression level of the PPA2 gene are selected from the following primer pair: the upstream primer sequence of the primer pair is shown in SEQ ID NO: 1, and the downstream primer sequence is shown in SEQ ID NO: 2.

6. The use according to claim 4, characterized in that the reagent for detecting the expression level of PPA2 protein is selected from PPA2 monoclonal antibody and/or PPA2 polyclonal antibody.

7. The use according to claim 4, characterized in that the prognostic evaluation index is selected from one or more of the risk of colorectal cancer cell metastasis and survival rate.