CN105648042B - Uses of Human YEATS4 Gene and Related Drugs - Google Patents

Abstract

The invention discloses the use of human YEATS4 gene and related medicines. The invention discloses the application of human YEATS4 gene in tumor treatment, tumor diagnosis and drug preparation. The present invention further constructs human YEATS4 gene small molecule interfering RNA, human YEATS4 gene interfering nucleic acid construct, and human YEATS4 gene interfering lentivirus and discloses their uses. The siRNA or the nucleic acid construct comprising the siRNA sequence and the lentivirus provided by the present invention can specifically inhibit the expression of the human YEATS4 gene, especially the lentivirus, which can efficiently infect the target cells and efficiently inhibit the expression of the YEATS4 gene in the target cells. , thereby inhibiting the growth of tumor cells and promoting tumor cell apoptosis, which is of great significance in tumor therapy.

Description

Uses of Human YEATS4 Gene and Related Drugs

technical field

The present invention relates to the field of biotechnology, and more particularly to the use of human YEATS4 gene and related medicines.

Background technique

RNA interference (RNAi) refers to post-transcriptional gene silencing using short double-stranded RNA (dsRNA) composed of nucleotides. It can efficiently and specifically block the expression of specific genes in the body, leading to their degradation, thereby causing the silencing of specific genes in the organism and causing cells to show the loss of a certain gene phenotype. It is a commonly used research gene emerging in recent years. Functional, laboratory techniques for finding cures for diseases. Studies have shown that double-stranded RNA with a length of 21-23 nt can specifically cause RNAi at the transcriptional and post-transcriptional levels (Tuschl T, Zamore PD, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to23nucleotideintervals. Cell 2000;101:25-33.). Although cancer patients have undergone chemotherapy, radiotherapy and comprehensive treatment, the five-year survival rate is still very low. If the genes related to the onset and progression of tumors can be intervened, it will open up new ways for tumor treatment. In recent years, RNAi has become an effective strategy for gene therapy of tumors. RNAi technology can inhibit the expression of proto-oncogenes, mutated tumor suppressor genes, cell cycle-related genes, and anti-apoptosis-related genes to inhibit tumor progression (Uprichard, Susan L. The therapeutic potential of RNA interference. FEBS Letters 2005; 579: 5996-6007.).

YEATS4 (proliferating cell nuclear antigen-binding protein, PAF15) is a protein encoded by the YEATS4 gene, which is located in the 31 band (15q22.31) of the 22 region of the long arm of human chromosome 15. It contains 5 exons and encodes 111 amino acids. Among them, the 15th lysine and the 24th lysine are mono/diubiquitinated, and the 63rd lysine is monoubiquitinated (neXtProt/Ensembl/Reactome). It is mainly located in the nucleus, with a small amount of distribution in the perinuclear region, and is highly evolutionarily conserved among different species (NCBI Reference Sequence: NP_001025160.1). The YEATS4 protein was only expressed in the S and G2 phases of the cell cycle, peaked in the G2/M phase, and declined sharply at the end of mitosis. The expression of this protein is also tissue-specific, with high expression in liver, pancreas and placenta. YEATS4 plays a role in regulating DNA repair during DNA replication. When DNA is damaged, the 15th and 24th lysines of YEATS4 are monoubiquitinated, recruiting DNA polymerase to synthesize DNA across the damage gap to achieve repair. Overexpression of this protein promotes the repair of DNA damage caused by UV irradiation and prevents cell death, while inhibition of its expression hinders DNA replication and cell survival [Simpson F, Lammerts van Bueren K, Butterfield N et al. The PCNA-associated factor YEATS4/p15(PAF) binds the potential tumor suppressor product p33ING1b. Exp Cell Res 2006;312:73–85. Turchi L, Fareh M, Aberdam E et al. ATF3 and p15PAF are novel gatekeepers ofgenomic integrity upon UV stress. Cell Death Differ 2009 16:728–37. van Bueren KL, Bennetts JS, Fowles LF, Berkman JL, Simpson F, Wicking C. Murine embryonic expression of the gene for the UV-responsive protein p15 (PAF). Gene ExprPatterns 2007;7:47–50 .].

There are few reports on YEATS4 in tumor-related fields. In order to study the correlation between YEATS4 and tumors, the present invention selects a colon cancer cell model, and uses RNAi as a means to study the role of YEATS4 in the occurrence and development of colon cancer.

SUMMARY OF THE INVENTION

The purpose of the present invention is to disclose therapeutic methods and medicines related to human YEATS4 gene, and to study the role of YEATS4 gene in tumor cell survival and apoptosis by means of RNA interference (RNAi).

In the first aspect of the present invention, the role of YEATS4 gene in the occurrence and development of tumors is studied by means of RNA interference, and a method for inhibiting or reducing tumor cell growth, proliferation, differentiation and/or survival is disclosed, and the method includes: A molecule capable of specifically inhibiting the transcription or translation of the YEATS4 gene, or the expression or activity of the YEATS4 protein is administered to tumor cells, thereby inhibiting the growth, proliferation, differentiation and/or survival of tumor cells.

The tumor cells are selected from tumor cells whose growth is associated with the expression or activity of the YEATS4 protein. Preferably, the tumor cells are selected from colon cancer.

In the method of inhibiting or reducing tumor cell growth, proliferation, differentiation and/or survival, the molecule is administered in an amount sufficient to reduce the transcription or translation of the YEATS4 gene, or to reduce the expression or activity of the YEATS4 protein. Further, the expression of the YEATS4 gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

The molecules may be selected from, but not limited to, nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

The nucleic acids include, but are not limited to, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, endoribonuclease III-produced small interfering RNA (esiRNA), or short hairpin RNA (shRNA).

The double-stranded RNA, ribozyme, esiRNA or shRNA contains the promoter sequence of the YEATS4 gene or the information sequence of the YEATS4 gene.

Further, the double-stranded RNA is small interfering RNA (siRNA). The small interfering RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer together, and the sequence of the first strand is continuous with 15-27 in the YEATS4 gene The nucleotide sequences are basically the same. The small interfering RNA can specifically bind to the mRNA fragment encoded by the target sequence, and specifically silence the expression of the human YEATS4 gene.

Further, the first strand sequence of the small interfering RNA is substantially the same as the target sequence in the YEATS4 gene. Preferably, the target sequence in the YEATS4 gene contains the sequence shown in any of SEQ ID NOs: 1-5.

The target sequence in the YEATS4 gene is the fragment in the YEATS4 gene corresponding to the mRNA fragment complementary to the small molecule interfering RNA when the small molecule interfering RNA specifically silences the expression of the YEATS4 gene.

Preferably, the YEATS4 gene is derived from human.

The first aspect of the present invention also discloses the use of the isolated human YEATS4 gene in the preparation or screening of tumor therapeutic drugs, or in the preparation of tumor diagnosis drugs.

Further, the tumor is selected from colon cancer.

The use of the isolated YEATS4 gene for the preparation or screening of tumor therapeutic drugs includes two aspects: first, the YEATS4 gene is used as the target of the drug or preparation on tumor cells to prepare tumor therapeutic drugs or preparations; The YEATS4 gene is used as the target of drugs or preparations for tumor cells to be used to screen tumor therapeutic drugs or preparations.

The use of the YEATS4 gene as a drug or preparation targeting tumor cells in the preparation of tumor therapeutic drugs or preparations specifically refers to: using the YEATS4 gene as the target of RNA interference to develop drugs or preparations for tumor cells, thereby reducing Expression level of YEATS4 gene in tumor cells.

The use of the YEATS4 gene as a drug or preparation for the action target of tumor cells in the screening of tumor therapeutic drugs or preparations specifically refers to: using the YEATS4 gene as an object of action, the drugs or preparations are screened to find a gene that can inhibit or promote the human YEATS4 gene. The expressed drug is used as an alternative drug for tumor treatment. The YEATS4 gene small interfering RNA (siRNA) according to the present invention is obtained by screening the human YEATS4 gene as the target, and can be used as a drug with the effect of inhibiting the proliferation of tumor cells. In addition, such as antibody drugs and small molecule drugs, the YEATS4 gene and its protein can also be used as targets.

The use of the YEATS4 gene for the preparation of a tumor diagnostic drug refers to using the YEATS4 gene expression product as a tumor diagnostic index in the preparation of a tumor diagnostic drug.

The expression levels of YEATS4 gene in tumor tissue, normal tissue and surrounding normal tissue were detected by immunohistochemistry. It was found that the expression of YEATS4 gene in tumor tissues was significantly higher than that in normal tissues and surrounding normal tissues. It is suggested that YEATS4 gene may act as an oncogene and play an important role in the occurrence and development of tumors; the expression level of YEATS4 gene may become a marker of tumor diagnosis.

The tumor therapeutic drug is a molecule that can specifically inhibit the transcription or translation of the YEATS4 gene, or can specifically inhibit the expression or activity of the YEATS4 protein, thereby reducing the expression level of the YEATS4 gene in tumor cells, thereby inhibiting the proliferation and growth of tumor cells. , differentiation and/or survival purposes.

The tumor therapeutic drugs or tumor diagnostic drugs obtained by preparing or screening the isolated YEATS4 gene include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

The nucleic acids include, but are not limited to, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, endoribonuclease III-produced small interfering RNA (esiRNA), or short hairpin RNA (shRNA).

The administration amount of the tumor therapeutic drug is a dose sufficient to reduce the transcription or translation of the human YEATS4 gene, or to reduce the expression or activity of the human YEATS4 protein. so that the expression of the human YEATS4 gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

The method of using the aforementioned tumor therapeutic drugs to treat tumors mainly achieves the purpose of treatment by reducing the expression level of the human YEATS4 gene and inhibiting the proliferation of tumor cells. Specifically, during the treatment, a substance that can effectively reduce the expression level of the human YEATS4 gene is administered to the patient.

A second aspect of the present invention discloses an isolated nucleic acid molecule that reduces YEATS4 gene expression in tumor cells, the nucleic acid molecule comprising:

a) a double-stranded RNA containing a nucleotide sequence capable of hybridizing to the YEATS4 gene under stringent conditions; or

b) shRNA containing a nucleotide sequence capable of hybridizing to the YEATS4 gene under stringent conditions.

Further, the double-stranded RNA comprises a first strand and a second strand, and the first strand and the second strand are complementary to form an RNA dimer together, and the sequence of the first strand is the same as that in the YEATS4 gene 15- 27 consecutive nucleotide sequences are substantially identical. Preferably, the sequence of the first strand is substantially the same as 19-23 consecutive nucleotide sequences in the YEATS4 gene; more preferably, the sequence of the first strand is 19, 20 or 21 consecutive in the YEATS4 gene. The nucleotide sequences are basically the same.

Further, the double-stranded RNA comprises a first strand and a second strand, and the first strand and the second strand are complementary to form an RNA dimer together, and the sequence of the first strand is the same as that in the YEATS4 gene. The target sequences are substantially identical.

The lengths of the first strand and the second strand of the double-stranded RNA are both 15-27 nucleotides; preferably, the lengths are 19-23 nucleotides; 21 nucleotides.

Further, the double-stranded RNA is small interfering RNA (siRNA). Further, the sequence of the first strand of the small interfering RNA is shown in SEQ ID NO: 17, specifically 5'-CCAAUAGUUUACGGUAAUGUU-3'.

The siRNA shown in SEQ ID NO: 17 is a chain of the small interfering RNA against the human YEATS4 gene designed with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence, and the other chain, that is, the sequence of the second chain is the same as that of the human YEATS4 gene. Complementary to the first strand sequence, the siRNA can specifically silence the expression of endogenous YEATS4 gene in tumor cells.

Further, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sense strand segment and the antisense strand segment are complementary in sequence, and The sequence of the sense strand fragment is substantially the same as the 15-27 consecutive nucleotide sequences in the YEATS4 gene. The shRNA can be processed into small interfering RNA (siRNA) to specifically silence the expression of endogenous YEATS4 gene in tumor cells.

Further, the shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and the sense strand segment and the antisense strand segment are complementary in sequence, And the sequence of the sense strand fragment is basically the same as the target sequence in the YEATS4 gene.

Preferably, the sense strand fragment is substantially the same as 19-23 consecutive nucleotide sequences in the YEATS4 gene; more preferably, the sense strand fragment is 19, 20 or 21 consecutive nucleotides in the YEATS4 gene. The sequence is basically the same.

Further, the sequence of the stem-loop structure of the shRNA can be selected from any one of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.

Further, the sequence of the shRNA is shown in SEQ ID NO: 18, specifically: 5'-CCAAUAGUUUACGGUAAUGUUUUCAAGAGA AACAUUACCGUAAACUAUUGG-3'.

The shRNA can be converted into siRNA after enzymatic digestion, which can specifically silence the expression of endogenous human YEATS4 gene in tumor cells.

The interfering lentiviral vector encoding the gene fragment of the shRNA of the present invention contains any sequence of SEQ ID NO: 1-5 and its complementary sequence.

The first strand of the double-stranded RNA or the sense strand fragment of the shRNA is substantially the same as the target sequence in the YEATS4 gene, and the target sequence of the YEATS4 gene is that siRNA is used to specifically silence the expression of the YEATS4 gene. Fragments in the YEATS4 gene corresponding to mRNA fragments recognized and silenced by siRNA.

Preferably, the target sequence in the YEATS4 gene contains any sequence of SEQ ID NOs: 1-5.

Further, the YEATS4 gene is derived from human.

The third aspect of the present invention discloses a YEATS4 gene interference nucleic acid construct, which contains a gene fragment encoding the shRNA in the isolated nucleic acid molecule of the present invention, and can express the shRNA.

The human YEATS4 gene interfering nucleic acid construct can be obtained by cloning the gene fragment encoding the aforementioned human YEATS4 gene shRNA into a known vector. Further, the YEATS4 gene interference nucleic acid construct is a YEATS4 gene interference lentiviral vector.

The YEATS4 gene interference lentiviral vector of the present invention is obtained by cloning the DNA fragment encoding the aforementioned YEATS4 gene shRNA into a known vector, and the known vector is mostly a lentiviral vector. After the infectious virus particles are infected with tumor cells, the shRNA of the present invention is then transcribed, and the siRNA is finally obtained through steps such as enzymatic cleavage processing, which is used to specifically silence the expression of the YEATS4 gene.

Further, the YEATS4 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a detectable marker in tumor cells; preferably, the detectable marker such as green fluorescence protein (GFP).

Further, the lentiviral vector can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshRNA, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/ Any of V5-GW/lacZ.

The embodiment of the present invention specifically lists a human YEATS4 gene interference lentiviral vector constructed with pGCSIL-GFP as a vector, named pGCSIL-GFP-YEATS4-siRNA.

The isolated nucleic acid molecule of the present invention can be used to prepare a medicament for preventing or treating tumor, and the tumor is colon cancer.

The YEATS4 gene siRNA of the present invention can be used to inhibit the proliferation of tumor cells, and further can be used as a drug or preparation for treating tumors. YEATS4 gene interference lentiviral vector can be used to prepare the YEATS4 gene siRNA. When used as a medicament or formulation for the treatment of tumors, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The specific dosage should also take into account factors such as the route of administration, the patient's health status, etc., which are all within the skill of the skilled physician.

In a fourth aspect of the present invention, a YEATS4 gene interference lentivirus is disclosed, which is formed by viral packaging of the aforementioned YEATS4 gene interference lentiviral vector with the assistance of a lentiviral packaging plasmid and a cell line. The lentivirus can infect tumor cells and produce small interfering RNA targeting the YEATS4 gene, thereby inhibiting the proliferation of colon cancer cells. The YEATS4 gene interfering lentivirus can be used to prepare medicines for preventing or treating tumors.

The fifth aspect of the present invention discloses a pharmaceutical composition for preventing or treating tumors, the effective substance of which contains the aforementioned isolated nucleic acid molecule, one or more of the YEATS4 gene interference nucleic acid construct or the YEATS4 gene interference lentivirus combination of species.

Further, the pharmaceutical composition contains 1-99 wt% of the double-stranded RNA, shRNA, YEATS4 gene interference nucleic acid construct or YEATS4 gene interference lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

In preparing these compositions, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or enclosed in a carrier which can be in the form of a capsule or sachet. When an excipient acts as a diluent, it can be a solid, semi-solid or liquid material that acts as an excipient, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulations may also include: wetting agents, emulsifiers, preservatives (eg, methyl and propyl hydroxybenzoates), sweetening agents, and the like.

The invention also discloses the application of the pharmaceutical composition in preparing a tumor therapeutic drug for treating colon cancer.

The application of the pharmaceutical composition provides a method for tumor treatment, specifically a method for preventing or treating tumors in a subject, comprising administering an effective dose of the pharmaceutical composition to the subject. Further, the tumor is selected from colon cancer.

When the pharmaceutical composition is used to prevent or treat tumors in a subject, an effective dose of the pharmaceutical composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the tumor part is suppressed.

The object of the method may be a human being.

A sixth aspect of the present invention discloses a kit for reducing YEATS4 gene expression in tumor cells, the kit comprising: the isolated nucleic acid molecule present in a container, a YEATS4 gene interference nucleic acid construct, and /or the YEATS4 gene interferes with the lentivirus.

To sum up, the present invention has designed five RNAi target sequences for the human YEATS4 gene, and constructed the corresponding YEATS4 RNAi vector, wherein the RNAi vector pGCSIL-GFP-YEATS4-siRNA of the coding sequence SEQ ID NO: 1 can significantly down-regulate the YEATS4 gene. Expression at the mRNA and protein levels. Using lentivirus (Lv for short) as a genetic manipulation tool to carry the RNAi vector pGCSIL-GFP-YEATS4-siRNA, the RNAi sequence targeting the YEATS4 gene can be efficiently introduced into human colon cancer RKO cells, and the expression level of the YEATS4 gene can be reduced. Significantly inhibit the proliferation of the above tumor cells. Therefore, lentivirus-mediated gene silencing of YEATS4 is a potential clinical non-surgical treatment for malignant tumors.

The siRNA or the nucleic acid construct comprising the siRNA sequence and the lentivirus provided by the present invention can specifically inhibit the expression of the human YEATS4 gene, especially the lentivirus, which can efficiently infect the target cells and efficiently inhibit the expression of the YEATS4 gene in the target cells. , promoting cell apoptosis, reducing the invasion and metastasis of tumor cells, etc., thereby inhibiting the growth of tumor cells and promoting tumor cell apoptosis, which is of great significance in tumor therapy.

Description of drawings

Figure 1: pGCSIL-GFP plasmid DNA map

Figure 2: The expression level of YEATS4 mRNA was significantly decreased after 4 days of YEATS4-RNAi lentivirus infection of colon cancer cells

Figure 3: YEATS4-RNAi lentivirus infects colon cancer cells for 4 days, causing cell proliferation inhibition

Detailed ways

The present invention relates to a group of small molecule interfering RNA (siRNA) sequences, RNA interference vectors and RNA interference lentiviruses targeting human YEATS4 gene. The coding region sequence of human YEATS4 mRNA is selected as the target site of siRNA, and the siRNA target site sequence is designed according to the consecutive 10-30 (preferably 15-27, more preferably 19-23) base sequences in the target site. Through gene cloning, a nucleic acid construct expressing the above siRNA is constructed, and the lentivirus expressing the above siRNA is packaged. Cell experiments proved that the above siRNA sequence can specifically silence the expression of endogenous YEATS4 gene in human tumor cells.

The inventors of the present invention have found through extensive and in-depth research that the YEATS4 gene is significantly highly expressed in tumor tissues; the inventors have found that using RNAi to down-regulate the expression of the human YEATS4 gene can effectively inhibit the proliferation of tumor cells and promote cell apoptosis It can effectively control the growth process of tumors, and this research result shows that the YEATS4 gene is a proto-oncogene and can be used as a target for tumor therapy. The inventors further synthesized and tested a variety of siRNAs against the YEATS4 gene, and screened out siRNAs that can effectively inhibit the expression of YEATS4 and thereby inhibit the proliferation and growth of colon cancer RKO cells, and the present invention was completed on this basis.

The present invention provides a series of small interfering RNA (siRNA) sequences that interfere with the human YEATS4 gene, and constructs a lentivirus that can specifically silence the expression of the YEATS4 gene. The present invention finds that the small interfering RNA and RNAi lentivirus designed for the human YEATS4 gene can stably and specifically downregulate the expression of the YEATS4 gene, and effectively inhibit the proliferation of human tumor cells. The present invention shows that the YEATS4 gene can promote the growth of tumor cells, and is expected to become a target for early diagnosis and treatment of tumors. Moreover, silencing the expression of YEATS4 gene by RNAi can be used as an effective means to inhibit tumor development.

The design idea of the present invention is:

In the present invention, a human YEATS4 gene RNAi lentivirus is screened and obtained by the following methods: extracting the human YEATS4 gene sequence from Genbank; predicting the siRNA site; synthesizing an effective siRNA sequence targeting the YEATS4 gene, containing an enzyme cutting site at both ends, Double-stranded DNA Oligo at the end; the lentiviral vector was double-enzyme digested and then ligated with double-stranded DNA Oligo to construct an RNAi plasmid expressing the siRNA sequence of the YEATS4 gene; the RNAi plasmid and the auxiliary vector (PackingMix, Sigma-aldrich Company) required for lentiviral packaging were combined Human embryonic kidney cells 293T were transfected to generate recombinant lentiviral particles, and then a lentivirus that efficiently silenced the YEATS4 gene could be prepared.

Based on the above method, the present invention provides five effective targets for interfering with the YEATS4 gene (specifically shown in SEQ ID NOs: 1-5), and constructs a lentivirus that specifically interferes with the human YEATS4 gene.

At the same time, the invention also discloses a human YEATS4 gene RNAi lentivirus (YEATS4-RNAi) and its preparation and application.

This study found that the use of lentivirus-mediated RNAi method can effectively inhibit the proliferation of tumor cells after reducing the expression of YEATS4 gene in tumor cells. This study shows that YEATS4 gene is a proto-oncogene that can promote tumor cell proliferation and has important biological functions in tumorigenesis and development. YEATS4 gene can be a target for tumor therapy. Lentivirus-mediated YEATS4 gene-specific silencing It can be used as a new means of tumor treatment.

The present invention is further described below in conjunction with the examples. It should be understood that the embodiments are only used to illustrate the present invention, but not to limit the scope of the present invention. In the embodiment, the experimental method without specifying the specific conditions and the reagent without specifying the formula are all according to the conventional conditions, such as [US] Sambrook.J et al.; Huang Peitang et al. A Guide to Molecular Cloning Assays, Third Edition. Beijing: The conditions described in Science Press 2002 or the conditions suggested by the manufacturer were performed or configured.

Example 1 Preparation of RNAi Lentivirus for Human YEATS4 Gene

1. Screening of effective siRNA targets against the human YEATS4 gene

Retrieve YEATS4 gene information from Genbank; design effective siRNA targets for YEATS4 gene. Table 1 lists five of the effective siRNA target sequences against the YEATS4 gene.

Table 1 siRNA target sequences targeting human YEATS4 gene

2. Preparation of Lentiviral Vectors

Synthesize double-stranded DNA Oligo sequences (Table 2) with sticky ends containing Age I and EcoR I restriction endonucleases against the siRNA target (take SEQ ID NO: 1 as an example); use Age I and EcoR I restriction endonucleases Act on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., Figure 1) to linearize it, and identify the digested fragments by agarose gel electrophoresis.

Table 2 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I restriction sites at both ends

Use T4 DNA ligase to linearize the double-enzyme digestion (enzyme digestion system shown in Table 4, 37 ° C, reaction 1h) and the purified double-stranded DNA Oligo ligation, in an appropriate buffer system (ligation system shown in Table 5) ligation at 16°C overnight, and the ligation product was recovered. The ligation product was transformed into fresh E. coli competent cells prepared by calcium chloride (transformation operation reference: Molecular Cloning Experiment Guide, Second Edition, pages 55-56). Dip on the surface of the colony that grows from the ligated transformation product, dissolve it in 10 μl LB medium, mix well and take 1 μl as a template; design universal PCR primers upstream and downstream of the RNAi sequence in the lentiviral vector, upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA -3' (SEQ ID NO: 10); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 11), carry out the PCR identification experiment (the PCR reaction system is shown in Table 6-1, and the reaction conditions are shown in Table 6- 2). The clones identified by PCR were sequenced and compared, and the correct clones were successfully constructed to express the RNAi vector against SEQ ID NO: 1, which was named pGCSIL-GFP-YEATS4-siRNA.

The pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed, and the negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 12). When constructing the pGCSIL-GFP-Scr-siRNA negative control plasmid, the double-stranded DNA Oligo sequence (Table 3) with the sticky ends of Age I and EcoR I restriction sites at both ends was synthesized for the Scr siRNA target. The other construction methods, identification methods and Conditions are the same as pGCSIL-GFP-YEATS4-siRNA.

Table 3 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I restriction sites at both ends

The vector that was linearized by double-enzyme digestion by T4 DNA ligase (the digestion system is shown in Table 4, 37°C, reaction for 1h)

Table 4 pGCSIL-GFP plasmid digestion reaction system

reagent Volume (μl) pGCSIL-GFP plasmid (1μg/μl) 2.0 10×buffer 5.0 100×BSA 0.5 Age I(10U/μl) 1.0 EcoR I (10U/μl) 1.0 dd H₂O 40.5 Total 50.0

Table 5 Vector DNA and double-stranded double-stranded DNA Oligo ligation reaction system

reagent Positive control (μl) Self-ligation control (μl) Connectivity group (μl) Linearized vector DNA (100ng/μl) 1.0 1.0 1.0 Annealed dsDNA Oligo (100ng/μl) 1.0 - 1.0 10 x T4 phage DNA ligase buffer 1.0 1.0 1.0 T4 phage DNA ligase 1.0 1.0 1.0 dd H₂O 16.0 17.0 16.0 Total 20.0 20.0 20.0

Table 6-1 PCR reaction system

reagent Volume (μl) 10×buffer 2.0 dNTPs (2.5mM) 0.8 upstream primer 0.4 downstream primer 0.4 Taq polymerase 0.2 template 1.0 ddH₂O 15.2 Total 20.0

Table 6-2 PCR reaction system program settings

3. Packaging YEATS4-siRNA Lentivirus

The DNA of the RNAi plasmid pGCSIL-GFP-YEATS4-siRNA was extracted with a plasmid extraction kit from Qiagen, and prepared into a 100 ng/μl stock solution.

24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were digested with trypsin, and the cell density was adjusted to 1.5×10 ⁵ cells/ml in DMEM complete medium containing 10% fetal bovine serum, and seeded in 6-well plates , 37 ° C, 5% CO ₂ incubator. When the cell density reaches 70%-80%, it can be used for transfection. 2h before transfection, the original medium was aspirated, and 1.5 ml of fresh complete medium was added. According to the instructions of the MISSION Lentiviral Packaging Mix kit from Sigma-aldrich, add 20 μl of Packing Mix (PVM), 12 μl of PEI, and 400 μl of serum-free DMEM medium to a sterilized centrifuge tube, take 20 μl of the above-extracted plasmid DNA, add to the above PVM/PEI/DMEM mixture.

The above-mentioned transfection mixture was incubated at room temperature for 15 min, transferred to the medium of human embryonic kidney cells 293T cells, and cultured in a 37° C., 5% CO ₂ incubator for 16 h. The culture medium containing the transfection mixture was discarded, washed with PBS solution, and 2 ml of complete medium was added. Since the lentiviral vector itself carries the reporter gene of green fluorescent protein, the expression of GFP in the two groups of cells can be observed under a fluorescence microscope after 24 hours to determine whether the lentiviral plasmid has been packaged by 293T cells. After culturing for 48 hours, the cell supernatant was collected, and the lentivirus was purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore). The steps were as follows: (1) Centrifuge at 4000g for 10 min at 4°C to remove cell debris; (2) 0.45 μm Filter the supernatant into a 40ml ultracentrifuge tube; (3) Centrifuge at 4000g for 10-15min to the required virus concentration volume; (4) After centrifugation, separate the filter cup and the filtrate collection cup below, The filter cup is upside down on the sample collection cup, and the centrifugal force does not exceed 1000g for 2 minutes; (5) Remove the centrifuge cup from the sample collection cup, and the virus concentrate in the sample collection cup is. Store the virus concentrate in aliquots at -80°C. The sequence of the first strand of the siRNA contained in the virus concentrate is shown in SEQ ID NO:18. The packaging process of the control lentivirus was the same as that of the YEATS4-siRNA lentivirus, only the pGCSIL-GFP-Scr-siRNA vector was used instead of the pGCSIL-GFP-YEATS4-siRNA vector.

Example 2 Detection of silencing efficiency of YEATS4 gene by real-time fluorescence quantitative RT-PCR

Colon cancer RKO cells in logarithmic growth phase were digested with trypsin, and a cell suspension (the number of cells was about 5×10 ⁴ /ml) was inoculated into a 6-well plate, and cultured until the cell confluence reached about 30%. According to the multiplicity of infection (MOI, RKO: 10) value, an appropriate amount of the virus prepared in Example 1 was added, the medium was replaced after culturing for 24 hours, and the cells were collected after the infection time reached 4 days. Total RNA was extracted according to Invitrogen's Trizol operating instructions. According to the M-MLV operating instructions of Promega company, reverse transcription of RNA to obtain cDNA (reverse transcription reaction system is shown in Table 7, 42 ° C for 1 h, and then in a 70 ° C water bath for 10 min to inactivate reverse transcriptase).

Real-time quantitative detection was performed using a TP800 Real time PCR instrument (TAKARA). Primers for the YEATS4 gene were as follows: upstream primer 5'-TCATAGAACTCTGAAACCACTGTC-3' (SEQ ID NO: 13) and downstream primer 5'-CCTGTAACCCTGTATCATTTGCTA-3' (SEQ ID NO: 14). Taking housekeeping gene GAPDH as internal reference, the primer sequences are as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 15) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 16). The reaction system was configured according to the ratio in Table 8.

Table 7 Reverse transcription reaction system

reagent Volume (μl) 5×RT buffer 4.0 10mM dNTPs 2.0 RNasin 0.5 M-MLV-RTase 1.0 DEPC H₂O 3.5 Total 11.0

Table 8 Real-time PCR reaction system

reagent Volume (μl) SYBR premix ex taq: 10.0 Upstream primer (2.5μM): 0.5 Downstream primer (2.5μM): 0.5 cDNA 1.0 ddH₂O 8.0 Total 20.0

The set program was two-step Real-time PCR: pre-denaturation at 95°C for 15s; subsequent denaturation at 95°C for 5s for each step; annealing and extension at 60°C for 30s; a total of 45 cycles were performed. The absorbance was read each time during the extension phase. After PCR, denature at 95°C for 1 min, and then cool to 55°C to fully bind the DNA double strands. Start from 55°C to 95°C, increase 0.5°C in each step, hold for 4s, read the absorbance value at the same time, and make a melting curve. The expression abundance of infected YEATS4 mRNA was calculated by 2- ^ΔΔCt analysis. Cells infected with control virus (Lv-Scr-siRNA) served as controls. The experimental results (Fig. 2) showed that the expression level of mRNA in colon cancer RKO cells was down-regulated by 62.9%.

Example 3 Detection of proliferation ability of tumor cells infected with YEATS4-siRNA lentivirus

Colon cancer RKO cells in logarithmic growth phase were digested with trypsin, and a cell suspension (the number of cells was about 5×10 ⁴ /ml) was inoculated into a 6-well plate, and cultured until the cell confluence reached about 30%. According to the multiplicity of infection (MOI, RKO: 10), an appropriate amount of virus was added, and the medium was replaced after culturing for 24 hours. After the infection time reached 4 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 ⁴ /ml), and the cells were seeded in a 96-well plate at a cell density of about 2000 cells/well. 5 replicate wells in each group, 100 μl per well. After laying the plate, place it in a 37°C, 5% CO ₂ incubator for cultivation. Starting from the second day after plating, the plate was detected and read once a day with the Cellomics instrument (Thermo Fisher), and the plate was detected and read continuously for 5 days. By adjusting the input parameters of Cellomics arrayscan, the number of cells with green fluorescence in each scanning well plate was accurately calculated, the data was statistically drawn, and the cell proliferation curve was drawn (the results are shown in Figure 3). The results showed that the proliferation rate of each tumor in the lentivirus infection group was significantly slowed down after 5 days of cell culture in vitro, which was much lower than the proliferation rate of tumor cells in the control group, and the number of viable cells decreased by 67.9%, indicating that YEATS4 gene silencing led to tumor cell proliferation. ability is inhibited.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form or substance. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, the Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. All those skilled in the art, without departing from the spirit and scope of the present invention, can utilize the above-disclosed technical content to make some changes, modifications and equivalent changes of evolution, all belong to the present invention. Equivalent embodiments; at the same time, any modification, modification and evolution of any equivalent changes made to the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solutions of the present invention.

Claims (11)

1. The application of interfering RNA aiming at the YEATS4 gene in the preparation of a colon cancer treatment drug, wherein the YEATS4 gene target sequence is shown as SEQ ID NO:1 is shown.

2. An isolated nucleic acid molecule that reduces expression of the YEATS4 gene in a colon cancer cell, the nucleic acid molecule comprising:

a) a double-stranded RNA comprising a nucleotide sequence capable of hybridizing to the YEATS4 gene, the double-stranded RNA comprising a first strand and a second strand, the first strand and the second strand being complementary to each other to form an RNA dimer, and the sequence of the first strand being identical to a target sequence in the YEATS4 gene, the YEATS4 gene target sequence being set forth in SEQ ID NO:1 is shown in the specification; or

b) shRNA containing a nucleotide sequence capable of hybridizing with a YEATS4 gene, wherein the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, the sequence of the sense strand segment is identical to a target sequence in the YEATS4 gene, and the target sequence of the YEATS4 gene is shown in SEQ ID NO:1 is shown.

3. The isolated nucleic acid molecule of claim 2, wherein the double-stranded RNA is a small interfering RNA having a first strand with a sequence as set forth in SEQ ID NO: shown at 17.

4. The isolated nucleic acid molecule of claim 2, wherein the sequence of said shRNA is as set forth in SEQ ID NO:18, respectively.

5. A YEATS4 gene interfering nucleic acid construct containing a gene segment encoding the shRNA in the isolated nucleic acid molecule of any one of claims 2-4, capable of expressing the shRNA.

6. The YES 4 gene interfering nucleic acid construct of claim 5, wherein the YEATS4 gene interfering nucleic acid construct is an interfering lentiviral vector.

7. The YEATS4 gene interfering nucleic acid construct of claim 6, wherein the interfering lentiviral vector is obtained by cloning a gene segment encoding the shRNA into a lentiviral vector selected from the group consisting of: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.

8. A YEATS4 gene interference lentivirus, which is formed by virus packaging of the YEATS4 gene interference nucleic acid construct of any one of claims 5-7 with the help of lentivirus packaging plasmid and cell line.

9. A pharmaceutical composition for treating tumor, wherein the main effective substance comprises the isolated nucleic acid molecule of any one of claims 2-4, the YEATS4 gene interference nucleic acid construct of any one of claims 5-7, or the YEATS4 gene interference lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.

10. Use of the isolated nucleic acid molecule of any one of claims 2 to 4, the YEATS4 gene-interfering nucleic acid construct of any one of claims 5 to 7, or the YEATS4 gene-interfering lentivirus of claim 8 for the preparation of a medicament for the treatment of tumors in the treatment of colon cancer.

11. A kit for reducing expression of the YEATS4 gene in a tumor cell, the kit comprising: an isolated nucleic acid molecule according to any one of claims 2 to 4, a YEATS4 gene interfering nucleic acid construct according to any one of claims 5 to 7, or a YEATS4 gene interfering lentivirus according to claim 8, in a container.

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