Uses of human POLE2 gene and related products

Abstract

This invention belongs to the field of biomedical research and specifically relates to the use of the human POLE2 gene as a target in the preparation of gastric cancer therapeutics or diagnostic drugs. Through extensive and in-depth research, the present invention has discovered that downregulating the expression of the human POLE2 gene using RNAi can effectively inhibit the proliferation of gastric cancer cells, promote apoptosis, and effectively control the growth process of gastric cancer. The siRNA provided by the present invention, or a nucleic acid construct or lentivirus containing the siRNA sequence, can specifically inhibit the proliferation and growth of gastric cancer cells, thereby treating gastric cancer and opening up new avenues for gastric cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical research, and specifically relates to the use of human POLE2 gene and related products. Background Art

[0002] Studies have found that the POLE2 gene is associated with lung cancer and colorectal cancer. For example, a study conducted a biological analysis of gene expression profiles in squamous cell lung cancer and normal tissues, and found that different differentially expressed genes are involved in multiple pathways, including tumor occurrence, development, and immune infiltration of squamous cell lung cancer. Among the differentially expressed genes, POLE2 was found to be a key gene that is closely related to the prognosis of patients with squamous cell lung cancer. The higher the expression level of the POLE2 gene, the worse the prognosis of lung cancer patients. It can be used as a prognostic biomarker for lung cancer. In another study, by detecting the background expression level of the POLE2 gene in three different lung adenocarcinoma cell lines, A549, NCI-H1299, and NCI-H1975, it was found that the gene was highly expressed in all three lung cancer cell lines. In A549 and NCI-H1299 cells, downregulating the POLE2 gene significantly inhibited tumor cell proliferation and induced tumor cell apoptosis. On the other hand, by analyzing the whole-genome exome sequencing data of 1,006 colorectal cancer patients and 1,609 healthy people, it was found that some genes are closely related to colorectal cancer, including the POLE2 gene, which laid a certain foundation for future research on colorectal cancer.

[0003] There are currently no reports on the use of POLE2 gene in the treatment of gastric cancer. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide uses of human POLE2 gene and related products.

[0005] In order to achieve the above-mentioned and other related purposes, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides the use of the human POLE2 gene as a target in the preparation of a gastric cancer therapeutic drug or a gastric cancer diagnostic drug.

[0007] The use of the human POLE2 gene as a target in the preparation of a gastric cancer treatment drug specifically involves screening drugs or formulations targeting the POLE2 gene to identify drugs that can inhibit human POLE2 gene expression as potential gastric cancer treatment candidates. For example, the POLE2 gene small interfering RNA (siRNA) described herein is obtained by screening against the human POLE2 gene and can be used as a drug that inhibits gastric cancer cell proliferation. In addition, other drugs, such as antibody drugs and small molecule drugs, can also target the POLE2 gene.

[0008] The use of the human POLE2 gene as a target for the preparation of gastric cancer diagnostic drugs specifically refers to: using the POLE2 gene expression product as a gastric cancer diagnostic indicator in the preparation of gastric cancer diagnostic drugs.

[0009] Immunohistochemistry was used to examine the expression of the POLE2 gene in tumor tissue, normal tissue, and surrounding normal tissue. The study found that POLE2 expression was significantly higher in gastric cancer tissue than in normal tissue and surrounding normal tissue. This suggests that POLE2 gene expression may serve as a diagnostic marker for tumors.

[0010] The gastric cancer therapeutic drug is a molecule that can specifically inhibit the transcription or translation of the POLE2 gene, or can specifically inhibit the expression or activity of the POLE2 protein, thereby reducing the expression level of the POLE2 gene in gastric cancer cells and achieving the purpose of inhibiting the proliferation, growth, differentiation and / or survival of gastric cancer cells.

[0011] The gastric cancer therapeutic drugs or gastric cancer diagnostic drugs prepared by the POLE2 gene include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, peptides, proteins or interfering lentiviruses.

[0012] The nucleic acid includes, but is not limited to, antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA produced by endoribonuclease III, or short hairpin RNA (shRNA).

[0013] The amount of the gastric cancer therapeutic drug administered is sufficient to reduce the transcription or translation of the human POLE2 gene, or to reduce the expression or activity of the human POLE2 protein, so that the expression of the human POLE2 gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

[0014] The method of treating gastric cancer using the aforementioned gastric cancer therapeutic drug is mainly to achieve the purpose of treatment by reducing the expression level of the human POLE2 gene to inhibit the proliferation of gastric cancer cells. Specifically, during treatment, a substance that can effectively reduce the expression level of the human POLE2 gene is administered to the patient.

[0015] In one embodiment, the target sequence of the POLE2 gene is shown in SEQ ID NO: 1, specifically: 5'-GATTGTTCTTGGAATGATA-3'.

[0016] In a second aspect, the present invention provides the use of a POLE2 inhibitor in preparing a product having at least one of the following effects:

[0017] Treat gastric cancer;

[0018] Inhibit the proliferation of gastric cancer cells;

[0019] Inhibit the growth of gastric cancer.

[0020] The product must include a POLE2 inhibitor and use the POLE2 inhibitor as the active ingredient for the aforementioned efficacy.

[0021] In the product, the active ingredient that exerts the aforementioned function may be only a POLE2 inhibitor, or may contain other molecules that can exert the aforementioned function.

[0022] That is, the POLE2 inhibitor is the only active ingredient or one of the active ingredients of the product.

[0023] The product can be a single-component substance or a multi-component substance.

[0024] The form of the product is not particularly limited and can be in the form of solid, liquid, gel, semi-fluid, aerosol or other substances.

[0025] The product is mainly targeted at mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0026] The products include but are not limited to medicines, health products, food, etc.

[0027] The POLE2 inhibitor can be a nucleic acid molecule, an antibody, or a small molecule compound.

[0028] As listed in the examples of the present invention, the POLE2 inhibitor can be a nucleic acid molecule that reduces the expression of the POLE2 gene in gastric cancer cells, specifically, double-stranded RNA or shRNA.

[0029] In a third aspect, the present invention provides a method for treating gastric cancer, comprising administering a POLE2 inhibitor to a subject.

[0030] The subject can be a mammal or a mammalian gastric cancer cell. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, or the like. The primate is preferably a monkey, an ape, or a human. The gastric cancer cell can be an isolated gastric cancer cell.

[0031] The subject may be a patient suffering from gastric cancer or an individual expecting treatment for gastric cancer, or the subject may be ex vivo gastric cancer cells from a patient suffering from gastric cancer or an individual expecting treatment for gastric cancer.

[0032] The POLE2 inhibitor can be administered to a subject before, during, or after receiving treatment for gastric cancer.

[0033] The fourth aspect of the present invention discloses a nucleic acid molecule for reducing the expression of POLE2 gene in gastric cancer cells, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.

[0034] Wherein, the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the POLE2 gene;

[0035] The shRNA contains a nucleotide sequence capable of hybridizing with the POLE2 gene.

[0036] Furthermore, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand complement each other to form an RNA dimer, and the sequence of the first strand is substantially identical to the target sequence in the POLE2 gene.

[0037] The target sequence in the POLE2 gene is a fragment in the POLE2 gene corresponding to the mRNA fragment recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used to specifically silence the expression of the POLE2 gene.

[0038] Furthermore, the target sequence of the double-stranded RNA is shown in SEQ ID NO: 1. Specifically, it is 5'-GATTGTTCTTGGAATGATA-3'. Furthermore, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2. Specifically, it is 5'-GAUUGUUCUUGGAAUGAUA-3'.

[0039] Furthermore, the double-stranded RNA is small interfering RNA (siRNA).

[0040] SEQ ID NO: 2 is one chain of a small interfering RNA targeting the human POLE2 gene, which is designed with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence. The sequence of the other chain, i.e., the second chain, is complementary to the sequence of the first chain. This siRNA can specifically silence the expression of the endogenous POLE2 gene in gastric cancer cells.

[0041] The shRNA includes a sense chain segment and an antisense chain segment, and a stem-loop structure connecting the sense chain segment and the antisense chain segment. The sequences of the sense chain segment and the antisense chain segment are complementary, and the sequence of the sense chain segment is substantially identical to the target sequence in the POLE2 gene.

[0042] Furthermore, the target sequence of the shRNA is shown in SEQ ID NO: 1.

[0043] The shRNA can be processed by enzyme cleavage to become small interfering RNA (siRNA), thereby playing a role in specifically silencing the expression of endogenous POLE2 gene in gastric cancer cells.

[0044] Furthermore, 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.

[0045] Furthermore, the sequence of the shRNA is shown in SEQ ID NO: 3, specifically 5'-CCGGGCGAUUGUUCUUGGAAUGAUACUCGAGUAUCAUUCCAAGAACAAUCGCUUUUU-3'.

[0046] Furthermore, the POLE2 gene is derived from humans.

[0047] In a fifth aspect, the present invention discloses a POLE2 gene interference nucleic acid construct, which contains a gene segment encoding the shRNA in the aforementioned nucleic acid molecule and is capable of expressing the shRNA.

[0048] The POLE2 gene interfering nucleic acid construct can be obtained by cloning the gene fragment encoding the aforementioned human POLE2 gene shRNA into a known vector.

[0049] Furthermore, the POLE2 gene interference nucleic acid construct is a POLE2 gene interference lentiviral vector.

[0050] The POLE2 gene interference lentiviral vector disclosed in the present invention is obtained by cloning a DNA fragment encoding the aforementioned POLE2 gene shRNA into a known vector, most of which are lentiviral vectors. The POLE2 gene interference lentiviral vector is packaged into infectious viral particles, which infect gastric cancer cells and then transcribe the shRNA of the present invention. Through steps such as enzymatic processing, the siRNA is finally obtained, which is used to specifically silence the expression of the POLE2 gene.

[0051] Furthermore, the POLE2 gene interference lentiviral vector further contains a promoter sequence and / or a nucleotide sequence encoding a marker that can be detected in gastric cancer cells; preferably, the detectable marker is such as green fluorescent protein (GFP).

[0052] Furthermore, the lentiviral vector can be 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, 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-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-CMV-Tag KO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DES T, any of pLenti6-GW / U6-laminshrna, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP or pLenti6.2 / N-Lumio / V5-GW / lacZ.

[0053] The embodiment of the present invention specifically lists a human POLE2 gene interference lentiviral vector constructed using pGCSIL-GFP as a vector, and is named pGCSIL-GFP-POLE2-siRNA.

[0054] The POLE2 gene siRNA of the present invention can be used to inhibit the proliferation of gastric cancer cells and can further be used as a drug or formulation for treating gastric cancer. A POLE2 gene interfering lentiviral vector can be used to prepare the POLE2 gene siRNA. When used as a drug or formulation for treating gastric cancer, 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 and the patient's health status, all of which are within the skill of a skilled physician.

[0055] In a sixth aspect, the present invention discloses a POLE2 gene-interfering lentivirus. This lentivirus is produced by packaging the aforementioned POLE2 gene-interfering nucleic acid construct with the aid of a lentiviral packaging plasmid and cell line. This lentivirus can infect gastric cancer cells and produce small interfering RNA targeting the POLE2 gene, thereby inhibiting the proliferation of gastric cancer cells. This POLE2 gene-interfering lentivirus can be used to prepare a drug for the prevention or treatment of gastric cancer.

[0056] In a seventh aspect, the present invention provides uses of the aforementioned nucleic acid molecule, or the aforementioned POLE2 gene interfering nucleic acid construct, or the aforementioned POLE2 gene interfering lentivirus for preparing a drug for preventing or treating gastric cancer, or for preparing a kit for reducing POLE2 gene expression in gastric cancer cells.

[0057] The use of the drug for preventing or treating gastric cancer provides a method for treating gastric cancer, specifically a method for preventing or treating gastric cancer in a subject, comprising administering an effective dose of the drug to the subject.

[0058] Furthermore, when the drug is used to prevent or treat gastric cancer in a subject, an effective dose of the drug is administered to the subject. Using this method, the growth, proliferation, recurrence, and / or metastasis of the gastric cancer is inhibited. Furthermore, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the growth, proliferation, recurrence, and / or metastasis of the gastric cancer is inhibited.

[0059] The subject of the method can be a human.

[0060] In an eighth aspect, the present invention provides a composition for preventing or treating gastric cancer, wherein the composition comprises:

[0061] The aforementioned nucleic acid molecule; and / or, the aforementioned POLE2 gene interference nucleic acid construct; and / or, the aforementioned POLE2 gene interference lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

[0062] The composition may be a pharmaceutical composition.

[0063] When the composition is used to prevent or treat gastric cancer in a subject, an effective dose of the composition is administered to the subject. Using this method, the growth, proliferation, recurrence, and / or metastasis of the gastric cancer is inhibited. Furthermore, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the growth, proliferation, recurrence, and / or metastasis of the gastric cancer is inhibited.

[0064] The form of the composition is not particularly limited and can be in various forms such as solid, liquid, gel, semi-fluid, aerosol, etc.

[0065] The composition is mainly targeted at mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0066] In summary, the present invention designs an RNAi target sequence for the human POLE2 gene and constructs a corresponding POLE2 RNAi vector, wherein the RNAi vector pGCSIL-GFP-POLE2-siRNA can significantly downregulate the expression of the POLE2 gene at both the mRNA and protein levels. Using lentivirus (abbreviated as Lv) as a genetic manipulation tool to carry the RNAi vector pGCSIL-GFP-POLE2-siRNA can efficiently introduce RNAi sequences targeting the POLE2 gene into gastric cancer AGS cells, reducing the expression level of the POLE2 gene and significantly inhibiting the proliferation of the above-mentioned tumor cells. Therefore, lentivirus-mediated POLE2 gene silencing is a potential clinical non-surgical treatment for malignant tumors.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] After extensive and in-depth research, the present invention has discovered that downregulating the expression of the human POLE2 gene using RNAi can effectively inhibit the proliferation of gastric cancer cells and promote apoptosis, effectively controlling the progression of gastric cancer. The siRNA provided by the present invention, or a nucleic acid construct or lentivirus containing the siRNA sequence, can specifically inhibit the proliferation and growth of gastric cancer cells, thereby treating gastric cancer and opening up new avenues for gastric cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 : Vector enzyme digestion electrophoresis.

[0070] Figure 2 : Electrophoresis identification of positive clones connected with shRNA fragments.

[0071] Figure 3 : RT-PCR detection of target gene knockdown efficiency at the mRNA level in AGS cells. (P<0.05)

[0072] Figure 4 Ceigo automated cell analysis revealed that POLE2 gene knockdown inhibits gastric cancer cell proliferation. (The cell line is AGS cells, and cell counts were performed 1, 2, 3, 4, and 5 days after viral infection.)

[0073] In the accompanying drawings,

[0074] **, P < 0.01 compared with the shCtrl group and the target gene shRNA lentivirus-treated group.

[0075] *, compared with the target gene shRNA lentivirus-treated group, 0.01≤P<0.05. DETAILED DESCRIPTION

[0076] The POLE2 gene is significantly overexpressed in some tumor cells. Through extensive and in-depth research, the inventors of the present invention discovered that downregulating the expression of the human POLE2 gene using RNAi can effectively inhibit tumor cell proliferation and control tumor growth. This research indicates that the POLE2 gene is a proto-oncogene and a target for tumor therapy. The inventors further synthesized and tested multiple siRNAs targeting the POLE2 gene, identifying siRNAs that effectively inhibited POLE2 expression and, consequently, the proliferation and growth of human gastric cancer AGS cells. This led to the completion of the present invention.

[0077] POLE2 inhibitors

[0078] Refers to a molecule that has an inhibitory effect on POLE2. Inhibitory effects on POLE2 include, but are not limited to, inhibiting the expression or activity of POLE2.

[0079] Inhibiting POLE2 activity means reducing POLE2 activity. Preferably, compared to before inhibition, POLE2 activity is reduced by at least 10%, more preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0080] Inhibiting POLE2 expression may specifically be inhibiting the transcription or translation of the POLE2 gene. Specifically, it may mean preventing the transcription of the POLE2 gene, or reducing the transcriptional activity of the POLE2 gene, or preventing the translation of the POLE2 gene, or reducing the translation level of the POLE2 gene.

[0081] Those skilled in the art can use conventional methods to regulate the gene expression of POLE2, such as gene knockout, homologous recombination, interfering RNA, etc.

[0082] The inhibition of POLE2 gene expression can be verified by detecting the expression level through PCR and Western Blot.

[0083] Preferably, compared with the wild type, POLE2 gene expression is reduced by at least 10%, more preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, even more preferably by at least 90%, and most preferably, POLE2 gene is not expressed at all.

[0084] Small molecule compounds

[0085] In the present invention, it refers to a compound composed of several or dozens of atoms and with a molecular mass below 1000.

[0086] Preparation of medicine for preventing or treating gastric cancer

[0087] Nucleic acid molecules that reduce POLE2 gene expression in gastric cancer cells; and / or POLE2 gene-interfering nucleic acid constructs; and / or POLE2 gene-interfering lentiviruses can be used as active ingredients to prepare drugs for preventing or treating gastric cancer. Typically, in addition to the active ingredient, the drug also includes one or more pharmaceutically acceptable carriers or excipients, depending on the dosage form.

[0088] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.

[0089] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0090] In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited and can be prepared into injections, oral liquids, tablets, capsules, dripping pills, sprays, etc., and can be prepared by conventional methods. The choice of pharmaceutical dosage form should match the mode of administration.

[0091] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0092] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0093] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0094] Example 1 Preparation of RNAi Lentivirus Targeting Human POLE2 Gene

[0095] 1. Screening of effective siRNA targets for the human POLE2 gene

[0096] The POLE2 (NM_002692) gene information was retrieved from Genbank; effective siRNA target sites for the POLE2 gene were designed. Table 1-1 lists the screened effective siRNA target sequences for the POLE2 gene.

[0097] Table 1-1 siRNA target sequences targeting human POLE2 gene

[0098] SEQ ID NO TargetSeq(5'-3') 1 GATTGTTCTTGGAATGATA

[0099] 2. Preparation of Lentiviral Vectors

[0100] A double-stranded DNA oligo sequence containing Age I and EcoR I restriction sites at both ends was synthesized for the siRNA target (using SEQ ID NO: 1 as an example) (Table 1-2). The pGCSIL-GFP vector (provided by Shanghai Gene Medical Technology Co., Ltd.) was linearized using Age I and EcoR I restriction endonucleases, and the digested fragments were identified by agarose gel electrophoresis.

[0101] Table 1-2 Double-stranded DNA oligos with Age I and EcoR I restriction sites at both ends

[0102]

[0103] Ligate the double-digested, linearized vector DNA (enzyme digestion system shown in Table 1-4, reaction at 37°C, 1 hour) with the purified double-stranded DNA oligo using T4 DNA ligase. Ligate overnight at 16°C in an appropriate buffer system (ligation system shown in Table 1-5). Recover the ligation product. Transform the ligation product into fresh competent E. coli cells prepared with calcium chloride (transformation procedures refer to: Molecular Cloning Laboratory Manual, 2nd Edition, pages 55-56). Blot the surface of the bacterial colony grown from the ligation transformation product, dissolve it in 10 μl of LB medium, mix well, and use 1 μl as 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: 6); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7). Perform PCR identification (PCR reaction system and reaction conditions shown in Table 1-6 and Table 1-7). The clones identified as positive by PCR were sequenced and aligned. The clones with correct alignment were the successfully constructed vectors expressing RNAi against SEQ ID NO: 1 and named as pGCSIL-GFP-POLE2-siRNA.

[0104] The pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed. The negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). To construct the pGCSIL-GFP-Scr-siRNA negative control plasmid, a double-stranded DNA oligo sequence containing Age I and EcoR I restriction sites at both ends was synthesized for the Scr siRNA target (Tables 1-3). The remaining construction methods, identification methods, and conditions were the same as those for pGCSIL-GFP-POLE2-siRNA.

[0105] Table 1-3 Double-stranded DNA oligos with Age I and EcoR I restriction sites at both ends

[0106]

[0107] Table 1-4 pGCSIL-GFP plasmid restriction enzyme digestion reaction system

[0108]

[0109]

[0110] Table 1-5 Vector DNA and double-stranded DNA Oligo ligation reaction system

[0111] Reagents Positive control (μl) Self-ligation control (μl) Connectome (μl) Linearized vector DNA (100 ng / μl) 1.0 1.0 1.0 Annealed double-stranded DNA oligo (100 ng / μl) 1.0 - 1.0 10×T4 phage DNA ligase buffer 1.0 1.0 1.0 T4 phage DNA ligase 1.0 1.0 1.0 <![CDATA[dd H2O]]> 16.0 17.0 16.0 Total 20.0 20.0 20.0

[0112] Table 1-6-1 PCR reaction system

[0113] Reagents Volume (μl) 10×buffer 2.0 dNTPs (2.5 mM) 0.8 Upstream primer 0.4 Downstream primer 0.4 Taq polymerase 0.2 template 1.0 <![CDATA[ddH2O]]> 15.2 Total 20.0

[0114] Table 1-7 PCR reaction system program settings

[0115]

[0116] 3. Packaging POLE2-siRNA Lentivirus

[0117] The DNA of RNAi plasmid pGCSIL-GFP-POLE2-siRNA was extracted using Qiagen's plasmid extraction kit and prepared into a 100 ng / μl storage solution.

[0118] 24 h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and the cell density was adjusted to 1.5×10 5 cells / ml, seeded in a 6-well plate, and cultured in a 37°C, 5% CO2 incubator. Cells can be used for transfection when the cell density reaches 70%-80%. 2 hours before transfection, aspirate the original culture medium and add 1.5 ml of fresh complete culture medium. Following the instructions of the Sigma-Aldrich MISSION Lentiviral Packaging Mix kit, add 20 μl of Packing Mix (PVM), 12 μl of PEI, and 400 μl of serum-free DMEM medium to a sterile centrifuge tube. Take 20 μl of the extracted plasmid DNA and add it to the PVM / PEI / DMEM mixture.

[0119] The transfection mixture was incubated at room temperature for 15 minutes, transferred to the culture medium of human embryonic kidney 293T cells, and cultured in a 37°C, 5% CO2 incubator for 16 hours. The culture medium containing the transfection mixture was discarded, washed with PBS solution, and 2 ml of complete culture medium was added, and the culture was continued for 48 hours. The cell supernatant was collected and purified and concentrated using a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) as follows: (1) Centrifuge at 4°C, 4000g for 10 minutes to remove cell debris; (2) Filter the supernatant through a 0.45 μm filter into a 40 ml ultracentrifuge tube; (3) Centrifuge at 4000g for 10-15 minutes to the required virus concentration volume; (4) After the centrifugation, separate the filter cup from the filtrate collection cup below, turn the filter cup upside down on the sample collection cup, and centrifuge for 2 minutes at a centrifugal force not exceeding 1000g; (5) Remove the centrifuge cup from the sample collection cup, and the sample collection cup contains the virus concentrate. The viral concentrate was aliquoted and stored at -80°C. The sequence of the first strand of the siRNA contained in the viral concentrate is shown in SEQ ID NO: 2. The packaging process for the control lentivirus was the same as that for the POLE2-siRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector was used instead of the pGCSIL-GFP-POLE2-siRNA vector.

[0120] The results of vector enzyme electrophoresis are as follows Figure 1 As shown,

[0121] Lane 1: 1kb Marker: from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp

[0122] Lane 2: Vector plasmid linearized by double enzyme digestion with Age I and EcoR I

[0123] Lane 3: vector plasmid without enzyme digestion

[0124] The electrophoresis identification results of the positive clones connected to the shRNA fragment are as follows Figure 2 As shown, lane 1: negative control (ddH2O), excluding false positive results caused by exogenous nucleic acid contamination in the system

[0125] Lane 2: Self-ligation control (empty vector self-ligation control group)

[0126] Lane 3: 250bp Marker: from top to bottom: 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp

[0127] Lanes 4-8: Monoclonal pGCSIL-GFP-POLE2-siRNA-1, 2, 3, 4, 5

[0128] The above results showed that pGCSIL-GFP-POLE2-siRNA was successfully constructed.

[0129] Example 2 Detection of gene silencing efficiency by real-time fluorescence quantitative RT-PCR

[0130] Human gastric cancer AGS cells in the logarithmic growth phase were trypsinized and prepared into a cell suspension (cell number was about 5×10 4 Cells were seeded with 500 μg of lentivirus (100 μg / ml) in 6-well plates and cultured until the cell confluency reached approximately 30%. Based on the multiplicity of infection (MOI, AGS: 20), an appropriate amount of the lentivirus prepared in Example 1 was added. After 24 hours of culture, the medium was replaced. After 5 days of infection, the cells were harvested. Total RNA was extracted according to the Invitrogen Trizol® operating instructions. RNA was reverse transcribed to obtain cDNA according to the Promega M-MLV® operating instructions (see Table 2-1 for the reverse transcription reaction system; incubate at 42°C for 1 hour, then incubate in a 70°C water bath for 10 minutes to inactivate the reverse transcriptase).

[0131] Real-time quantitative detection was performed using a TP800 Real-time PCR instrument (TAKARA). The primers for the POLE2 gene were as follows: upstream primer 5'-TGAGAAGCAACCCTTGTCATC-3' (SEQ ID NO: 11) and downstream primer 5'-TCATCAACAGACTGACTGCATTC-3' (SEQ ID NO: 12). The housekeeping gene GAPDH was used as an internal reference, and the primer sequences were as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 13) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 14). The reaction system was prepared according to the ratios in Table 2-2.

[0132] Table 2-1 Reverse transcription reaction system

[0133] Reagents Volume (μl) 5×RT buffer 4.0 10mM dNTPs 2.0 RNasin 0.5 M-MLV-RTase 1.0 <![CDATA[DEPC H2O]]> 3.5 Total 11.0

[0134] Table 2-2 Real-time PCR reaction system

[0135] Reagents Volume (μl) SYBR premix ex taq 10.0 Upstream primer (2.5 μM 0.5 Downstream primer (2.5 μM) 0.5 cDNA 1.0 <![CDATA[ddH2O]]> 8.0 Total 20.0

[0136] The program was set to a two-step real-time PCR: initial denaturation at 95°C for 15 seconds; subsequent denaturation steps at 95°C for 5 seconds; and annealing and extension at 60°C for 30 seconds, for a total of 45 cycles. The absorbance was read during each extension step. After PCR, denaturation was performed at 95°C for 1 minute, followed by cooling to 55°C to allow full binding of the DNA duplex. The temperature was increased by 0.5°C from 55°C to 95°C, held for 4 seconds, while absorbance was read to generate a melting curve. A 2- ΔΔCt The expression abundance of POLE2 mRNA was calculated by the analysis method. Cells infected with the control virus were used as controls. Figure 3 As shown, the expression level of POLE2 mRNA in human gastric cancer AGS cells was downregulated by 72.6%.

[0137] Example 3 Detection of the proliferation ability of tumor cells infected with POLE2-siRNA lentivirus

[0138] Human gastric cancer AGS cells in the logarithmic growth phase were trypsinized and prepared into a cell suspension (cell number was about 5×10 4 / ml) were inoculated into 6-well plates and cultured until the cell confluence reached approximately 30%. According to the multiplicity of infection (MOI, AGS: 20), an appropriate amount of virus was added. After 24 hours of culture, the medium was replaced. After the infection time reached 5 days, the cells in the logarithmic growth phase of each experimental group were collected. The cells were resuspended in complete medium to form a cell suspension (2×10 4 / ml), inoculated into 96-well plates at a cell density of approximately 3000 cells / well. Each group had 5 replicate wells, 100 μl per well. After laying the plates, they were placed in a 37°C, 5% CO2 incubator for culture. Starting from the second day after laying the plates, the plates were read once a day using a Ceigo instrument (Nexcelom), and the readings were continued for 5 days. By adjusting the input parameters of Ceigo, the number of cells with green fluorescence in each scanned well plate was accurately calculated, and the data was statistically plotted to draw a cell proliferation curve (the results are shown in Figure 2). Figure 4 The results showed that after 5 days of in vitro culture, the proliferation rate of each tumor in the lentivirus-infected group was significantly slowed, far lower than that of the tumor cells in the control group, and the number of viable cells decreased by 67%, indicating that POLE2 gene silencing inhibited the proliferation ability of human gastric cancer AGS cells.

[0139] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention. Sequence Listing <110> Shanghai GeneCare Gene Medical Technology Co., Ltd. <120> Uses of human POLE2 gene and related products <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 gattgttctt ggaatgata 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <400> 2 gauuguucuu ggaaugaua 19 <210> 3 <211> 57 <212> RNA <213> Artificial Sequence <400> 3 ccgggcgauu guucuuggaa ugauacucga guaucauucc aagaacaauc gcuuuuu 57 <210> 4 <211> 58 <212> DNA <213> Artificial Sequence <400> 4 ccgggcgatt gttcttggaa tgatactcga gtatcattcc aagaacaatc gctttttg 58 <210> 5 <211> 58 <212> DNA <213> Artificial Sequence <400> 5 aattcaaaaa gcgattgttc ttggaatgat actcgagtat cattccaaga acaatcgc 58 <210> 6 <211> twenty four <212> DNA <213> Artificial Sequence <400> 6 cctatttccc atgattcctt cata 24 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 gtaatacggt tatccacgcg 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 ttctccgaac gtgtcacgt 19 <210> 9 <211> 54 <212> DNA <213> Artificial Sequence <400> 9 ccggttctcc gaacgtgtca cgtctcgaga cgtgacacgt tcggagaatt tttg 54 <210> 10 <211> 54 <212> DNA <213> Artificial Sequence <400> 10 aattcaaaaa ttctccgaac gtgtcacgtc tcgagacgtg acacgttcgg agaa 54 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <400> 11 tgagaagcaa cccttgtcat c 21 <210> 12 <211> twenty three <212> DNA <213> Artificial Sequence <400> 12 tcatcaacag actgactgca ttc 23 <210> 13 <211> twenty one <212> DNA <213> Artificial Sequence <400> 13 tgacttcaac agcgacaccc a 21 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <400> 14 caccctgttg ctgtagccaa a 21

Claims

1. Use of a POLE2 inhibitor in the preparation of a product having at least one of the following effects: Treat gastric cancer; Inhibit the proliferation of gastric cancer cells; Inhibit gastric cancer growth; The POLE2 inhibitor is a nucleic acid molecule that reduces the expression of the POLE2 gene in gastric cancer cells; the nucleic acid molecule is shRNA or double-stranded RNA; the double-stranded RNA is siRNA; the target sequence of the shRNA or siRNA is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that Also includes one or more of the following characteristics: 1) The double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand complement each other to form an RNA dimer, and the sequence of the first strand is as shown in SEQ ID NO: 2; 2) The nucleotide sequence of the shRNA is shown in SEQ ID NO:

3.

3. Use of a POLE2 gene interfering nucleic acid construct in the preparation of a product for treating gastric cancer, characterized in that: The POLE2 gene interference nucleic acid construct contains a gene segment encoding the shRNA in the nucleic acid molecule according to any one of claims 1 to 2, and can express the shRNA.

4. Use of a POLE2 gene interference lentivirus in the preparation of a product for treating gastric cancer, characterized in that: The POLE2 gene interfering lentivirus is prepared by viral packaging of the interfering nucleic acid construct according to claim 3 with the assistance of a lentiviral packaging plasmid and a cell line.

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