Use of human capn7 gene and related products

Abstract

The present application belongs to the field of biological medicine research, and particularly relates to the use of human CAPN7 gene as a target in the preparation of a gastric cancer treatment drug or a gastric cancer diagnosis drug. Extensive and in-depth research has found that down-regulating the expression of human CAPN7 gene by using an RNAi method can effectively inhibit the metastasis of gastric cancer cells and effectively control the growth process of gastric cancer. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the present application can specifically inhibit the metastasis and growth of gastric cancer cells, thereby treating gastric cancer and opening up a new direction for the treatment of gastric cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine research, and particularly relates to the use of human CAPN7 gene and related products. BACKGROUND

[0002] CAPN7 is a member of the calcium-dependent cysteine protease family, which is a heterodimer composed of an invariant small subunit and a variable large subunit. The large subunit has a cysteine protease domain, and both subunits have a calcium-binding domain. Studies have shown that in endometrial cancer stromal cells, CAPN7 can promote cell invasion and metastasis through MMP2 (Reprod Biol Endocrinol. 2013 Jul 15; 11: 64. doi: 10.1186 / 1477-7827-11-64.). CAPN7 can down-regulate the expression of β3-integrin by degrading HOXA 10, thereby affecting embryo implantation (Cell Death Dis. 2018 Feb 19; 9 (3): 291. doi: 10.1038 / s41419-018-0317-3.). CAPN7 can also participate in the degradation of epidermal growth factor receptor (FEBS J. 2014 Aug; 281 (16): 3642-55. doi: 10.1111 / febs.12886. Epub 2014 Jul 8.). LINC00657 can promote colorectal cancer cell invasion and metastasis through the PI3K / AKT pathway and the expression of CAPN7 (Eur Rev Med Pharmacol Sci. 2018 Oct; 22 (19): 6315-6323. doi: 10.26355 / eurrev_201810_16042.). There is no report on the use of CAPN7 gene for the treatment of gastric cancer. SUMMARY

[0003] In order to overcome the problems existing in the prior art, the purpose of the present application is to provide the use of human CAPN7 gene and related products.

[0004] In order to achieve the above-mentioned purpose and other related purposes, the present application adopts the following technical solutions:

[0005] In the first aspect of the present application, the use of human CAPN7 gene as a target in the preparation of a gastric cancer treatment drug or in the preparation of a gastric cancer diagnosis drug is provided.

[0006] The human CAPN7 gene as a target for preparing a gastric cancer treatment drug refers to taking the CAPN7 gene as an object of action to screen drugs or preparations to find drugs that can inhibit the expression of the human CAPN7 gene as a candidate drug for treating gastric cancer. The CAPN7 gene small interfering RNA (siRNA) as described in the present application is obtained by screening the human CAPN7 gene as an object of action, and can be used as a drug having an inhibitory effect on the metastasis of gastric cancer cells. In addition to this, drugs such as antibody drugs, small molecule drugs, etc. can also take the CAPN7 gene as an object of action.

[0007] The human CAPN7 gene as a target for preparing a gastric cancer treatment drug refers to taking the CAPN7 gene as an object of action to screen drugs or preparations to find drugs that can inhibit the expression of the human CAPN7 gene as a candidate drug for treating gastric cancer. The CAPN7 gene small interfering RNA (siRNA) as described in the present application is obtained by screening the human CAPN7 gene as an object of action, and can be used as a drug having an inhibitory effect on the metastasis of gastric cancer cells. In addition to this, drugs such as antibody drugs, small molecule drugs, etc. can also take the CAPN7 gene as an object of action.

[0008] The gastric cancer treatment drug can specifically inhibit the transcription or translation of the CAPN7 gene, or can specifically inhibit the expression or activity of the CAPN7 protein, thereby reducing the expression level of the CAPN7 gene in gastric cancer cells, and achieving the purpose of inhibiting the growth, differentiation, metastasis and / or survival of gastric cancer cells.

[0009] The gastric cancer treatment drug or gastric cancer diagnostic drug prepared by the CAPN7 gene includes but is not limited to nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0010] The nucleic acid includes but is not limited to antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNAs prepared by endoribonuclease III, or short hairpin RNAs (shRNAs).

[0011] The administration amount of the gastric cancer treatment drug is a dose sufficient to reduce the transcription or translation of the human CAPN7 gene, or a dose sufficient to reduce the expression or activity of the human CAPN7 protein. The expression of the human CAPN7 gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

[0012] The method for treating gastric cancer by using the aforementioned gastric cancer treatment drug mainly inhibits the proliferation of gastric cancer cells by reducing the expression level of the human CAPN7 gene to achieve the purpose of treatment. Specifically, when treating, a substance that can effectively reduce the expression level of the human CAPN7 gene is administered to the patient.

[0013] In one embodiment, the target sequence of the CAPN7 gene is as shown in SEQ ID NO: 1. Specifically: 5'-TGCGATTTATCCAGTTGAA-3'.

[0014] In a second aspect, the present application provides use of a CAPN7 inhibitor in the manufacture of a product having at least one of the following effects:

[0015] treating gastric cancer;

[0016] inhibiting metastasis of gastric cancer cells;

[0017] inhibiting growth of gastric cancer.

[0018] The product necessarily comprises the CAPN7 inhibitor, and the CAPN7 inhibitor is the effective component for the aforementioned effects.

[0019] The effective component for the aforementioned effects in the product can only be the CAPN7 inhibitor, or can comprise other molecules that can have the aforementioned effects.

[0020] That is, the CAPN7 inhibitor is the only effective component or one of the effective components of the product.

[0021] The product can be a single-component substance, or a multi-component substance.

[0022] The product has no special form, and can be in the form of a solid, a liquid, a gel, a semi-liquid, an aerosol, or the like.

[0023] The product is mainly targeted at mammals. The mammals are preferably rodents, even-toed ungulates, odd-toed ungulates, lagomorphs, primates, and the like. The primates are preferably monkeys, apes, or humans.

[0024] The product includes, but is not limited to, a drug, a health product, a food, and the like.

[0025] The CAPN7 inhibitor can be a nucleic acid molecule, an antibody, or a small molecule compound.

[0026] As exemplified in the embodiments of the present application, the CAPN7 inhibitor can be a nucleic acid molecule that reduces the expression of a CAPN7 gene in gastric cancer cells. Specifically, it can be double-stranded RNA or shRNA.

[0027] In a third aspect, the present application provides a method for treating gastric cancer, which comprises administering a CAPN7 inhibitor to a subject.

[0028] The subject can be a mammal or a gastric cancer cell of a mammal. The mammal is preferably a rodent, an even-toed ungulate, an odd-toed ungulate, a lagomorph, a primate, and the like. The primate is preferably a monkey, an ape, or a human. The gastric cancer cell can be an ex vivo gastric cancer cell.

[0029] The subject can be a patient suffering from gastric cancer or an individual expecting treatment of gastric cancer. Alternatively, the subject is an ex vivo gastric cancer cell of a patient suffering from gastric cancer or an individual expecting treatment of gastric cancer.

[0030] The CAPN7 inhibitor can be administered to the subject before, during or after treatment of gastric cancer.

[0031] The fourth aspect of the present application discloses a nucleic acid molecule for reducing expression of CAPN7 gene in gastric cancer cells, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.

[0032] The double-stranded RNA comprises a nucleotide sequence capable of hybridizing with the CAPN7 gene.

[0033] The shRNA comprises a nucleotide sequence capable of hybridizing with the CAPN7 gene.

[0034] Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is substantially identical to the target sequence in the CAPN7 gene.

[0035] The target sequence in the CAPN7 gene is a fragment in the CAPN7 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 CAPN7 gene.

[0036] Further, the target sequence of the double-stranded RNA is shown in SEQ ID NO: 1. Specifically, 5'-TGCGATTTATCCAGTTGAA-3'. Further, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2. Specifically, 5'-UGCGAUUUAUCCAGUUGAA-3'.

[0037] Further, the double-stranded RNA is small interfering RNA (siRNA).

[0038] SEQ ID NO: 2 is one strand of small interfering RNA designed for human CAPN7 gene with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence, and the sequence of the other strand, i.e. the second strand, is complementary to the sequence of the first strand. The siRNA can specifically silence the expression of endogenous CAPN7 gene in gastric cancer cells.

[0039] The shRNA comprises a sense strand fragment and an antisense strand fragment, and a stem loop structure connecting the sense strand fragment and the antisense strand fragment, the sequence of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is substantially identical to the target sequence in the CAPN7 gene.

[0040] Further, the target sequence of the shRNA is shown as SEQ ID NO: 1.

[0041] The shRNA can be processed into small interfering RNA (siRNA) after enzyme digestion, and then play a role in specifically silencing the expression of the endogenous CAPN7 gene in the gastric cancer cells.

[0042] 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.

[0043] Further, the sequence of the shRNA is shown as SEQ ID NO: 3. Specifically, 5'-UGCGAUUUAUCCAGUUUGAACUCGAGUUCAACUGGAUAAAUCGCA-3'.

[0044] Further, the CAPN7 gene is derived from human.

[0045] In the fifth aspect of the present application, a CAPN7 gene interfering nucleic acid construct is disclosed, which contains a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule, and can express the shRNA.

[0046] The CAPN7 gene interfering nucleic acid construct can be obtained by cloning the gene fragment encoding the shRNA of the aforementioned human CAPN7 gene into a known vector.

[0047] Further, the CAPN7 gene interfering nucleic acid construct is a CAPN7 gene interfering lentivirus vector.

[0048] The CAPN7 gene interfering lentivirus vector disclosed in the present application is obtained by cloning the DNA fragment encoding the shRNA of the aforementioned CAPN7 gene into a known vector, and the known vector is mostly a lentivirus vector. After the CAPN7 gene interfering lentivirus vector is packaged into infectious virus particles, it infects gastric cancer cells, and then transcribes the shRNA disclosed in the present application, and finally obtains the siRNA through enzyme digestion and other steps, which is used for specifically silencing the expression of the CAPN7 gene.

[0049] Further, the CAPN7 gene interfering lentivirus vector also contains a promoter sequence and / or a nucleotide sequence encoding a detectable marker in the gastric cancer cells; preferably, the detectable marker is green fluorescent protein (GFP).

[0050] Further, the lentivirus vector can be selected from any one 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-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 / V5-GW / lacZ.

[0051] The present application specifically lists the human CAPN7 gene interference lentivirus vector constructed by using pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-CAPN7-siRNA.

[0052] The CAPN7 gene siRNA of the present application can be used to inhibit the proliferation of gastric cancer cells, and can be further used as a drug or preparation for treating gastric cancer. The CAPN7 gene interference lentivirus vector can be used to prepare the CAPN7 gene siRNA. When used as a drug or preparation for treating gastric cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The specific dose should also consider the administration route, patient health status and other factors, which are within the skill of a skilled physician.

[0053] In the sixth aspect of the present application, a CAPN7 gene interference lentivirus is disclosed, which is formed by virus packaging of the aforementioned CAPN7 gene interference nucleic acid construct with the assistance of lentivirus packaging plasmid and cell line. The lentivirus can infect gastric cancer cells and produce small interfering RNA against CAPN7 gene, thereby inhibiting the proliferation of gastric cancer cells. The CAPN7 gene interference lentivirus can be used to prepare a drug for preventing or treating gastric cancer.

[0054] In a seventh aspect of the present application, the use of the aforementioned nucleic acid molecule, or the aforementioned CAPN7 gene interfering nucleic acid construct, or the aforementioned CAPN7 gene interfering lentivirus, for the preparation of a medicament for preventing or treating gastric cancer, or for the preparation of a kit for reducing the expression of CAPN7 gene in gastric cancer cells.

[0055] The use of the medicament 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 amount of the medicament to the subject.

[0056] Further, when the medicament is used for preventing or treating gastric cancer in a subject, an effective amount of the medicament needs to be administered to the subject. By this method, the growth, proliferation, recurrence and / or metastasis of the gastric cancer 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 gastric cancer is inhibited.

[0057] The subject of the method can be a human.

[0058] In an eighth aspect of the present application, a composition for preventing or treating gastric cancer is provided, the effective substance of which comprises:

[0059] The aforementioned nucleic acid molecule; and / or, the aforementioned CAPN7 gene interfering nucleic acid construct; and / or, the aforementioned CAPN7 gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

[0060] The composition can be a pharmaceutical composition.

[0061] When the composition is used for preventing or treating gastric cancer in a subject, an effective amount of the composition needs to be administered to the subject. By this method, the growth, proliferation, recurrence and / or metastasis of the gastric cancer 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 gastric cancer is inhibited.

[0062] The form of the composition is not particularly limited, and can be various forms of solid, liquid, gel, semi-liquid, aerosol, etc.

[0063] The subject targeted by the composition is mainly a mammal. The mammal is preferably a rodent, an even-toed ungulate, an odd-toed ungulate, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a human.

[0064] In summary, the present application designs the RNAi target sequence for human CAPN7 gene, constructs the corresponding CAPN7 RNAi vector, wherein the RNAi vector pGCSIL-GFP-CAPN7-siRNA can significantly down-regulate the expression of CAPN7 gene at mRNA level and protein level. Using lentivirus (abbreviated as Lv) as a gene operation tool to carry the RNAi vector pGCSIL-GFP-CAPN7-siRNA can efficiently introduce the RNAi sequence for CAPN7 gene into gastric cancer AGS cells, reduce the expression level of CAPN7 gene, and significantly inhibit the proliferation ability of the tumor cells. Therefore, the lentivirus-mediated CAPN7 gene silencing is a potential clinical non-surgical treatment for malignant tumors.

[0065] Compared with the prior art, the present application has the following beneficial effects:

[0066] The present application has found through extensive and in-depth research that down-regulating the expression of human CAPN7 gene by using the RNAi method can effectively inhibit the metastasis of gastric cancer cells and effectively control the growth process of gastric cancer, which indicates that CAPN7 gene is a proto-oncogene and can be used as a target for tumor treatment. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the present application can specifically inhibit the growth and metastasis of gastric cancer, thereby treating gastric cancer and opening up a new direction for the treatment of gastric cancer. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 : RT-PCR detection of mRNA level of AGS cell target gene reduction efficiency.

[0068] Figure 2 : Statistical graph of metastasis ability of tumor cells infected with lentivirus.

[0069] In the drawings,

[0070] The column chart represents the average value of three experiments, and the error line represents the standard deviation (SD).

[0071] **, shCtrl compared with the target gene shRNA lentivirus treatment group, P<0.01. DETAILED DESCRIPTION

[0072] The application proves the role of CAPN7 gene in the occurrence of gastric cancer from the perspective of cell function. After the target gene shRNA lentivirus is constructed and transfected into gastric cancer cells, the expression of the target gene at the mRNA and protein levels in the two groups of gastric cancer cell lines is detected compared with the transfection control lentivirus. Then, cell proliferation, apoptosis and other tests are carried out through cell function experiments. The results show that compared with the control group, the inhibition degree of gastric cancer cell proliferation in the shRNA group is obviously higher than that in the control group, and the increase degree of cell apoptosis rate is higher than that in the control group.

[0073] According to the above research results, further exploration and development of new diagnosis and treatment methods for the gene can provide more choices for the diagnosis and treatment of gastric cancer patients.

[0074] CAPN7 inhibitor

[0075] It refers to a molecule that has an inhibitory effect on CAPN7. Having an inhibitory effect on CAPN7 includes but is not limited to inhibiting the expression or activity of CAPN7.

[0076] Inhibiting the activity of CAPN7 refers to reducing the activity of CAPN7. Preferably, the activity of CAPN7 is reduced by at least 10% compared with before inhibition, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0077] Inhibiting the expression of CAPN7 can specifically inhibit the transcription or translation of the CAPN7 gene. Specifically, it can refer to: not transcribing the CAPN7 gene, or reducing the transcription activity of the CAPN7 gene, or not translating the CAPN7 gene, or reducing the translation level of the CAPN7 gene.

[0078] Those skilled in the art can use conventional methods to regulate the expression of the CAPN7 gene, such as gene knockout, homologous recombination, interference RNA, etc.

[0079] The inhibition of the expression of the CAPN7 gene can be verified by PCR and Western Blot detection of the expression amount.

[0080] Preferably, compared with the wild type, the expression of the CAPN7 gene is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, more preferably by at least 90%, and most preferably the CAPN7 gene is completely not expressed.

[0081] Small molecule compound

[0082] In the present application, it refers to a compound composed of several or dozens of atoms with a molecular mass of less than 1000.

[0083] Preparation of a drug for preventing or treating gastric cancer

[0084] A nucleic acid molecule which reduces the expression of CAPN7 gene in gastric cancer cells; and / or, a CAPN7 gene interfering nucleic acid construct; and / or, a CAPN7 gene interfering lentivirus can be used as an active ingredient to prepare a medicament for preventing or treating gastric cancer. In general, the medicament can include one or more pharmaceutically acceptable carriers or excipients, in addition to the active ingredient, as necessary according to the different dosage forms.

[0085] "Pharmaceutically acceptable" means that the molecules and compositions, when administered to animals or humans, do not produce adverse, allergic, or other untoward reactions with respect to the efficacy of the active ingredient.

[0086] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, i.e., they can be blended with the active ingredient without causing a substantial degradation of the efficacy of the medicament under normal conditions. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; 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; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffer solutions, etc. These substances are used as necessary to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0087] In the present invention, the pharmaceutical dosage form is not particularly limited unless otherwise specified, and can be prepared into a dosage form such as a needle, an oral solution, a tablet, a capsule, a dripping pill, a spray, etc., by a conventional method. The pharmaceutical dosage form should be selected to match the administration method.

[0088] 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 following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are used to describe the specific embodiments, not to limit the scope of the present invention. The test methods in the following examples, unless otherwise specified, are generally performed under conventional conditions, or under the conditions recommended by the respective manufacturers.

[0089] When the embodiments give numerical ranges, it is understood that unless the present invention indicates otherwise, every numerical range's two endpoints and any number between the two endpoints are optional. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to specific methods, devices, materials, etc. described in the embodiments, any methods, devices, and materials similar or equivalent to those described in the embodiments of the present invention can be used in the practice of the present invention, according to the knowledge of the person skilled in the art and the description of the present invention.

[0090] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all use conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0091] Example 1 Preparation of lentivirus for RNAi of human CAPN7 gene

[0092] 1. Screening of effective siRNA target points for human CAPN7 gene

[0093] CAPN7 (NM_014296) gene information was retrieved from Genbank; effective siRNA target points for CAPN7 gene were designed. Table 1-1 lists the effective siRNA target point sequences screened for CAPN7 gene.

[0094] Table 1-1 siRNA target point sequences targeting human CAPN7 gene

[0095] SEQ ID NO TargetSeq (5'-3') 1 TGCGATTTATCCAGTTGAA

[0096] 2. Preparation of lentivirus vector

[0097] Double-stranded DNA Oligo sequences containing cohesive ends with Age I and EcoR I enzyme cutting sites at both ends were synthesized for siRNA target points (taking SEQ ID NO: 1 as an example) (Table 1-2); pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) was linearized by Age I and EcoR I restriction endonucleases, and the enzyme cutting fragments were identified by agarose gel electrophoresis.

[0098] Table 1-2 Double-stranded DNA Oligo containing cohesive ends with Age I and EcoR I enzyme cutting sites at both ends

[0099]

[0100] The linearized vector DNA and purified double-stranded DNA Oligo were ligated by T4 DNA ligase, and the ligation reaction was performed in a suitable buffer system (ligation system as shown in Table 1-5) at 16°C overnight. The ligation product was recovered and transformed into fresh E. coli competent cells prepared by calcium chloride (transformation operation reference: Guide to Molecular Cloning, 2nd Edition, pp. 55-56). The ligation and transformation product was spread on a bacterial colony surface, dissolved in 10 μl of LB medium, mixed, and 1 μl was used as a template. Universal PCR primers were designed upstream and downstream of the RNAi sequence in the lentiviral vector, the upstream primer sequence was 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6), and the downstream primer sequence was 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7). PCR identification experiments were performed (PCR reaction system as shown in Table 1-6, reaction conditions as shown in Table 1-7). The positive clones of PCR identification were sequenced and compared, and the correct clones were successfully constructed as the expression RNAi vector for SEQ ID NO: 1, named pGCSIL-GFP-CAPN7-siRNA.

[0101] The pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed, and the negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When constructing 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 (Table 1-3) for the Scr siRNA target, and the rest of the construction method, identification method and conditions were the same as those of pGCSIL-GFP-CAPN7-siRNA.

[0102] Table 1-3 Double-stranded DNA Oligo containing Age I and EcoR I restriction sites at both ends

[0103]

[0104] Table 1-4 pGCSIL-GFP plasmid digestion reaction system

[0105] Reagent Volume (μl) pGCSIL-GFP plasmid (1 μg / μl) 2.0 10 x buffer 5.0 100 x BSA 0.5 Age I (10 U / μl) 1.0 EcoR I (10 U / μl) 1.0 dd H2O 40.5 Total 50.0

[0106] Table 1-5 Vector DNA and double-stranded DNA Oligo ligation reaction system

[0107] Reagent Positive control (μl) Self-ligation control (μl) Ligation group (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 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

[0108] Table 1-6 PCR reaction system

[0109] Reagent Volume (μl) 10 x buffer 2.0 dNTPs (2.5 mM) 0.8 Upstream primer 0.4 Downstream primer 0.4 Taq polymerase 0.2 Template 1.0 ddH2O 15.2 Total 20.0

[0110] Table 1-7 PCR reaction system program setting

[0111]

[0112] 3. Package CAPN7-siRNA lentivirus

[0113] Extract the DNA of RNAi plasmid pGCSIL-GFP-CAPN7-siRNA with Qiagen plasmid extraction kit, and prepare 100 ng / μl storage solution.

[0114] 24 h before transfection, digest the logarithmic growth phase human embryonic kidney 293T cells with trypsin, adjust the cell density to 1.5 x 10 5 5CO2 incubator. When the cell density reaches 70%-80%, it can be used for transfection. 2 h before transfection, aspirate the original culture medium, and add 1.5 ml of fresh complete culture medium. According to the instructions of the MISSION Lentiviral Packaging Mix kit of Sigma-aldrich company, add Packing Mix (PVM) 20 μl, PEI 12 μl, serum-free DMEM medium 400 μl, and 20 μl of the above extracted plasmid DNA to the above PVM / PEI / DMEM mixture.

[0115] The transfection mixture was incubated at room temperature for 15 min, and then transferred to the culture medium of human embryonic kidney 293T cells, which were incubated at 37°C in a 5% CO2 incubator for 16 h. The culture medium containing the transfection mixture was discarded, and the cells were washed with PBS and incubated in 2 ml of complete medium for another 48 h. The cell supernatant was collected, and the lentivirus was purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) according to the following steps: (1) centrifugation at 4°C and 4000 g for 10 min to remove cell debris; (2) filtration of the supernatant through a 0.45 μm filter into a 40 ml ultracentrifuge tube; (3) centrifugation at 4000 g for 10-15 min to the desired volume of concentrated virus; (4) after centrifugation, the filter cup was separated from the lower filtrate collection cup, and the filter cup was inverted on the sample collection cup and centrifuged at a centrifugal force of no more than 1000 g for 2 min; (5) the centrifugal cup was removed from the sample collection cup, and the concentrated virus solution was obtained in the sample collection cup. The concentrated virus solution was aliquoted and stored at -80°C. The sequence of the first strand of siRNA contained in the concentrated virus solution is shown in SEQ ID NO: 2. The packaging process of the control lentivirus was the same as that of CAPN7-siRNA lentivirus, except that the pGCSIL-GFP-CAPN7-siRNA vector was replaced by the pGCSIL-GFP-Scr-siRNA vector.

[0116] Example 2 Detection of the silencing efficiency of the gene by real-time fluorescent quantitative RT-PCR

[0117] Human gastric cancer AGS cells in the logarithmic growth phase were trypsinized to prepare a cell suspension (cell number: about 5 x 10 4 / ml) which was inoculated into a 6-well plate and cultured until the cell confluence reached about 30%. An appropriate amount of the lentivirus prepared in Example 1 was added according to the multiplicity of infection (MOI, AGS: 10), and the culture medium was replaced after 24 h. The cells were collected after 5 days of infection. Total RNA was extracted according to the Trizol operation manual of Invitrogen. cDNA was obtained by reverse transcription of RNA according to the M-MLV operation manual of Promega (reverse transcription reaction system: see Table 2-1, 42°C for 1 h, and then inactivation of reverse transcriptase by water bath in a 70°C water bath for 10 min).

[0118] Real time PCR was performed by using TP800 Real time PCR instrument (TAKARA). The primers of CAPN7 gene were as follows: upstream primer 5'-TGGAAAGTGGAGTGGTCAGA-3' (SEQ ID NO: 11) and downstream primer 5'-ATTGCCTTGGTCCTCGTAGC-3' (SEQ ID NO: 12). 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 proportion in Table 2-2.

[0119] Table 2-1 Reverse transcription reaction system

[0120] Reagent Volume (μl) 5 x RT buffer 4.0 10 mM dNTPs 2.0 RNasin 0.5 M-MLV-RTase 1.0 DEPC H2O 3.5 Total 11.0

[0121] Table 2-2 Real-time PCR reaction system

[0122] Reagent 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 ddH2O 8.0 Total 20.0

[0123] The program was set as two-step Real-time PCR: pre-denaturation at 95°C for 30 s; then denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s; a total of 40 cycles. The absorbance value was read at each extension stage. After PCR, the DNA was denatured at 95°C for 15 s, and then cooled to 60°C to allow the DNA double strands to fully bind. From 60°C to 95°C, each step increased by 0.5°C, and the absorbance value was read for 4 s to make a melting curve. The 2- ΔΔCt The expression abundance of CAPN7 mRNA in the cells infected with lentivirus was analyzed by using the method. The cells infected with control virus were used as a control. The experimental results are shown in Figure 1 , indicating that the expression level of CAPN7 mRNA in human gastric cancer AGS cells was down-regulated by 59%.

[0124] Example 8 Detection of metastasis level of tumor cells infected with lentivirus

[0125] AGS tumor cells in the logarithmic growth phase were trypsinized to prepare a cell suspension (the cell number was about 5x10 4 / ml) which was inoculated in a 6-well plate and cultured until the cell confluence reached about 30%. According to the multiplicity of infection (MOI=10), an appropriate amount of virus was added, and the culture medium was replaced after 24 h. After 3 days of infection, the cells in the logarithmic growth phase were collected. First, the OrisTM block alcohol was sterilized by immersion, dried, and placed in a 96-well plate; then 5x104 The next day, the OrisTM block was carefully removed and the cells were gently washed 2-3 times with serum-free medium and then incubated with low serum medium (0.5% FBS). The cells were incubated in a 37℃, 5% CO2 incubator and the appropriate time point was selected according to the pre-experiment. By adjusting the input parameters of the analysis settings, the area of green fluorescent cells in each scan of the well plate was accurately calculated.

[0126] The results of cell metastasis are shown in Figure 2 As shown in Table 1, after the expression of CAPN7 gene was reduced by RNA interference (shCAPN7 group) compared with the corresponding control interference (shCtrl group), the metastasis ability of tumor cells was 58% of the control group.

[0127] The above description is only a preferred embodiment of the present application, and is not intended to limit the present application in any form or substance. It should be noted that, for ordinary skilled persons in the art, without departing from the method of the present application, some improvements and supplements can also be made, which should be considered as the protection scope of the present application. For those skilled in the art, without departing from the spirit and scope of the present application, some changes, modifications and equivalent changes made by using the disclosed technical content are equivalent embodiments of the present application; at the same time, any equivalent changes, modifications and evolution of the above-mentioned embodiments according to the essential technology of the present application are still within the scope of the technical solutions of the present application. SEQUENCE LISTING <110> Shanghai Jikai Gene Medicine Science and Technology Co., Ltd. <120> Use of human CAPN7 gene and related products <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 tgcgatttat ccagttgaa 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <400> 2 ugcgauuuau ccaguugaa 19 <210> 3 <211> 45 <212> RNA <213> Artificial Sequence <400> 3 ugcgauuuau ccaguuugaa cucgaguuca acuggauaaa ucgca 45 <210> 4 <211> 54 <212> DNA <213> Artificial Sequence <400> 4 ccggtgcgat ttatccagtt gaactcgagt tcaactggat aaatcgcatt tttg 54 <210> 5 <211> 54 <212> DNA <213> Artificial Sequence <400> 5 aattcaaaaa tgcgatttat ccagttgaac tcgagttcaa ctggataaat cgca 54 <210> 6 <211> 24 <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> 20 <212> DNA <213> Artificial Sequence <400> 11 tggaaagtgg agtggtcaga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 attgccttgg tcctcgtagc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <400> 13 TGA CTT CAA CAG CGA CAC CC 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <400> 14 CAC CCT GTT GCT GTA GCC AA 21

Claims

1. Use of CAPN7-RNAi lentivirus in the preparation of a drug for treating gastric cancer, wherein the CAPN7-RNAi lentivirus comprises a coding sequence of double-stranded RNA or shRNA, the CAPN7-RNAi lentivirus targets a human CAPN7 gene, and a target sequence of the CAPN7-RNAi lentivirus is shown as SEQ ID NO:

1.

2. Use of a CAPN7 inhibitor in the preparation of a product having at least one of the following effects: treating gastric cancer; inhibiting metastasis of gastric cancer cells; the CAPN7 inhibitor is a CAPN7-RNAi lentivirus, a target sequence of the CAPN7 inhibitor is shown as SEQ ID NO: 1, and the CAPN7-RNAi lentivirus comprises a coding sequence of double-stranded RNA or shRNA.

3. Use according to claim 2, characterized in that, Further comprising one or more of the following features: 1) the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and a sequence of the first strand is shown as SEQ ID NO: 2; 2) a nucleotide sequence of the shRNA is shown as SEQ ID NO:

3.

4. Use of CAPN7-RNAi lentivirus in the preparation of a kit for reducing expression of a CAPN7 gene in gastric cancer cells, wherein a target sequence of the CAPN7-RNAi lentivirus effective against a CAPN7 gene is shown as SEQ ID NO: 1, and the CAPN7-RNAi lentivirus comprises a coding sequence of double-stranded RNA or shRNA.

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