Use of human cfap65 gene and related products

Abstract

This invention belongs to the field of biomedical research, specifically relating to the use of the human CFAP65 gene as a target in the preparation of therapeutic or diagnostic drugs for gastric cancer. Extensive and in-depth research has revealed that downregulating the expression of the human CFAP65 gene using RNAi methods can effectively inhibit the proliferation of gastric cancer cells and effectively control the growth process of gastric cancer. The siRNA or nucleic acid constructs containing the siRNA sequence, or lentiviruses provided by this invention, can specifically inhibit the proliferation rate and growth of gastric cancer cells, thereby treating gastric cancer and opening up new directions for gastric cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical research, specifically relating to the uses of the human CFAP65 gene and related products. Background Technology

[0002] CFAP65 is a protein-coding gene. Diseases associated with CFAP65 include spermatogenesis failure and nonsyndrome male infertility due to sperm motility disorders. This gene likely plays a role in sperm motility, and the protein it encodes is likely a transmembrane protein with a putative coil-coil domain. An ortholog of this gene is involved in the Rose-comb mutation, a large chromosomal inversion that leads to comb-like morphology changes and sperm motility defects (Wang W, et al. Biallelic mutations in CFAP65 lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations. J Med Genet. 2019. PMID:31501240). Human mitochondrial transcription factor A (TFAM) is associated with promoting tumor growth and invasion. TFAM activates mitochondrial DNA (mtDNA) transcription and affects nuclear gene expression through retrograde mitochondrial signaling. Knocking out CFAP65 (cilia and flagella-associated protein 65) or PCK1 (cytosolic phosphoenolpyruvate carboxylkinase) can rescue the effects of TFAM depletion on cell morphology and proliferation. Studies have found that PCK1 acts downstream of CFAP65 in calcium-mediated retrograde signaling. Furthermore, depletion of 2',3'-dideoxycytidine mtDNA is sufficient to induce CFAP65 and PCK1 expression and inhibit cell proliferation. Therefore, the TFAM-mtDNA-calcium-CFAP65-PCK1 axis is involved in mitochondrial retrograde signaling, affecting tumor cell differentiation and proliferation (Transcriptomic Analysis of Mitochondrial TFAM Depletion Changing Cell Morphology and Proliferation). However, reports on the role of CFAP65 in tumor-related fields are scarce. Summary of the Invention

[0003] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide the use of the human CFAP65 gene and related products.

[0004] To achieve the above-mentioned objectives and other related objectives, the present invention adopts the following technical solution:

[0005] In a first aspect, the present invention provides the use of the human CFAP65 gene as a target in the preparation of a gastric cancer therapeutic drug or in the preparation of a gastric cancer diagnostic drug.

[0006] The use of the human CFAP65 gene as a target in the preparation of gastric cancer therapeutic drugs specifically refers to screening drugs or formulations by targeting the CFAP65 gene to identify those that can inhibit human CFAP65 gene expression as candidate drugs for gastric cancer treatment. For example, the CFAP65 gene small interfering RNA (siRNA) described in this invention was obtained by screening using the human CFAP65 gene as a target and can be used as a drug with inhibitory effects on the proliferation of gastric cancer cells. In addition, antibody drugs, small molecule drugs, and other similar drugs can also target the CFAP65 gene.

[0007] The use of the human CFAP65 gene as a target for the preparation of gastric cancer diagnostic drugs specifically refers to using the CFAP65 gene expression product as a gastric cancer diagnostic indicator in the preparation of gastric cancer diagnostic drugs.

[0008] The gastric cancer treatment drug is a molecule that can specifically inhibit the transcription or translation of the CFAP65 gene, or specifically inhibit the expression or activity of the CFAP65 protein, thereby reducing the expression level of the CFAP65 gene in gastric cancer cells and achieving the purpose of inhibiting the proliferation, growth, differentiation and / or survival of gastric cancer cells.

[0009] The gastric cancer treatment or diagnostic drugs prepared through the CFAP65 gene include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, peptides, proteins, or interfering lentiviruses.

[0010] The nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA or short hairpin RNA (shRNA) prepared by ribonuclease III.

[0011] The dosage of the gastric cancer treatment drug is sufficient to reduce the transcription or translation of the human CFAP65 gene, or sufficient to reduce the expression or activity of the human CFAP65 protein, so as to reduce the expression of the human CFAP65 gene by at least 50%, 80%, 90%, 95%, or 99%.

[0012] The aforementioned method of treating gastric cancer with the aforementioned drugs primarily aims to inhibit the proliferation of gastric cancer cells by reducing the expression level of the human CFAP65 gene. Specifically, during treatment, substances that effectively reduce the expression level of the human CFAP65 gene are administered to the patient.

[0013] In one embodiment, the target sequence of the CFAP65 gene is shown in SEQ ID NO: 1. Specifically, it is: 5'-CCTTGAAACTCCAGAAGAT-3'.

[0014] A second aspect of the invention provides the use of a CFAP65 inhibitor in the preparation of a product having at least one of the following effects:

[0015] Treatment of stomach cancer;

[0016] Inhibits the proliferation rate of gastric cancer cells;

[0017] Inhibits the growth of gastric cancer.

[0018] The product must include a CFAP65 inhibitor, and the CFAP65 inhibitor is the active ingredient for the aforementioned effects.

[0019] In the product, the active ingredient that performs the aforementioned function may be only a CFAP65 inhibitor, or it may contain other molecules that can perform the aforementioned function.

[0020] That is, the CFAP65 inhibitor is the sole active ingredient or one of the active ingredients in the product.

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

[0022] There are no special restrictions on the form of the product; it can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0023] The product is primarily targeted at mammals. Preferred mammals include rodents, even-toed ungulates, perissodactyls, lagomorphs, and primates. Preferred primates include monkeys, apes, or humans.

[0024] The products mentioned include, but are not limited to, medicines, health products, and food.

[0025] The CFAP65 inhibitor can be a nucleic acid molecule, antibody, or small molecule compound.

[0026] As illustrated in the embodiments of the present invention, the CFAP65 inhibitor can be a nucleic acid molecule that reduces the expression of the CFAP65 gene in gastric cancer cells. Specifically, it can be double-stranded RNA or shRNA.

[0027] A third aspect of the present invention provides a method for treating gastric cancer by administering a CFAP65 inhibitor to a subject.

[0028] The target organism can be a mammal or gastric cancer cells from a mammal. The mammal is preferably a rodent, even-toed ungulate, perissodactyl, lagomorph, or primate. The primate is preferably a monkey, ape, or human. The gastric cancer cells can be isolated gastric cancer cells.

[0029] The subject can be a patient suffering from gastric cancer or an individual expecting treatment for gastric cancer. Alternatively, the subject can be isolated gastric cancer cells from a patient or an individual expecting treatment for gastric cancer.

[0030] The CFAP65 inhibitor can be administered to subjects before, during, and after gastric cancer treatment.

[0031] The fourth aspect of this invention discloses a nucleic acid molecule that reduces the expression of the CFAP65 gene in gastric cancer cells, said nucleic acid molecule comprising double-stranded RNA or shRNA.

[0032] The double-stranded RNA contains a nucleotide sequence capable of hybridizing with the CFAP65 gene.

[0033] The shRNA contains a nucleotide sequence that can hybridize with the CFAP65 gene.

[0034] Furthermore, the double-stranded RNA comprises a first strand and a second strand, which are complementary to form an RNA dimer, and the sequence of the first strand is substantially the same as the target sequence in the CFAP65 gene.

[0035] The target sequence in the CFAP65 gene is the segment in the CFAP65 gene that is recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used to specifically silence the expression of the CFAP65 gene.

[0036] Furthermore, the target sequence of the double-stranded RNA is shown in SEQ ID NO: 1, specifically 5'-CCTTGAAACTCCAGAAGAT-3'. Even further, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2, specifically 5'-CCUUGAAACUCCAGAAGAU-3'.

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

[0038] SEQ ID NO: 2 is a strand of a small interfering RNA designed with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence, targeting the human CFAP65 gene. The other strand, i.e., the second strand, is complementary to the first strand sequence. This siRNA can specifically silence the expression of the endogenous CFAP65 gene in gastric cancer cells.

[0039] The shRNA comprises a sense strand and an antisense strand, and a stem-loop structure connecting the sense strand and the antisense strand. The sequences of the sense strand and the antisense strand are complementary, and the sequence of the sense strand is substantially the same as the target sequence in the CFAP65 gene.

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

[0041] The shRNA, after being processed by enzyme digestion, can become small interfering RNA (siRNA), which can then specifically silence the expression of the endogenous CFAP65 gene in gastric cancer cells.

[0042] Furthermore, the stem-loop sequence of the shRNA can be selected from any of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC.

[0043] Furthermore, the sequence of the shRNA is shown in SEQ ID NO: 3. Specifically, it is 5'-AUCCUUGAAACUCCAGAAGAUCUCGAGAUCUUCUGGAGUUUCAAGGAU-3'.

[0044] Furthermore, the CFAP65 gene is derived from humans.

[0045] In a fifth aspect, the present invention discloses a CFAP65 gene interference nucleic acid construct containing a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule, and capable of expressing the shRNA.

[0046] The CFAP65 gene interference nucleic acid construct can be obtained by cloning the gene fragment encoding the aforementioned human CFAP65 gene shRNA into a known vector.

[0047] Furthermore, the CFAP65 gene interference nucleic acid construct is a CFAP65 gene interference lentiviral vector.

[0048] The CFAP65 gene interference lentiviral vector disclosed in this invention is obtained by cloning a DNA fragment encoding the aforementioned CFAP65 gene shRNA into a known vector, most of which are lentiviral vectors. After the CFAP65 gene interference lentiviral vector is packaged into infectious viral particles, it infects gastric cancer cells and then transcribes the shRNA of this invention. Through enzyme digestion and other steps, the siRNA is finally obtained and used to specifically silence the expression of the CFAP65 gene.

[0049] Furthermore, the CFAP65 gene interference lentiviral vector also contains a promoter sequence and / or a nucleotide sequence encoding a detectable marker in gastric cancer cells; preferably, the detectable marker is green fluorescent protein (GFP).

[0050] Furthermore, 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, pL... 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.

[0051] The embodiments of the present invention specifically illustrate a human CFAP65 gene interference lentiviral vector constructed using pGCSIL-GFP as the vector, named pGCSIL-GFP-CFAP65-siRNA.

[0052] The CFAP65 gene siRNA of this invention can be used to inhibit the proliferation of gastric cancer cells, and further can be used as a drug or preparation for treating gastric cancer. A CFAP65 gene interference lentiviral vector can be used to prepare the CFAP65 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 dosage should also consider factors such as the route of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0053] In a sixth aspect, this invention discloses a CFAP65 gene-interfering lentivirus, which is prepared by viral packaging of the aforementioned CFAP65 gene-interfering nucleic acid construct with the assistance of a lentiviral packaging plasmid and a cell line. This lentivirus can infect gastric cancer cells and produce small interfering RNA targeting the CFAP65 gene, thereby inhibiting the proliferation of gastric cancer cells. This CFAP65 gene-interfering lentivirus can be used to prepare drugs for the prevention or treatment of gastric cancer.

[0054] In a seventh aspect, the present invention provides the use of the aforementioned nucleic acid molecule, or the aforementioned CFAP65 gene interference nucleic acid construct, or the aforementioned CFAP65 gene interference lentivirus, for the purpose of: preparing a drug for the prevention or treatment of gastric cancer, or preparing a kit for reducing the expression of the CFAP65 gene in gastric cancer cells.

[0055] The application of the drug for the prevention or treatment of gastric cancer provides a method for the treatment of gastric cancer, specifically a method for the prevention or treatment of gastric cancer in a subject, comprising administering an effective dose of the drug to the subject.

[0056] Furthermore, when the drug is used to prevent or treat gastric cancer in a subject, an effective dose of the drug needs to be administered to the subject. Using this method, the growth, proliferation, recurrence, and / or metastasis of the gastric cancer are 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 are inhibited.

[0057] The object of the method can be a person.

[0058] An eighth aspect of the present invention provides a composition for the prevention or treatment of gastric cancer, wherein the active ingredient comprises:

[0059] The aforementioned nucleic acid molecules; and / or, the aforementioned CFAP65 gene interference nucleic acid constructs; and / or, the aforementioned CFAP65 gene interference lentiviruses, as well as pharmaceutically acceptable vectors, diluents, or excipients.

[0060] The composition may be a pharmaceutical composition.

[0061] When the composition is used to prevent or treat gastric cancer in a subject, an effective dose of the composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence, and / or metastasis of the gastric cancer are 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 are inhibited.

[0062] The composition is not particularly limited in form and can be in various forms such as solid, liquid, gel, semi-fluid, or aerosol.

[0063] The composition is primarily intended for use with mammals. Preferred mammals include rodents, even-toed ungulates, perissodactyls, lagomorphs, and primates. Preferred primates include monkeys, apes, or humans.

[0064] In summary, this invention designed an RNAi target sequence for the human CFAP65 gene and constructed a corresponding CFAP65 RNAi vector. The RNAi vector pGCSIL-GFP-CFAP65-siRNA significantly downregulated the expression of the CFAP65 gene at both the mRNA and protein levels. Using lentivirus (Lv) as a gene manipulation tool to carry the RNAi vector pGCSIL-GFP-CFAP65-siRNA enabled the targeted and efficient introduction of the CFAP65 gene-targeting RNAi sequence into gastric cancer AGS cells, reducing the expression level of the CFAP65 gene and significantly inhibiting the proliferation of these tumor cells. Therefore, lentivirus-mediated CFAP65 gene silencing is a potential non-surgical clinical treatment for malignant tumors.

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

[0066] This invention, through extensive and in-depth research, has discovered that downregulating the expression of the human CFAP65 gene using RNAi methods can effectively inhibit the proliferation of gastric cancer cells and effectively control the growth process of gastric cancer. This research indicates that the CFAP65 gene is a proto-oncogene and can serve as a target for tumor therapy. The siRNA or nucleic acid constructs containing this siRNA sequence, or lentiviruses provided by this invention, can specifically inhibit the proliferation rate of gastric cancer cells and suppress gastric cancer growth, thereby treating gastric cancer and opening up new directions for gastric cancer treatment. Attached Figure Description

[0067] Figure 1 RT-PCR was used to detect the reduction efficiency of target genes at the mRNA level in AGS cells.

[0068] Figure 2 Automated analysis of Celigo cells revealed that CFAP65 gene reduction inhibited the proliferation of gastric cancer cells. (The cell line was AGS cells, and cell counts were performed on days 1, 2, 3, 4, and 5 post-viral infection.)

[0069] In the attached diagram,

[0070] The bar chart represents the average of three experiments, and the error bars represent the standard deviation (SD).

[0071] **,shCtrl compared with the lentivirus treatment group containing the target gene shRNA, P<0.01. Detailed Implementation

[0072] This invention demonstrates the role of the CFAP65 gene in gastric cancer development from a cellular functional perspective. A lentivirus containing the target gene shRNA was constructed and transfected into gastric cancer cells. The expression levels of the target gene at the mRNA and protein levels in the two gastric cancer cell lines were detected and compared with a control lentivirus transfected with shRNA. Subsequently, cell proliferation and other functional assays were performed. The results showed that the shRNA group exhibited significantly greater inhibition of gastric cancer cell proliferation compared to the control group.

[0073] Based on the above research results, further exploration and development of new diagnostic and therapeutic methods targeting this gene could provide more options for the diagnosis and treatment of gastric cancer patients.

[0074] CFAP65 inhibitors

[0075] This refers to molecules that have an inhibitory effect on CFAP65. Inhibitory effects on CFAP65 include, but are not limited to, inhibiting the expression or activity of CFAP65.

[0076] Inhibiting CFAP65 activity means reducing CFAP65 activity. Preferably, the CFAP65 activity is reduced by at least 10% compared to before inhibition, more preferably by at least 30%, even more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0077] Inhibiting CFAP65 expression can specifically involve inhibiting the transcription or translation of the CFAP65 gene. Specifically, this can mean preventing the CFAP65 gene from being transcribed, reducing the transcriptional activity of the CFAP65 gene, preventing the CFAP65 gene from being translated, or reducing the translation level of the CFAP65 gene.

[0078] Those skilled in the art can use conventional methods to regulate the gene expression of CFAP65, such as gene knockout, homologous recombination, and interfering RNA.

[0079] The inhibition of CFAP65 gene expression can be verified by detecting expression levels using PCR and Western Blot.

[0080] Preferably, compared with the wild type, CFAP65 gene expression is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, even better by at least 70%, and still better by at least 90%, and most preferably by no expression of the CFAP65 gene at all.

[0081] Small molecule compounds

[0082] In this invention, it refers to compounds composed of several or dozens of atoms with a molecular mass of less than 1000.

[0083] Preparation of drugs for the prevention or treatment of gastric cancer

[0084] Drugs for the prevention or treatment of gastric cancer can be prepared using nucleic acid molecules that reduce CFAP65 gene expression in gastric cancer cells; and / or, CFAP65 gene interference nucleic acid constructs; and / or CFAP65 gene interference lentiviruses as active ingredients. Typically, in addition to the active ingredient, the drug may include one or more pharmaceutically acceptable carriers or excipients, depending on the specific dosage form required.

[0085] "Pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0086] A "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, meaning it can be miscible with it without significantly reducing the drug's efficacy 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 gum 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, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0087] In this invention, unless otherwise specified, the drug dosage form is not particularly limited and can be formulated as injections, oral liquids, tablets, capsules, pellets, sprays, etc., and can be prepared by conventional methods. The choice of drug dosage form should be matched with the route of administration.

[0088] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0089] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

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

[0091] Example 1: Preparation of RNAi Lentiviral Targeting the Human CFAP65 Gene

[0092] 1. Screening for effective siRNA targets against the human CFAP65 gene.

[0093] Gene information for CFAP65 (NM_194302) was retrieved from GenBank; effective siRNA targets targeting the CFAP65 gene were designed. Table 1-1 lists the selected effective siRNA target sequences targeting the CFAP65 gene.

[0094] Table 1-1 siRNA target sequences targeting the human CFAP65 gene

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

[0096] 2. Preparation of lentiviral vectors

[0097] Double-stranded DNA oligo sequences (Table 1-2) with Age I and EcoRI restriction sites at both ends were synthesized targeting the siRNA target (SEQ ID NO: 1 as an example). The pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) was linearized by acting Age I and EcoRI restriction endonucleases, and the restriction fragments were identified by agarose gel electrophoresis.

[0098] Table 1-2 Double-stranded DNA Oligo with Age I and EcoRI restriction sites at both ends

[0099]

[0100] The vector DNA, which had been linearized by double digestion (digestion system as shown in Table 1-4, 37℃, 1h reaction), and purified double-stranded DNA were ligated using T4 DNA ligase. The ligation was carried out overnight at 16℃ in an appropriate buffer system (ligation system as shown in Table 1-5), and the ligation product was recovered. The ligation product was transformed into fresh *E. coli* competent cells prepared with calcium chloride (transformation procedure reference: *Molecular Cloning: A Laboratory Manual*, 2nd edition, pp. 55-56). A small amount of the transformed bacterial clone was dipped into 10 μl of LB medium, mixed well, 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'-CCAAGTGACCAGCGGATTGAC-3' (SEQ ID NO: 6); the downstream primer sequence was 5'-GGGTTGAGGCAGCAAAAGATG-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). Clones that were positive by PCR were sequenced and compared. Clones that matched correctly were identified as the successfully constructed expression vector for RNAi targeting SEQ ID NO: 1, and named pGCSIL-GFP-CFAP65-siRNA.

[0101] A negative control plasmid, pGCSIL-GFP-Scr-siRNA, was constructed. The target sequence of the negative control siRNA 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 EcoRI restriction sites at both ends was synthesized targeting the Scr siRNA (Table 1-3). All other construction methods, identification methods, and conditions were the same as for pGCSIL-GFP-CFAP65-siRNA.

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

[0103]

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

[0105] reagents Volume (μl) pGCSIL-GFP plasmid (1 μg / μl) 2.0 10×buffer 5.0 100×BSA 0.5 Age I (10 U / μl) 1.0 EcoRI (10 U / μl) 1.0 <![CDATA[dd H2O]]> 40.5 Total 50.0

[0106] Table 1-5 Oligo Ligation Reaction System for Vector DNA and Double-Stranded DNA

[0107] reagents Positive control (μl) Self-ligation control (μl) Connecting 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×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

[0108] Table 1-6 PCR Reaction System

[0109] reagents 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 <![CDATA[ddH2O]]> 15.2 Total 20.0

[0110] Table 1-7 PCR Reaction System Program Settings

[0111]

[0112] 3. Packaging CFAP65-shRNA lentivirus

[0113] DNA was extracted from the RNAi plasmid pGCSIL-GFP-CFAP65-siRNA using a plasmid extraction kit from Qiagen and prepared into a 100 ng / μl stock solution.

[0114] 24 hours before transfection, logarithmically growing human embryonic kidney 293T cells were digested with trypsin, and the cell density was adjusted to 1.5 × 10⁶ cells / year in DMEM complete medium containing 10% fetal bovine serum. 5 Cells / ml were seeded into 6-well plates and cultured at 37°C in a 5% CO2 incubator. Cells were ready for transfection when the cell density reached 70%-80%. Two hours before transfection, the original culture medium was aspirated, and 1.5 ml of fresh complete culture medium was added. Following the instructions of the Sigma-Aldrich MISSION Lentiviral Packaging Mix kit, 20 μl of Packing Mix (PVM), 12 μl of PEI, and 400 μl of serum-free DMEM medium were added to a sterile centrifuge tube. 20 μl of the extracted plasmid DNA was then added to the PVM / PEI / DMEM mixture.

[0115] The above transfection mixture was incubated at room temperature for 15 min and then transferred to the culture medium of human embryonic kidney 293T cells. It was cultured at 37°C in a 5% CO2 incubator for 16 h. The culture medium containing the transfection mixture was discarded, washed with PBS solution, and 2 ml of complete culture medium was added. The cells were cultured for another 48 h. The cell supernatant was collected and the lentivirus was purified and concentrated using a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) as follows: (1) Centrifuge at 4°C and 4000g for 10 min to remove cell debris; (2) Filter the supernatant into a 40 ml ultracentrifuge tube using a 0.45 μm filter; (3) Centrifuge at 4000g for 10-15 min to the required concentrated virus volume; (4) After centrifugation, separate the filter cup and the filtrate collection cup below. Invert the filter cup onto the sample collection cup and centrifuge for 2 min with a centrifugal force not exceeding 1000g; (5) Remove the centrifuge cup from the sample collection cup. The sample collection cup contains the concentrated virus solution. The viral concentrate was aliquoted and stored at -80°C. The first strand sequence of the siRNA contained in the viral concentrate is shown in SEQ ID NO:2. The packaging process of the control lentivirus was the same as that of the CFAP65-shRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector was used instead of the pGCSIL-GFP-CFAP65-siRNA vector.

[0116] Example 2: Detection of gene silencing efficiency using real-time quantitative RT-PCR

[0117] Human gastric cancer AGS cells in the logarithmic growth phase were digested with pancreatic enzymes to prepare a cell suspension (approximately 5 × 10⁻⁶ cells). 4 Cells were seeded in 6-well plates at a concentration of approximately 30% ( / ml) and cultured until confluence reached approximately 30%. Based on the multiplicity of infection (MOI, AGS: 10), an appropriate amount of the lentivirus prepared in Example 1 was added. After culturing for 24 hours, the medium was changed, and cells were collected after 5 days of infection. Total RNA was extracted according to the Invitrogen Trizol instructions. cDNA was obtained by reverse transcription of the RNA according to the Promega M-MLV instructions (reverse transcription reaction system shown in Table 2-1, reaction at 42°C for 1 hour, followed by inactivation of reverse transcriptase by water bath at 70°C for 10 minutes).

[0118] Real-time quantitative PCR was performed using a TP800 Real-time PCR instrument (TAKARA). The primers for the CFAP65 gene were as follows: upstream primer 5'-CCAAGTGACCAGCGGATTGAC-3' (SEQ ID NO: 11) and downstream primer 5'-GGGTTGAGGCAGCAAAAGATG-3' (SEQ ID NO: 12). The housekeeping gene GAPDH was used as an internal control, with the following primer sequences: 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 proportions in Table 2-2.

[0119] Table 2-1 Reverse Transcription Reaction System

[0120] reagents Volume (μl) 5×RT buffer 4.0 10mM dNTPs 2.0 RNasin 0.4 M-MLV-RTase 1.0 RNase-Free 2.6 Total 10.0

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

[0122] 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

[0123] The program was set as a two-step Real-time PCR: pre-denaturation at 95°C for 30 s; followed by each subsequent denaturation step at 95°C for 5 s; annealing extension at 60°C for 30 s; for a total of 40 cycles. Absorbance was read during each extension phase. After PCR, denaturation was performed at 95°C for 15 s, then cooled to 60°C to allow for complete DNA double-strand binding. From 60°C to 95°C, the temperature was increased by 0.5°C at each step, held for 4 s, and absorbance was read simultaneously to construct a melting curve. A 2- ΔΔCt The expression abundance of CFAP65 mRNA in cells infected with lentivirus was calculated using analytical methods. Cells infected with control virus were used as a control. Experimental results are as follows: Figure 1 As shown, the expression level of CFAP65 mRNA in human gastric cancer AGS cells was downregulated by 70.4%.

[0124] Example 3: Detection of the proliferation ability of tumor cells infected with CFAP65-shRNA lentivirus

[0125] Human gastric cancer AGS cells in the logarithmic growth phase were digested with pancreatic enzymes to prepare a cell suspension (approximately 5 × 10⁻⁶ cells). 4 Cells were seeded in 6-well plates at a concentration of approximately 30% ( / ml) and cultured until confluence reached approximately 30%. Based on the multiplicity of infection (MOI, AGS: 10), an appropriate amount of virus was added, and the culture medium was changed after 24 hours. After 5 days of infection, cells in the logarithmic growth phase were collected from each experimental group. The cells were resuspended in complete culture medium to form a cell suspension (2 × 10⁻⁶). 4Cells were seeded at a density of approximately 1500 cells / well in 96-well plates. Five replicates were performed per group, with 100 μl per well. After seeding, the plates were incubated at 37°C in a 5% CO2 incubator. Starting the day after seeding, the plates were analyzed and read daily using a Celigo instrument (Nexcelom) for five consecutive days. By adjusting the input parameters in the analysis settings, the number of green fluorescent cells in each scan was accurately calculated, and the data were statistically plotted to generate cell proliferation curves (results are shown in Figure 1). Figure 2 (As shown). The results showed that after 5 days of in vitro cell culture, the proliferation rate of tumors in the lentivirus-infected group was significantly slowed down, far lower than that of tumor cells in the control group, and the number of viable cells decreased by 59.3%, indicating that silencing of the CFAP65 gene inhibited the proliferation of human gastric cancer AGS cells.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention. sequence list <110> Shanghai Jikai Gene Medical Technology Co., Ltd. <120> Uses and related products of the human CFAP65 gene <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 ccttgaaact ccagaagat 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <400> 2 ccuugaaacu ccagaagau 19 <210> 3 <211> 48 <212> RNA <213> Artificial Sequence <400> 3 auccuugaaa cuccagaaga ucucgagauc uucuggaguu ucaaggau 48 <210> 4 <211> 58 <212> DNA <213> Artificial Sequence <400> 4 ccggatcctt gaaactccag aagatctcga gatcttctgg agtttcaagg attttttg 58 <210> 5 <211> 58 <212> DNA <213> Artificial Sequence <400> 5 aattcaaaaa atccttgaaa ctccagaaga tctcgagatc ttctggagtt tcaaggat 58 <210> 6 <211> twenty one <212> DNA <213> Artificial Sequence <400> 6 ccaagtgacc agcggattga c 21 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 gggttgaggc agcaaaagat g 21 <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 ccaagtgacc agcggattga c 21 <210> 12 <211> twenty one <212> DNA <213> Artificial Sequence <400> 12 gggttgaggc agcaaaagat g 21 <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 CFAP65 inhibitors in the preparation of products possessing at least one of the following functionalities: Treatment of stomach cancer; Inhibits the proliferation rate of gastric cancer cells; Inhibits the growth of gastric cancer The CFAP65 inhibitor is a CFAP65-siRNA lentivirus with an effective siRNA target sequence for the CFAP65 gene, such as SEQ ID NO:

1.

2. The use according to claim 1, characterized in that, The CFAP65-siRNA lentivirus includes double-stranded RNA or shRNA, wherein the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand being complementary to form an RNA dimer, and the sequence of the first strand is shown in SEQ ID NO: 2; or, the nucleotide sequence of the shRNA is shown in SEQ ID NO: 3.

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