Use of human igfl3 gene and related products

Abstract

This invention belongs to the field of biomedical research, specifically relating to the use of the human IGFL3 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 IGFL3 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 IGFL3 gene and related products. Background Technology

[0002] IGFL3 (Insulin growth factor-like family member 3) is a protein-coding gene. Diseases associated with insulin-like growth factor 3 include unilateral or bilateral testicular disorders and cryptorchidism. IGFL2 is an important accessory gene of this gene.

[0003] There are currently no reports of using the IGFL3 gene for the treatment of gastric cancer. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide the use of the human IGFL3 gene and related products.

[0005] To achieve the above-mentioned objectives and other related objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of the human IGFL3 gene as a target in the preparation of a gastric cancer therapeutic drug or in the preparation of a gastric cancer diagnostic drug.

[0007] The use of the human IGFL3 gene as a target in the preparation of gastric cancer therapeutic drugs specifically refers to screening drugs or formulations by targeting the IGFL3 gene to identify those that can inhibit human IGFL3 gene expression as candidate drugs for gastric cancer treatment. For example, the IGFL3 gene small interfering RNA (siRNA) described in this invention was obtained by screening using the human IGFL3 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 IGFL3 gene.

[0008] The use of the human IGFL3 gene as a target for the preparation of gastric cancer diagnostic drugs specifically refers to the application of the IGFL3 gene expression product as a gastric cancer diagnostic indicator in the preparation of gastric cancer diagnostic drugs.

[0009] The gastric cancer treatment drug is a molecule that can specifically inhibit the transcription or translation of the IGFL3 gene, or specifically inhibit the expression or activity of the IGFL3 protein, thereby reducing the expression level of the IGFL3 gene in gastric cancer cells and achieving the purpose of inhibiting the proliferation, growth, differentiation and / or survival of gastric cancer cells.

[0010] The gastric cancer treatment or diagnostic drugs prepared through the IGFL3 gene include, but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, peptides, proteins, or interfering lentiviruses.

[0011] 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.

[0012] The dosage of the gastric cancer treatment drug is sufficient to reduce the transcription or translation of the human IGFL3 gene, or sufficient to reduce the expression or activity of the human IGFL3 protein, so as to reduce the expression of the human IGFL3 gene by at least 50%, 80%, 90%, 95%, or 99%.

[0013] 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 IGFL3 gene. Specifically, during treatment, substances that effectively reduce the expression level of the human IGFL3 gene are administered to the patient.

[0014] In one embodiment, the target sequence of the IGFL3 gene is shown in SEQ ID NO: 1 and SEQ ID NO: 2. Specifically:

[0015] 5'-TGAGGGTTCTGGGTATGAA-3' (SEQ ID NO: 1);

[0016] 5'-GTGGGAACAAGATCTACAA-3' (SEQ ID NO: 2).

[0017] A second aspect of the invention provides the use of an IGFL3 inhibitor in the preparation of a product having at least one of the following effects:

[0018] Treatment of stomach cancer;

[0019] Inhibits the proliferation rate of gastric cancer cells;

[0020] Inhibits the growth of gastric cancer.

[0021] The product must include an IGFL3 inhibitor, and use the IGFL3 inhibitor as the active ingredient for the aforementioned effects.

[0022] In the product, the effective ingredient that performs the aforementioned function may be only an IGFL3 inhibitor, or it may contain other molecules that can perform the aforementioned function.

[0023] That is, the IGFL3 inhibitor is the sole active ingredient or one of the active ingredients in the product.

[0024] The product can be a single-component substance or a multi-component substance.

[0025] 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.

[0026] 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.

[0027] The products mentioned include, but are not limited to, medicines, health products, and food.

[0028] The IGFL3 inhibitor can be a nucleic acid molecule, antibody, or small molecule compound.

[0029] As illustrated in the embodiments of the present invention, the IGFL3 inhibitor can be a nucleic acid molecule that reduces the expression of the IGFL3 gene in gastric cancer cells. Specifically, it can be double-stranded RNA or shRNA.

[0030] A third aspect of the present invention provides a method for treating gastric cancer by administering an IGFL3 inhibitor to a subject.

[0031] 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.

[0032] 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.

[0033] The IGFL3 inhibitor can be administered to subjects before, during, and after gastric cancer treatment.

[0034] The fourth aspect of this invention discloses a nucleic acid molecule that reduces the expression of the IGFL3 gene in gastric cancer cells, said nucleic acid molecule comprising double-stranded RNA or shRNA.

[0035] The double-stranded RNA contains a nucleotide sequence capable of hybridizing with the IGFL3 gene.

[0036] The shRNA contains a nucleotide sequence that can hybridize with the IGFL3 gene.

[0037] 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 IGFL3 gene.

[0038] The target sequence in the IGFL3 gene is the segment in the IGFL3 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 IGFL3 gene.

[0039] Furthermore, the target sequences of the double-stranded RNA are shown in SEQ ID NO: 1 and SEQ ID NO: 2. Specifically: 5'-TGAGGGTTCTGGGTATGAA-3' (SEQ ID NO: 1); 5'-GTGGGAACAAGATCTACAA-3' (SEQ ID NO: 2).

[0040] Furthermore, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 3 and SEQ ID NO: 4. Specifically:

[0041] 5'-UGAGGGGUUCUGGGUAUGAA-3' (SEQ ID NO: 3);

[0042] 5'-GUGGGAACAAGAUCUACAA-3' (SEQ ID NO: 4).

[0043] Furthermore, the double-stranded RNA is a small interfering RNA (siRNA).

[0044] SEQ ID NO: 3 is a single strand of a small interfering RNA (siRNA) targeting the human IGFL3 gene, designed with the sequence shown in SEQ ID NO: 1 as the RNA interference target sequence. The second strand is complementary to the first strand. SEQ ID NO: 4 is a single strand of a small interfering RNA (siRNA) targeting the human IGFL3 gene, designed with the sequence shown in SEQ ID NO: 2 as the RNA interference target sequence. The second strand is complementary to the first strand. These siRNAs can specifically silence the expression of the endogenous IGFL3 gene in gastric cancer cells.

[0045] 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 IGFL3 gene.

[0046] Furthermore, the target sequence of the shRNA is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0047] The shRNA, after being processed by enzyme digestion, can become small interfering RNA (siRNA), which can then specifically silence the expression of the endogenous IGFL3 gene in gastric cancer cells.

[0048] 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.

[0049] Furthermore, the sequences of the shRNA are shown in SEQ ID NO: 5 and SEQ ID NO: 6. Specifically, they are 5'-UGAGGGUUCUGGGUAUGAACUCGAGUUCAUACCCAGAACCCUCA-3' (SEQ ID NO: 5); 5'-GUGGGAACAAGAUCUACAACUCGAGUUGUAGAUCUUGUUCCCAC-3' (SEQ ID NO: 6).

[0050] Furthermore, the IGFL3 gene is derived from humans.

[0051] In a fifth aspect, the present invention discloses an IGFL3 gene interference nucleic acid construct containing a gene fragment encoding the shRNA in the aforementioned nucleic acid molecule, which can express the shRNA.

[0052] The aforementioned IGFL3 gene interference nucleic acid construct can be obtained by cloning the gene fragment encoding the aforementioned human IGFL3 gene shRNA into a known vector.

[0053] Furthermore, the IGFL3 gene interference nucleic acid construct is an IGFL3 gene interference lentiviral vector.

[0054] The IGFL3 gene interference lentiviral vector disclosed in this invention is obtained by cloning a DNA fragment encoding the aforementioned IGFL3 gene shRNA into a known vector, most of which are lentiviral vectors. After the IGFL3 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 IGFL3 gene.

[0055] Furthermore, the IGFL3 gene-interfering 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).

[0056] 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.

[0057] The embodiments of the present invention specifically illustrate the human IGFL3 gene interference lentiviral vectors constructed using pGCSIL-GFP as the vector, named pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2, and the corresponding lentiviruses named IGFL3-siRNA lentivirus 1 and IGFL3-siRNA lentivirus 2, respectively.

[0058] The IGFL3 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. An IGFL3 gene interference lentiviral vector can be used to prepare the IGFL3 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.

[0059] In a sixth aspect, this invention discloses an IGFL3 gene-interfering lentivirus, which is prepared by viral packaging of the aforementioned IGFL3 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 IGFL3 gene, thereby inhibiting the proliferation of gastric cancer cells. This IGFL3 gene-interfering lentivirus can be used to prepare drugs for the prevention or treatment of gastric cancer.

[0060] In a seventh aspect, the present invention provides the use of the aforementioned nucleic acid molecule, or the aforementioned IGFL3 gene interference nucleic acid construct, or the aforementioned IGFL3 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 IGFL3 gene in gastric cancer cells.

[0061] 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.

[0062] 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.

[0063] The object of the method can be a person.

[0064] An eighth aspect of the present invention provides a composition for the prevention or treatment of gastric cancer, wherein the active ingredient comprises:

[0065] The aforementioned nucleic acid molecules; and / or, the aforementioned IGFL3 gene interference nucleic acid constructs; and / or, the aforementioned IGFL3 gene interference lentiviruses, as well as pharmaceutically acceptable vectors, diluents, or excipients.

[0066] The composition may be a pharmaceutical composition.

[0067] 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, and recurrence 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, and recurrence of the gastric cancer are inhibited.

[0068] The composition is not particularly limited in form and can be in various forms such as solid, liquid, gel, semi-fluid, or aerosol.

[0069] 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.

[0070] In summary, this invention designed an RNAi target sequence for the human IGFL3 gene and constructed corresponding IGFL3 RNAi vectors. The RNAi vectors pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2 significantly downregulated the expression of the IGFL3 gene at both the mRNA and protein levels. Using lentivirus (Lv) as a gene manipulation tool to carry the RNAi vectors pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2, the RNAi sequence targeting the IGFL3 gene can be efficiently introduced into gastric cancer AGS cells, reducing the expression level of the IGFL3 gene and significantly inhibiting the proliferation of these tumor cells. Therefore, lentivirus-mediated IGFL3 gene silencing is a potential non-surgical clinical treatment for malignant tumors.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] This invention, through extensive and in-depth research, has discovered that downregulating the expression of the human IGFL3 gene using RNAi can effectively inhibit the proliferation of gastric cancer cells and promote apoptosis, thereby effectively controlling 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 of gastric cancer cells and suppress gastric cancer growth, thus treating gastric cancer and opening up new directions for gastric cancer treatment. Attached Figure Description

[0073] Figure 1 RT-PCR was used to detect the reduction efficiency of target genes at the mRNA level in AGS cells.

[0074] Figure 2 Automated analysis of Celigo cells revealed that the reduction of the IGFL3 gene 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.)

[0075] In the attached diagram,

[0076] All values ​​are the averages of three experiments, and the error bars represent the standard deviation (SD).

[0077] **,shCtrl compared with the lentivirus treatment group containing the target gene shRNA, P<0.01. Detailed Implementation

[0078] This invention demonstrates the role of the IGFL3 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 both mRNA and protein levels were detected in the two gastric cancer cell lines compared to a control lentivirus. Subsequently, cellular functional experiments were conducted to detect cell proliferation and apoptosis. The results showed that the shRNA group exhibited significantly greater inhibition of gastric cancer cell proliferation compared to the control group.

[0079] 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.

[0080] IGFL3 inhibitors

[0081] This refers to molecules that have an inhibitory effect on IGFL3. Inhibitory effects on IGFL3 include, but are not limited to, inhibiting the expression or activity of IGFL3.

[0082] Inhibiting IGFL3 activity means reducing IGFL3 activity. Preferably, compared to before inhibition, IGFL3 activity is reduced by at least 10%, 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%.

[0083] Inhibiting IGFL3 expression can specifically involve inhibiting the transcription or translation of the IGFL3 gene. Specifically, this can mean preventing the IGFL3 gene from being transcribed, reducing the transcriptional activity of the IGFL3 gene, preventing the IGFL3 gene from being translated, or reducing the translation level of the IGFL3 gene.

[0084] Those skilled in the art can use conventional methods to regulate the gene expression of IGFL3, such as gene knockout, homologous recombination, and interfering RNA.

[0085] The inhibition of IGFL3 gene expression can be verified by detecting expression levels using PCR and Western Blot.

[0086] Preferably, compared with the wild type, IGFL3 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 IGFL3 gene at all.

[0087] Small molecule compounds

[0088] In this invention, it refers to compounds composed of several or dozens of atoms with a molecular mass of less than 1000.

[0089] Preparation of drugs for the prevention or treatment of gastric cancer

[0090] Drugs for the prevention or treatment of gastric cancer can be prepared using nucleic acid molecules that reduce IGFL3 gene expression in gastric cancer cells; and / or, IGFL3 gene interference nucleic acid constructs; and / or IGFL3 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.

[0091] "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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] Example 1: Preparation of RNAi Lentiviral Virus Targeting the Human IGFL3 Gene

[0098] 1. Screening for effective siRNA targets against the human IGFL3 gene.

[0099] Gene information for IGFL3 (NM_207393) was retrieved from GenBank; effective siRNA targets targeting the IGFL3 gene were designed. Table 1-1 lists the selected effective siRNA target sequences for the IGFL3 gene.

[0100] Table 1-1 siRNA target sequences targeting the human IGFL3 gene

[0101] SEQ ID NO TargetSeq(5'-3') 1 TGAGGGTTCTGGGTATGAA 2 GTGGGAACAAGATCTACAA

[0102] 2. Preparation of lentiviral vectors

[0103] Double-stranded DNA oligo sequences (Table 1-2) with Age I and EcoRI restriction sites at both ends were synthesized targeting siRNA (using SEQ ID NO: 1 and 2 as examples). 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 enzyme-digested fragments were identified by agarose gel electrophoresis.

[0104] Table 1-2 Double-stranded DNA Oligo containing Age I and EcoRI restriction sites at both ends

[0105]

[0106] The vector DNA, which had been linearized by double digestion (digestion system as shown in Table 1-4, 37℃, 1 h), 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 then transformed into fresh *E. coli* competent cells prepared with calcium chloride (transformation procedure reference: *Molecular Cloning: A Laboratory Manual*, 2nd edition, pp. 55-56). Dip the transformed bacterial clone into a sample, dissolve it in 10 μl of LB medium, mix well, and take 1 μl as a template. Design universal PCR primers for the upstream and downstream of the RNAi sequence of SEQ ID NO: 1 in the lentiviral vector. The upstream primer sequence is 5'-GAACAAGATCTACAACCCTTCAG-3' (SEQ ID NO: 11); the downstream primer sequence is 5'-GGGAGATGGGAGATAAGTGAC-3' (SEQ ID NO: 12). Design universal PCR primers for the upstream and downstream of the RNAi sequence of SEQ ID NO: 2 in the lentiviral vector. The upstream primer sequence is 5'-GAACAAGATCTACAACCCTTCAG-3' (SEQ ID NO: 13); the downstream primer sequence is 5'-GGGAGATGGGAGATAAGTGAC-3' (SEQ ID NO: 14). Perform PCR identification experiments (PCR reaction system is shown in Table 1-6, reaction conditions are shown in Table 1-7). Clones that were positive by PCR were sequenced and compared. Clones that were correctly matched were the successfully constructed expression RNAi vectors for SEQ ID NO: 1 and SEQ ID NO: 2, and were named pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2, respectively.

[0107] 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: 15). 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-IGFL3-siRNA-1.

[0108] Table 1-3 Double-stranded DNA Oligo containing Age I and EcoRI restriction sites at both ends

[0109]

[0110] Table 1-4 pGCSIL-GFP plasmid digestion reaction system

[0111] 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

[0112] Table 1-5 Oligo ligation reaction system for vector DNA and double-stranded DNA

[0113] 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

[0114] Table 1-6 PCR Reaction System

[0115] 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

[0116] Table 1-7 PCR Reaction System Program Settings

[0117]

[0118] 3. Packaging IGFL3-shRNA lentivirus

[0119] DNA was extracted from RNAi plasmids pGCSIL-GFP-IGFL3-siRNA-1 and pGCSIL-GFP-IGFL3-siRNA-2 using a plasmid extraction kit from Qiagen and prepared into a 100 ng / μl stock solution.

[0120] 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.

[0121] 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 for the control lentivirus was the same as that for the IGFL3-shRNA lentivirus, except that the pGCSIL-GFP-IGFL3-siRNA-1 vector was replaced with the pGCSIL-GFP-IGFL3-siRNA-1 vector.

[0122] Example 2: Detection of gene silencing efficiency using real-time quantitative RT-PCR

[0123] 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: 20), 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).

[0124] Real-time quantitative PCR was performed using a TP800 Real-time PCR instrument (TAKARA). Primers targeting the IGFL3 gene of lentiviral IGFL3-shRNA lentiviral 1 are as follows: upstream primer 5'-CAGTCTTCCTCCTCCAGTGTT-3' (SEQ ID NO: 18) and downstream primer 5'-CGGGAGATGGGAGATAAGTG-3' (SEQ ID NO: 19). Primers targeting the IGFL3 gene of lentiviral IGFL3-shRNA lentiviral 2 are as follows: upstream primer 5'-CAGTCTTCCTCCTCCAGTGTT-3' (SEQ ID NO: 20) and downstream primer 5'-CGGGAGATGGGAGATAAGTG-3' (SEQ ID NO: 21). Using the housekeeping gene GAPDH as an internal control, the primer sequences are as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 22) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 23). The reaction system was prepared according to the proportions in Table 2-2.

[0125] Table 2-1 Reverse Transcription Reaction System

[0126] 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

[0127] Table 2-2 Real-time PCR reaction system

[0128] 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

[0129] 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 IGFL3 mRNA in cells infected with lentivirus was calculated using analytical methods. Cells infected with control virus were used as a control. The experimental results are as follows: Figure 1 As shown, the expression level of IGFL3 mRNA in human gastric cancer AGS cells infected with IGFL3-shRNA lentivirus 1 (i.e., shIGFL3-1 group) was downregulated by 56.0%, and the expression level of IGFL3 mRNA in human gastric cancer AGS cells infected with IGFL3-shRNA lentivirus 2 (i.e., shIGFL3-2 group) was downregulated by 52.3%.

[0130] Example 3: Detection of the proliferation ability of tumor cells infected with IGFL3-shRNA lentivirus

[0131] 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: 20), 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). 4 Cells were seeded at a density of approximately 2000 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, much lower than that of tumor cells in the control group. The number of viable cells infected with IGFL3-shRNA lentivirus 1 (i.e., shIGFL3-siRNA-1 group) and IGFL3-shRNA lentivirus 2 (i.e., shIGFL3-siRNA-2 group) decreased by 64% and 56.4%, respectively, indicating that IGFL3 gene silencing inhibited the proliferation of human gastric cancer AGS cells.

[0132] 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 IGFL3 gene <160> twenty three <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 tgagggttct gggtatgaa 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 gtgggaacaa gatctacaa 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <400> 3 ugaggguucu ggguaugaa 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <400> 4 gugggaacaa gaucuacaa 19 <210> 5 <211> 44 <212> RNA <213> Artificial Sequence <400> 5 ugaggguucu ggguaugaac ucgaguucau acccagaacc cuca 44 <210> 6 <211> 44 <212> RNA <213> Artificial Sequence <400> 6 gugggaacaa gaucuacaac ucgaguugua gaucuuguuc ccac 44 <210> 7 <211> 58 <212> DNA <213> Artificial Sequence <400> 7 ccgggttgag ggttctgggt atgaactcga gttcataccc agaaccctca actttttg 58 <210> 9 <211> 58 <212> DNA <213> Artificial Sequence <400> 9 aattcaaaaa gttgagggtt ctgggtatga actcgagttc atacccagaa ccctcaac 58 <210> 9 <211> 58 <212> DNA <213> Artificial Sequence <400> 9 ccgggtgtgg gaacaagatc tacaactcga gttgtagatc ttgttcccac actttttg 58 <210> 10 <211> 58 <212> DNA <213> Artificial Sequence <400> 10 aattcaaaaa gtgtgggaac aagatctaca actcgagttg tagatcttgt tcccacac 58 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <400> 11 gaacaagatc tacaaccctt cag 23 <210> 12 <211> twenty one <212> DNA <213> Artificial Sequence <400> 12 gggagatggg agataagtga c 21 <210> 13 <211> twenty three <212> DNA <213> Artificial Sequence <400> 13 gaacaagatc tacaaccctt cag 23 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <400> 14 gggagatggg agataagtga c 21 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <400> 15 ttctccgaac gtgtcacgt 19 <210> 16 <211> 54 <212> DNA <213> Artificial Sequence <400> 16 ccggttctcc gaacgtgtca cgtctcgaga cgtgacacgt tcggagaatt tttg 54 <210> 17 <211> 54 <212> DNA <213> Artificial Sequence <400> 17 aattcaaaaa ttctccgaac gtgtcacgtc tcgagacgtg acacgttcgg agaa 54 <210> 18 <211> twenty one <212> DNA <213> Artificial Sequence <400> 18 cagtcttcct cctccagtgt t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 cgggagatgg gagataagtg 20 <210> 20 <211> twenty one <212> DNA <213> Artificial Sequence <400> 20 cagtcttcct cctccagtgt t 21 <210> twenty one <211> 20 <212> DNA <213> Artificial Sequence <400> twenty one cgggagatgg gagataagtg 20 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty two tgacttcaac agcgacaccc a 21 <210> twenty three <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty three caccctgttg ctgtagccaa a 21

Claims

1. Use of IGFL3 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 effective siRNA target sequence of the IGFL3 inhibitor against the IGFL3 gene is SEQ ID NO: 1 or 2, and the IGFL3 inhibitor is a lentivirus containing double-stranded RNA or shRNA.

2. The use according to claim 1, characterized in that, It also includes 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 being complementary to form an RNA dimer, and the sequence of the first strand is shown in SEQ ID NO: 3 or SEQ ID NO: 4; 2) The nucleotide sequence of the shRNA is shown in SEQ ID NO: 5 or SEQ ID NO: 6.

Images