CN111035762B

CN111035762B - Application of human EDDM3A gene and related product

Info

Publication number: CN111035762B
Application number: CN201911328232.9A
Authority: CN
Inventors: 王楠; 何显力; 王萌; 乔庆; 吴涛
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2021-09-28
Anticipated expiration: 2039-12-20
Also published as: CN111035762A

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human EDDM3A gene as a target in preparation of a gastric cancer treatment drug. The invention discovers that the proliferation of gastric cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the EDDM3A gene of a human is reduced by adopting an RNAi method, and the growth process of the gastric cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of gastric cancer cells, promote the apoptosis of gastric cancer cells, inhibit the cloning of gastric cancer cells, inhibit the transfer capacity of gastric cancer cells and inhibit the growth of gastric cancer cells, thereby treating gastric cancer and opening up a new direction for the treatment of gastric cancer.

Description

Application of human EDDM3A gene and related product

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human EDDM3A gene and a related product.

Background

EDDM3A (Epididymis secretor Sperm Binding Protein) is a Sperm Binding Protein secreted by epididymal epithelial cells. Testicular sperm initiate differentiation of spermatogenic stem cells but initially do not move progressively and therefore do not fertilize the ovum. The process of further sperm maturation in the testis requires exposure to the microenvironment of the epididymal cavity and undergoes a series of changes including modification of enzymes, loss of original components in epididymal secretions and increase of new glycoproteins. These modified proteins and enzymes are synthesized by epithelial cells lining the epididymal duct and secreted to the tip of the duct, where they come into contact with and may be absorbed by the sperm membrane. This process imparts some motility to the sperm.

A major epididymal-specific cDNA clone family, called EDDM3A, was isolated and identified by differential screening of human epididymal cDNA libraries. EDDM3A maps to chromosome 14q 11.2. Two different but homologous gene transcripts, HE3 α and HE3 β, were identified by EDDM3A in more detail sequence and PCR approach analysis. Southern blotting analysis of human genomic DNA revealed that at least three independent EDDM 3A-related genes were present in the human genome. Northern hybridization analysis shows that the EDDM3A gene product is specifically expressed in human epididymis. Furthermore, no other non-primate species, other than swine, have been identified as expressing homologous sequences in the epididymis (Kirchhoff C1, Pera I, Rust W, Ivell R. major human epididymis-specific gene product, HE3, is the first expression of a novel gene family.1994 Feb; 37(2): 130-7). HE3A (also known as epididymis protein 3A, epididymis secretion protein E3-ALPHA, human epididymis specific protein 3-ALPHA, HE3-ALPHA, EP3A, FAM12ALPHA, RAM1) is a human gene encoded by EDDM 3A. The protein encoded by EDDM3A had a number of amino acids of 147 and a mass of 17.6 kDa.

Reports on EDDM3A in tumor-related fields are not searched at present.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the application of the human EDDM3A gene and related products.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the invention, the use of the human EDDM3A gene as a target in the preparation of a gastric cancer treatment drug is provided.

The human EDDM3A gene as a target for preparing the gastric cancer treatment drug specifically comprises the following steps: the EDDM3A gene is used as an action object, and a medicament or a preparation is screened to find out a medicament capable of inhibiting the expression of the human EDDM3A gene as a candidate medicament for treating gastric cancer. The EDDM3A gene small interfering RNA (siRNA) is obtained by screening human EDDM3A gene serving as an action object and can be used as a medicine for inhibiting gastric cancer cell proliferation. In addition, EDDM3A gene can be used as an object of action, for example, an antibody drug, a small molecule drug, or the like.

The gastric cancer treatment drug is a molecule capable of specifically inhibiting the transcription or translation of EDDM3A gene, or specifically inhibiting the expression or activity of EDDM3A protein, so that the expression level of EDDM3A gene in gastric cancer cells is reduced, and the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the gastric cancer cells is achieved.

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

The administration amount of the gastric cancer treatment drug is a dose sufficient to reduce the transcription or translation of the human EDDM3A gene, or the expression or activity of the human EDDM3A protein. Such that the expression of the human EDDM3A gene is reduced by at least 50%, 80%, 90%, 95% or 99%.

The method for treating the gastric cancer by adopting the gastric cancer treatment drug achieves the treatment purpose by mainly reducing the expression level of the EDDM3A gene of a human body and inhibiting the proliferation of gastric cancer cells. Specifically, in treatment, a substance effective in reducing the expression level of the human EDDM3A gene is administered to the patient.

In one embodiment, the EDDM3A gene has a target sequence as set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-GGCTGTGTGTATACAGTAA-3' are provided.

In a second aspect of the invention, there is provided the use of an EDDM3A inhibitor in the manufacture of a product having at least one of the following effects:

treating gastric cancer;

inhibiting the proliferation rate of gastric cancer cells;

promoting the apoptosis of gastric cancer cells;

inhibiting the cloning of gastric cancer cells;

inhibiting the transfer capacity of gastric cancer cells;

inhibiting the growth of gastric cancer.

The product necessarily comprises an EDDM3A inhibitor, and an EDDM3A inhibitor as an active ingredient of the aforementioned effects.

In the product, the effective component for the above functions can be only an EDDM3A inhibitor, and can also comprise other molecules for the above functions.

That is, the EDDM3A inhibitor is the only active ingredient or one of the active ingredients of the product.

The product may be a single component material or a multi-component material.

The form of the product is not particularly limited, and can be various substance forms such as solid, liquid, gel, semifluid, aerosol and the like.

The product is primarily directed to mammals. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.

Such products include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.

The EDDM3A inhibitor can be a nucleic acid molecule, an antibody or a small molecule compound.

As exemplified in the examples herein, the EDDM3A inhibitor may be a nucleic acid molecule that reduces the expression of EDDM3A gene in gastric cancer cells. Specifically, it may be a double-stranded RNA or shRNA.

In a third aspect of the invention, there is provided a method of treating gastric cancer by administering an EDDM3A inhibitor to a subject.

The subject may be a mammal or a mammalian gastric cancer cell. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human. The gastric cancer cell may be an isolated gastric cancer cell.

The subject may be a patient suffering from gastric cancer or an individual in whom treatment is desired. Or isolated gastric cancer cells of a subject who is a gastric cancer patient or an individual expected to treat gastric cancer.

The EDDM3A inhibitor may be administered to a subject before, during, or after receiving treatment for gastric cancer.

The fourth aspect of the invention discloses a nucleic acid molecule for reducing the expression of EDDM3A gene in gastric cancer cells, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.

Wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the EDDM3A gene;

the shRNA contains a nucleotide sequence capable of hybridizing with the EDDM3A gene.

Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the EDDM3A gene.

The target sequence in the EDDM3A gene is a fragment in the EDDM3A gene corresponding to an mRNA fragment which is identified and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the EDDM3A gene.

Further, the target sequence of the double-stranded RNA is shown as SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-GGCTGTGTGTATACAGTAA-3' are provided. Further, the sequence of the first strand of the double-stranded RNA is shown as SEQ ID NO:2, respectively. Specifically 5'-GGCUGUGUGUAUACAGUAA-3'.

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

SEQ ID NO:2 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at one strand of small interfering RNA of the human EDDM3A gene, the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of endogenous EDDM3A gene in gastric cancer cells.

The shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is basically identical to a target sequence in the EDDM3A gene.

Further, the target sequence of the sh RNA is shown as SEQ ID NO:1 is shown.

The shRNA can become small interfering RNA (siRNA) after enzyme digestion and processing, and further plays a role in specifically silencing endogenous EDDM3A gene expression in gastric cancer cells.

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

Further, the sequence of the shRNA is shown as SEQ ID NO: 3, respectively. Specifically 5'-CAGGCUGUGUGUAUACAGUAACUCGAGUUACUGUAUACACACAGCCUG-3'.

Further, the EDDM3A gene is derived from a human.

In the fifth aspect of the invention, the EDDM3A gene interference nucleic acid construct contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The EDDM3A gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human EDDM3A gene shRNA into a known vector.

Further, the EDDM3A gene interference nucleic acid construct is an EDDM3A gene interference lentiviral vector.

The EDDM3A gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the EDDM3A gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the EDDM3A gene interference lentiviral vector is packaged into infectious viral particles by virus, and then infects gastric cancer cells to transcribe the shRNA, and the siRNA is finally obtained by the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the EDDM3A gene.

Further, the EDDM3A gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence which codes for a marker which can be detected in gastric cancer cells; preferably, the detectable label is Green Fluorescent Protein (GFP).

Further, the lentiviral vector may be selected from the group consisting of: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.

The embodiment of the invention specifically lists a human EDDM3A gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named as pGCSIL-GFP-EDDM 3A-siRNA.

The EDDM3A gene siRNA can be used for inhibiting the proliferation of gastric cancer cells, and further can be used as a medicine or a preparation for treating gastric cancer. The EDDM3A gene interference lentiviral vector can be used for preparing the EDDM3A gene siRNA. When used as a medicament or formulation for treating gastric cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The sixth aspect of the invention discloses an EDDM3A gene interference lentivirus, which is formed by virus packaging of the EDDM3A gene interference nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect gastric cancer cells and generate small interfering RNA aiming at EDDM3A gene, thereby inhibiting the proliferation of the gastric cancer cells. The EDDM3A gene interference lentivirus can be used for preparing medicines for preventing or treating gastric cancer.

In a seventh aspect of the invention, there is provided a use of the nucleic acid molecule, or the EDDM3A gene-interfering nucleic acid construct, or the EDDM3A gene-interfering lentivirus, wherein: is used for preparing a medicine for preventing or treating gastric cancer or a kit for reducing the expression of EDDM3A gene in gastric cancer cells.

The application of the medicament for preventing or treating the gastric cancer provides a method for treating the gastric cancer, in particular to a method for preventing or treating the gastric cancer in a subject, which comprises the step of administering an effective dose of the medicament to the subject.

Further, when the drug is used for preventing or treating gastric cancer in a subject, an effective dose of the drug needs to be administered to the subject. With 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.

The subject of the method may be a human.

In an eighth aspect of the present invention, there is provided a composition for preventing or treating gastric cancer, comprising, as active ingredients:

the aforementioned nucleic acid molecules; and/or, the aforementioned EDDM3A gene interfering nucleic acid construct; and/or the aforementioned EDDM3A gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.

The composition may be a pharmaceutical composition.

When the composition is used for preventing or treating gastric cancer in a subject, an effective dose of the composition needs to be administered to the subject. With 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.

The form of the composition is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, etc.

The subject to which the composition is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.

In conclusion, the RNAi target sequence of the human EDDM3A gene is designed, and the corresponding EDDM3A RNAi vector is constructed, wherein the RNAi vector pGCSIL-GFP-EDDM3A-siRNA can remarkably reduce the expression of the EDDM3A gene at the mRNA level and the protein level. Lentivirus (lentivirus, abbreviated as Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-EDDM3A-siRNA, so that an RNAi sequence aiming at the EDDM3A gene can be efficiently introduced into AGS (gastric adenocarcinoma cells) and MGC80-3 cells of gastric cancer cells in a targeted manner, the expression level of the EDDM3A gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated EDDM3A gene silencing is therefore a potential clinical non-surgical treatment modality for malignancies.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers that the proliferation of gastric cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the EDDM3A gene of a human is reduced by adopting an RNAi method, and the growth process of the gastric cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the proliferation rate of gastric cancer cells, promote the apoptosis of gastric cancer cells, inhibit the cloning of gastric cancer cells, inhibit the transfer capacity of gastric cancer cells and inhibit the growth of gastric cancer cells, thereby treating gastric cancer and opening up a new direction for the treatment of gastric cancer.

Drawings

FIG. 1-1: and detecting mRNA expression abundance comparison after the target gene is knocked down in the AGS cells by RT-PCR.

FIGS. 1-2: RT-PCR detection MGC80-3 target gene knockdown mRNA expression abundance comparison.

FIG. 2-1: and detecting the condition that AGS cell targets reduce protein level expression of EDDM3A gene by Western Blot.

FIG. 2-2: and detecting that MGC80-3 cell target points reduce protein level expression of EDDM3A genes by Western Blot.

FIG. 3-1: AGS cells are infected by shRNA lentivirus, and after 3 days, the cell number (upper graph, lower left graph) and the cell number change multiple (lower right graph) of the EDDM3A gene knock-down group and a control group are changed along with time.

FIG. 3-2: MGC80-3 cells were infected with shRNA lentivirus, and 3 days later, the number of cells (upper and lower left panels) and the fold change of the number of cells (lower right panel) in the EDDM3A gene knockdown group and the control group were plotted against time.

FIG. 4-1: AGS cells were analyzed by comparing absorbance at 490nm (left panel) and fold change in absorbance (right panel) with time in a microplate reader for each experimental group. OD490 here reflects the number of viable cells.

FIG. 4-2: and (3) the absorbance of each experimental group in MGC80-3 cells to 490nm wavelength in a microplate reader (left graph) and the time-dependent variation multiple of the absorbance (right graph). OD490 here reflects the number of viable cells.

FIG. 5-1: after AGS cells are infected by shRNA lentivirus, the EDDM3A gene knock-down group forms a clone number comparison with a control group.

FIG. 5-2: after MGC80-3 cells are infected by shRNA lentivirus, the EDDM3A gene knockdown group and a control group form a clone number comparison.

FIG. 6-1: AGS cells are infected by shRNA lentivirus, and after 5 days of culture, dot plots of apoptosis rates of the shEDDM3A group and a control group (shCtrl) are compared.

FIG. 6-2: AGS cells are infected by shRNA lentivirus, and after 5 days of culture, a statistical chart of apoptosis rate comparison of the shEDDM3A group and a control group (shCtrl) is obtained.

FIGS. 6-3: the shRNA lentivirus infects MGC80-3 cells, and after 5 days of culture, the apoptosis rate of the shEDDM3A group and the control group (shCtrl) is compared with a dot diagram.

FIGS. 6 to 4: the apoptosis rate of shEDDM3A group and a control group (shCtrl) is compared with a statistical chart after the shRNA lentivirus infects MGC80-3 cells and is cultured for 5 days.

FIG. 7-1: human gastric adenocarcinoma cells AGS each experimental group showed a comparison of the number of metastatic cells in the transwell chamber.

FIG. 7-2: human gastric adenocarcinoma cells AGS the number of transferred cells in the transwell chamber compared to the shCtrl change was compared for each experimental group. (3 experiments in total in the bar chart, 9 mean values of representative pictures per experiment)

FIGS. 7-3: human gastric carcinoma cells MGC 80-3A graph comparing the number of transferred cells in the transwell chamber of each experimental group.

FIGS. 7-4: human gastric carcinoma cells MGC80-3 the number of transferred cells in the transwell chamber was compared to the change in shCtrl.

FIG. 8-1: AGS cell scratch 24h migration pattern.

FIG. 8-2: AGS cell scratch 24h mobility statistics. (bar graph represents the average of 5 experiments)

FIGS. 8 to 3: MGC80-3 cell scratch 24h migration map.

FIGS. 8 to 4: MGC80-3 cell scratch 24h mobility statistics. (bar graph represents the average of 5 experiments)

In the drawings, there is shown in the drawings,

when not explicitly noted, the bar graph represents the mean of 3 experiments and the error bars represent the Standard Deviation (SD).

P <0.01 for shCtrl compared to target gene shRNA lentivirus treatment group.

And compared with the target gene shRNA lentivirus treatment group, the shCtrl is not less than 0.01 and P is less than 0.05.

Detailed Description

The invention proves the function of the EDDM3A gene in the generation of gastric cancer from the viewpoint of cell function. Different gastric cancer cells are transfected after constructing a target gene shRNA lentivirus, and the expression conditions of mRNA and protein level target genes in two groups of gastric cancer cell lines are respectively detected by comparing the target gene shRNA lentivirus with a transfection control lentivirus; and then cell proliferation, apoptosis and other detection are carried out through cytofunctional experiments, and the results show that compared with a control group, the proliferation inhibition degree of two gastric cancer cells in the shRNA group is obviously higher than that of the control group, and the increase degree of the apoptosis rate is higher than that of the control group.

EDDM3A inhibitor

Refers to a molecule having an inhibitory effect on EDDM 3A. Having inhibitory effects on EDDM3A include, but are not limited to: inhibiting the expression or activity of EDDM 3A.

Inhibition of EDDM3A activity refers to a decrease in EDDM3A activity. Preferably, EDDM3A activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% as compared to before inhibition.

The inhibition of the expression of EDDM3A specifically can be inhibition of the transcription or translation of EDDM3A gene, and specifically can be inhibition of the expression of EDDM3A gene: by not transcribing the gene for EDDM3A, by reducing the transcriptional activity of the gene for EDDM3A, by not translating the gene for EDDM3A, or by reducing the level of translation of the gene for EDDM 3A.

The regulation of EDDM3A gene expression can be performed by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.

The inhibition of EDDM3A gene expression was confirmed by PCR and Western Blot.

Preferably, the EDDM3A gene expression is reduced by at least 10%, preferably by at least 30%, even more preferably by at least 50%, even more preferably by at least 70%, even more preferably by at least 90%, most preferably the EDDM3A gene is not expressed at all, compared to the wild type.

Small molecule compounds

The invention refers to a compound which is composed of several or dozens of atoms and has the molecular mass of less than 1000.

Preparation of medicine for preventing or treating gastric cancer

Nucleic acid molecules that reduce EDDM3A gene expression in gastric cancer cells can be utilized; and/or, an EDDM3A gene interfering nucleic acid construct; and/or, the EDDM3A gene interferes lentivirus to be used as an effective component for preparing a medicament for preventing or treating gastric cancer. Generally, the medicament can comprise one or more pharmaceutically acceptable carriers or auxiliary materials besides the effective components according to the requirements of different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e., capable of being blended therewith without substantially diminishing the effectiveness of the drug under ordinary circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are 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; powdered gum 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; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1 preparation of RNAi lentivirus against the human EDDM3A Gene

1. Screening effective siRNA target point aiming at human EDDM3A gene

EDDM3A (NM-006683) gene information is called from Genbank; designing effective siRNA target point aiming at EDDM3A gene. The effective siRNA target sequences screened against the EDDM3A gene are listed in Table 1-1.

TABLE 1-1 siRNA target sequences targeting the human EDDM3A gene

SEQ ID NO	TargetSeq(5’-3’)
		1	GGCTGTGTGTATACAGTAA

2. Preparation of Lentiviral vectors

Synthesizing double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I enzyme cutting sites at two ends aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes Age I and EcoR I act on pGCSIL-GFP vector (provided by Shanghai Jikai Gene chemistry Co., Ltd.), linearize it, and identify the enzyme-cleaved fragments by agarose gel electrophoresis.

TABLE 1-2 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends

The vector DNA linearized by double digestion (digestion system shown in tables 1-4, 37 ℃ C., reaction 1h) and the purified double-stranded DNA Oligo were ligated by T4 DNA ligase at 16 ℃ C. overnight in an appropriate buffer system (ligation system shown in tables 1-5), and the ligation product was recovered. The ligation product was transformed into calcium chloride prepared fresh E.coli competent cells (transformation protocol reference: molecular cloning protocols second edition, pages 55-56). Dipping the surface of the clone of the strain growing out of the connected transformation product, dissolving the surface in 10 mul LB culture medium, uniformly mixing and taking 1 mul as a template; designing universal PCR primers at the upstream and downstream of RNAi sequence in the lentiviral vector, wherein the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); the sequence of the downstream primer is as follows: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), and PCR identification experiments were performed (PCR reaction system shown in tables 1-6, reaction conditions shown in tables 1-7). Sequencing and comparing the clones which are identified to be positive by the PCR, wherein the correctly compared clones are the clones which are successfully constructed and are directed at the nucleotide sequence shown in SEQ ID NO:1, named pGCSIL-GFP-EDDM 3A-siRNA.

pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When pGCSIL-GFP-Scr-siRNA negative control plasmid is constructed, double-stranded DNA Oligo sequences (tables 1-3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at the Scr siRNA target spot, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-EDDM 3A-siRNA.

TABLE 1-3 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends

TABLE 1-4 pGCSIL-GFP plasmid digestion reaction System

Reagent	Volume (μ l)
		pGCSIL-GFP plasmid (1. mu.g/. mu.l)	2.0
10×buffer	5.0
		100×BSA	0.5
Age I(10U/μl)	1.0
		EcoR I(10U/μl)	1.0
dd H₂O	40.5
		Total	50.0

TABLE 1-5 ligation reaction System of vector DNA and double-stranded DNA Oligo

Reagent	Positive control (μ l)	Self-contained control (μ l)	Connecting group (mu l)
				Linearized vector DNA (100 ng/. mu.l)	1.0	1.0	1.0
Annealed double stranded DNA Oligo (100 ng/. mu.l)	1.0	-	1.0
				10 XT 4 phage DNA ligase buffer	1.0	1.0	1.0
T4 phage DNA ligase	1.0	1.0	1.0
				dd H₂O	16.0	17.0	16.0
Total	20.0	20.0	20.0

TABLE 1-6-1 PCR reaction System

Reagent	Volume (μ l)
		10×buffer	2.0
dNTPs(2.5mM)	0.8
		Upstream primer	0.4
Downstream primer	0.4
		Taq polymerase	0.2
Form panel	1.0
		ddH₂O	15.2
Total	20.0

TABLE 1-7 PCR reaction System Programming

3. Packaging EDDM3A-shRNA lentivirus

The DNA of RNAi plasmid pGCSIL-GFP-EDDM3A-siRNA was extracted using a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l of stock solution.

Human embryonic kidney cells 293T in logarithmic growth phase were trypsinized 24h prior to transfectionCells, cell density adjusted to 1.5X 10 with DMEM complete medium containing 10% fetal bovine serum⁵Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator. The cell density can reach 70-80% to be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. Mu.l of Packing Mix (PVM), 12. mu.l of PEI, and 400. mu.l of serum-free DMEM medium were added to a sterilized centrifuge tube according to the instructions of the MISSION Lentiviral Packing Mix kit from Sigma-aldrich, and 20. mu.l of the above-mentioned extracted plasmid DNA was added to the above-mentioned PVM/PEI/DMEM mixture.

The transfection mixture was incubated at room temperature for 15min, transferred to medium of human embryonic kidney 293T cells at 37 ℃ with 5% CO₂Culturing for 16h in an incubator. The medium containing the transfection mixture was discarded, washed with PBS solution, 2ml of complete medium was added and incubation continued for 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: centrifuging at 4 deg.C and 4000g for 10min to remove cell debris; (2) filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; (3) centrifuging at 4000g for 10-15min to obtain the required virus concentration volume; (4) after the centrifugation is finished, separating the filter cup from the lower filtrate collecting cup, reversely buckling the filter cup on the sample collecting cup, and centrifuging for 2min until the centrifugal force is not more than 1000 g; (5) the centrifuge cup is removed from the sample collection cup, and the virus concentrate is obtained. Subpackaging the virus concentrated solution and storing at-80 ℃. The sequence of the first strand of siRNA contained in the virus concentrate is shown in SEQ ID NO. 2. The packaging procedure for the control lentivirus was identical to that of the EDDM3A-siRNA lentivirus, except that the pGCSIL-GFP-Scr-siRNA vector was used in place of the pGCSIL-GFP-EDDM3A-siRNA vector.

Example 2 detection of Gene silencing efficiency by real-time fluorescent quantitative RT-PCR

Human gastric adenocarcinoma AGS and human gastric carcinoma MGC80-3 cells in logarithmic growth phase are separately trypsinized to prepare cell suspension (the number of cells is about 5X 10)⁴/ml) were inoculated in 6-well plates, respectively, and cultured until the degree of cell confluence reached about 30%. Adding appropriate amount of adjuvant according to complex infection value (MOI: AGS is 10, MGC80-3 is 20)The culture medium was changed after 24 hours of culturing the lentivirus prepared in example 1 in an amount, and the cells were collected after the infection time reached 5 days. Total RNA was extracted according to the Trizol protocol of Invitrogen corporation. The RNA was reverse-transcribed to obtain cDNA according to the M-MLV protocol of Promega (reverse transcription reaction system shown in Table 2-1, reaction at 42 ℃ for 1 hour, and then reverse transcriptase was inactivated by water bath for 10min at 70 ℃ in a water bath).

Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (TAKARA). Primers for the AGS cell EDDM3A gene were as follows: an upstream primer 5'-CATTGTGGCGTAGATGGATA-3' (SEQ ID NO: 11) and a downstream primer 5'-ATAAATGTAAGCGGGGAGTG-3' (SEQ ID NO: 12). The primers for the MGC80-3 cell EDDM3A gene were as follows: an upstream primer: 5'-AAAAGAGGCTCTGAAAGGCAAG-3' (SEQ ID NO: 13) downstream primer: 5'-CGCTCCCCTTCTCATTGATGC-3' (SEQ ID NO: 14). The housekeeping gene GAPDH is used as an internal reference for both cells, and the primer sequences are as follows: an upstream primer 5'-TGACTTCAA CAGCGACACCCA-3' (SEQ ID NO: 15) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 16). The reaction system was prepared in the proportions shown in Table 2-2.

TABLE 2-1 reverse transcription reaction System

Reagent	Volume (μ l)
		5×RT buffer	4.0
10mM dNTPs	2.0
		RNasin	0.4
M-MLV-RTase	1.0
		RNase-Free H₂O	2.6
Total	10.0

TABLE 2-2 Real-time PCR reaction System

Reagent	Volume (μ l)
		SYBR premix ex taq	6.0
Primer MIX (5. mu.M)	0.3
		cDNA	0.6
ddH₂O	5.1
		Total	12.0

The program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 30 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃;a total of 40 cycles were performed. Each time reading the absorbance value during the extension phase. After completion of PCR, the DNA double strand was sufficiently bound by denaturation at 95 ℃ for 15 seconds and then cooling to 60 ℃. Melting curves were prepared by increasing the temperature from 60 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By using 2^-ΔΔCtThe analysis method calculates the expression abundance of the EDDM3A infected mRNA. Cells infected with the control virus served as controls. The results of the experiments are shown in FIGS. 1-1 and 1-2, which indicate that the expression levels of EDDM3A mRNA in human gastric adenocarcinoma AGS and human gastric carcinoma MGC80-3 cells were down-regulated by 99.8% and 52.4%, respectively.

Example 3 detection of Gene silencing efficiency by Western Blotting method

1. Extraction of Total cellular proteins

1) Control viruses and RNAi viruses against the EDDM3A interfering target were evaluated based on complex infection values (MOI: AGS 10, MGC80-3 20) infected cells of interest (AGS and MGC80-3 cells).

2) After 5 days of infection, cell samples were collected and washed twice with PBS. An appropriate amount of RIPA lysate was taken and PMSF was added to a final concentration of 1mM within a few minutes before use. (using RIPA lysate, instruction book: http:// www.beyotime.com/RIPA-lysine-buffer. htm).

3) Adding appropriate amount of RIPA lysate, and lysing on ice for 10-15 min. Cells were scraped off and transferred to a new EP tube, and then cells were sonicated (20 times at 40W, 1s each, 2s apart).

4) After centrifugation at 12000g for 15min at 4 ℃, the supernatant was removed and purified using BCA Protein Assay Kit (manufacturer: biyuntian, goods number: P0010S) to determine the protein concentration.

5) The protein concentration of each sample was adjusted to be consistent by adding fresh lysate, typically 2. mu.g/. mu.L. Then 6X padding buffer with the volume of 1/5 is added and mixed evenly, the mixture is boiled for 10min in a metal bath with the temperature of 100 ℃, and the mixture is stored for standby at the temperature of 80 ℃ after being centrifuged for a short time.

2.SDS-PAGE

1) Preparing glue: according to the molecular weight of the target protein, glue with different concentrations is prepared, and the specific system is shown in tables 3-1, 3-2 and 3-3:

TABLE 3-1 SDS-PAGE gels (8mL system)

TABLE 3-2 SDS-PAGE gels (10mL system)

TABLE 3 SDS-PAGE gels (different systems)

2) Loading: after the gel is solidified, the comb is pulled out, the electrophoresis buffer solution is used for cleaning the sample loading hole, and the prepared sample is loaded.

3) Electrophoresis: concentrating the gel at 80mA for 20 min; the separation gel was 120mA, 1 h.

3. Immunoblotting (Wet transfer)

After the electrophoresis is finished, the protein is transferred to the PVDF membrane by using a transfer electrophoresis device and electrotransfer for 150min under the constant current condition of 300mA at 4 ℃.

4. Antibody hybridization:

1) and (3) sealing: PVDF membrane was blocked with blocking solution (TBST solution containing 5% skim milk) at room temperature for 1h or overnight at 4 ℃.

2) Primary antibody incubation: antibodies were diluted in blocking solution (Anti-EDDM3A, SIGMA, 1: 200 dilution; Anti-GAPDH Santa-Cruz, 1: 2000 dilution), incubated with the blocked PVDF membrane at room temperature for 2h or overnight at 4 ℃ and washed 4 times with TBST for 8min each.

3) And (3) secondary antibody incubation: the corresponding secondary antibody (Anti-Rabbit IgG, Santa-Cruz, 1: 2000 dilution; Anti-Mouse IgG, Santa-Cruz, 1: 2000 dilution) was diluted with blocking solution, the PVDF membrane was incubated at room temperature for 1.5h and washed 4 times with TBST, 8min each.

X-ray development:

1) using CST corporation 20X

Reagent and 20X Peroxide #7003 kit, mixing solution A and solution B in the kit according to the proportion of 1:1, reversing and mixing evenly, and standing for a plurality of minutes for use.

2) Taking out the film, wiping the absorbent paper dry, spreading into a cassette, dripping a proper amount of ECL luminous liquid uniformly mixed in the step 1, spreading a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the cassette, and adjusting the exposure time to be 1s to a plurality of minutes (the exposure time needs to be tried for a plurality of times, and the exposure time is properly adjusted according to whether the naked eyes can see fluorescence and whether the fluorescence intensity is not proper).

3) Taking out the X-ray film, placing in developing solution, taking out after banding occurs, rinsing in clear water for several seconds, and placing in fixing solution for at least 2 min.

4) Taking out the X-ray film, drying and analyzing.

The results of the two cell lines are respectively shown in figures 2-1 and 2-2, and Western Blot experiments show that the target has a knocking-down effect on the endogenous expression of the EDDM3A gene, so that the target is an effective target.

Example 3 Celigo experiment to examine the proliferation potency of tumor cells infected with EDDM3A-shRNA lentivirus

Human gastric adenocarcinoma cell AGS and human gastric carcinoma cell MGC80-3 in logarithmic growth phase are separately trypsinized to prepare cell suspension (the number of cells is about 5X 10)⁴/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number (MOI: AGS is 10, MGC80-3 is 20), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting the cells of each experimental group in the logarithmic growth phase after the infection time reaches 3 days. Complete medium resuspension into cell suspension (2X 10)⁴Per ml) at a cell density of about 1500 per well, 96-well plates were seeded. Each set of 5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO₂Culturing in an incubator. The plate reading was performed once per day with Cellomics apparatus (Nexcelom) starting the next day after plating, and the plate reading was performed continuously for 5 days. The number of cells with green fluorescence in the well plate of each scanning is accurately calculated by adjusting the input parameters of analysis settings, and the data is statistically plotted to draw a cell proliferation curve for 5 days.

The results are shown in fig. 3-1 to fig. 3-2, and the results show that after two tumor cells in the lentivirus infection group (shEDDM3A group) are cultured in vitro for 5 days, the proliferation rate is remarkably reduced and is far lower than that of the tumor cells in the control group, the cell numbers of AGS and MGC80-3 viable cells are respectively reduced by 82.4% and 73.41%, and the gene silencing of EDDM3A causes the proliferation capacity of AGS of human gastric adenocarcinoma cells and MGC80-3 cells of human gastric adenocarcinoma cells to be inhibited.

Example 4 MTT assay to examine the proliferation potency of tumor cells infected with EDDM3A-shRNA lentivirus

Human gastric adenocarcinoma cell AGS and human gastric carcinoma cell MGC80-3 in logarithmic growth phase are separately trypsinized to prepare cell suspension (the number of cells is about 5X 10)⁴/ml) were inoculated in 6-well plates, respectively, and cultured until the degree of cell confluence reached about 30%. According to the respective infection number of different cells, appropriate amount of EDDM3A-shRNA lentivirus and the virus of a control group are added, the culture medium is replaced after 24h of culture, after trypsinization is carried out on each experimental group cell in logarithmic phase, the complete culture medium is resuspended into cell suspension, and counting is carried out. Determining the density of plated cells (1500 cells/well) in a 96-well plate according to the growth speed of the cells, repeating 3 wells of each group, uniformly plating, observing the cell density of each experimental group under a microscope after the cells are completely precipitated, fixing one group if the density is not uniform, finely adjusting the amount of the cells of other groups, plating again (for example, finding that the Con group has more cells, reducing the cell amount, plating again), and putting into a cell culture box for culture. Starting the day after plating, 20. mu.L of 5mg/mL MTT was added to the wells 4h before termination of the culture without changing the medium. After 4h, the culture was completely aspirated, and the formazan particles were dissolved by adding 100. mu.L of LDMSO, taking care not to aspirate the formazan particles at the bottom of the well plate. Oscillating for 2-5min with oscillator, and detecting OD value with enzyme labeling instrument 490/570 nm. And (6) carrying out data statistical analysis.

The results are shown in fig. 4-1 to 4-2, after 5 days of in vitro cell culture of each tumor in the lentivirus infection group (shEDDM3A group), the proliferation rate is significantly reduced, which is much lower than that of the tumor cells in the control group, and the numbers of active cells of AGS and MGC80-3 are respectively reduced by 33% and 80.69%, which indicates that the proliferation capacity of human gastric adenocarcinoma cells AGS and human gastric carcinoma cells MGC80-3 is inhibited due to the gene silencing of EDDM 3A.

Example 5 detection of the clonogenic Capacity of tumor cells infected with EDDM3A-shRNA lentivirus

Human gastric adenocarcinoma cells AGS and human gastric cancer cells MGC80-3 are respectively digested by pancreatin and inoculated into a 12-well plate, and the cell density is 10-15%. The next day was changed to fresh medium containing 5. mu.g/ml polybrene. EDDM3A-shRNA lentivirus is added into a culture plate according to the infection multiplicity (MOI: AGS is 10, MGC80-3 is 20), and the culture medium is replaced by fresh medium after 12-24h of infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 80%.

After the cells infected with the virus in the logarithmic growth phase are digested by pancreatin, the complete culture medium is re-suspended into cell suspension; after counting the cells, inoculating the cells into a 6-well plate (800 cells/well), continuously culturing the inoculated cells in an incubator for 7 days, changing the liquid every 3 days in the middle, and observing the cell state; photographing the cell clone under a fluorescent microscope before the experiment is terminated; at the end of the experiment, cells were fixed with paraformaldehyde, washed with PBS, Giemsa stained, and photographed.

As shown in fig. 5-1 to 5-2, compared with the control group (shCtrl group), after the RNA interference of the shEDDM3A group reduced the expression of the gene, the number of clones formed by human gastric adenocarcinoma AGS and human gastric carcinoma MGC80-3 cells was significantly reduced, and the clone volume was significantly reduced; it was shown that EDDM3A gene silencing results in a reduction in the ability of tumor cells to form clones. The plate cloning test detects that after the expression of the gene is reduced, the cloning capacity of the tumor cells is reduced.

Example 6 FACS detection of the level of apoptosis in tumor cells infected with EDDM3A-shRNA lentivirus

Human gastric adenocarcinoma cells AGS and human gastric cancer cells MGC80-3 are digested with pancreatin and inoculated into 12-well plate with cell density of 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. EDDM3A-shRNA lentivirus is added into a culture plate according to the infection complex number (AGS is 10, MGC80-3 is 20), and the culture medium is replaced by fresh medium after 12-24h of infection. Passage is carried out after 72h of infection, detection is carried out on 120h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

Will be in logarithmic growthAfter long-term cell trypsinization, the complete medium is resuspended into a cell suspension; collecting the supernatant in the same 5mL centrifuge tube, each group having three multiple holes (the number of cells is not less than 5 × 10 to ensure enough cells on the machine⁵Treatment). 1300rmp for 5min, discard the supernatant and wash the cell pellet with 4 ℃ pre-cooled PBS. The cell pellet was washed once with 1 Xbinding buffer (eBioscience, 88-8007-74), centrifuged at 1300rmp for 3min, and the cells were collected. 200 μ L of 1 XBinding buffer resuspended cell pellet. Add 10. mu.L Annexin V-APC (eBioscience, 88-8007) for staining, protected from light at room temperature for 10-15 min. According to the cell amount, 400-800. mu.L of 1 × binding buffer is added, and detection is carried out by an up-flow cytometer. The results were analyzed.

As shown in FIGS. 6-1 and 6-4, the change of the apoptosis ratio of tumor cells after the Annexin V single staining method detects the expression of the genes. It was found that the apoptosis ratio of both AGS and MGC80-3 tumor cells increased upon down-regulation of gene expression. Compared with a control group, the number of apoptotic tumor cells is increased remarkably after the RNA interference of the shEDDM3A group reduces the expression of genes; indicating that gene silencing leads to apoptosis of tumor cells.

Example 7 Transwell metastasis assay for the level of tumor cell invasion by EDDM 3A-shRNA-infecting lentivirus

The Transwell cells (Corning) were removed and the desired number of cells were placed in a new 24-well plate, 100. mu.L serum-free medium was added to the upper chamber and placed in an incubator at 37 ℃ for 1 h. Respectively preparing human gastric adenocarcinoma cell AGS infected with EDDM3A-shRNA and Scr-shRNA lentivirus and human gastric carcinoma cell MGC80-3 serum-free suspension, counting, adjusting cell number according to pre-experiment, generally 10⁵Perwell (24 well plate). The medium was carefully removed from the upper chamber and 100. mu.L of cell suspension was added, and 600. mu.L of medium containing 30% FBS was added to the lower chamber. At the same time, the cell suspension was used to spread an MTS 96 well plate, approximately 5000 cells were seeded per well, and OD570 was measured after seeding as a transfer reference. The incubation was carried out in an incubator at 37 ℃ for a period of time (the specific time was adjusted according to the preliminary experiment). Reversely covering the chamber on absorbent paper to remove the culture medium, lightly removing non-transferred cells in the chamber with cotton swab, dropping 2-3 drops of Giemsa staining solution to the lower surface of the membrane to stain the transferred cells for 3-5min, soaking and washing the chamber for several timesAnd air drying. Taking a picture by a microscope: for each transwell cell, fields of view were randomly selected and 4 pictures were taken at 100X and 9 pictures at 200X. Counting by 200X pictures, performing data analysis, and comparing the difference of cell transfer capacity of the experimental group and the control group: calculating the number of transferred cells (Migratory cells per field) of each group, obtaining a p value by T-Test analysis, and judging whether a significant difference (p) exists or not<0.05, there is a significant difference, otherwise there is no significant difference)

The invasion results of the two cells are shown in fig. 7-1 to fig. 7-4, and compared with the control group (shCtrl group), the RNA interference of the shEDDM3A group reduced the expression of EDDM3A gene, and then the transfer capacities of AGS and MGC80-3 tumor cells were reduced.

Example 8 scratch healing assay to detect the level of tumor cell invasion by EDDM 3A-shRNA-infecting lentiviruses

According to the group of experimental design, about 100 mul/well of human gastric adenocarcinoma cells AGS and human gastric adenocarcinoma cells MGC80-3 infecting EDDM3A-shRNA and Scar-shRNA lentivirus are respectively added into each hole of a 96-hole plate, a cell culture medium is a culture medium containing 10% FBS, and the criterion is that the degree of confluence of the cells reaches more than 90% in the next day. The next day, low-concentration serum medium (0.1% FBS) was changed, and the lower center of the 96-well plate was aligned with a scratching instrument and gently pushed upward to form scratches. Serum-free medium was used to gently rinse 2-3 times, and low-concentration serum medium (0.1% FBS) was added to photograph. 37 ℃ and 5% CO₂Culturing in an incubator, and taking pictures at proper time points (generally 0h, 8h, 16h, 24h and the like can be selected) according to pre-experiments. Pictures were taken by fluorescence microscopy (with the central shaded area of the 96 wells as reference, and the scratch in the middle of the picture). From the post-scratch pictures, the cell mobilities of each group were calculated.

The results of the two cells are shown in FIGS. 8-1 to 8-4, respectively, and compared with the control group (shCtrl group), the RNA interference of the shEDDM3A group reduced the expression of the EDDM3A gene, and then the metastatic capacity of AGS and MGC80-3 tumor cells was reduced.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

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Claims

1. Use of human EDDM3A gene as target in preparing medicine for treating gastric cancer.

Use of an EDDM3A inhibitor for the preparation of a product having at least one of the following effects:

treating gastric cancer;

inhibiting the proliferation rate of gastric cancer cells;

promoting the apoptosis of gastric cancer cells;

inhibiting the cloning of gastric cancer cells;

inhibiting the transfer capacity of gastric cancer cells;

inhibiting the growth of gastric cancer.

3. Use according to claim 2, further comprising one or more of the following features:

1) the EDDM3A inhibitor is a molecule having an inhibitory effect on EDDM 3A;

2) the EDDM3A inhibitor is the only effective component or one of the effective components of the product;

3) the EDDM3A inhibitor is selected from double-stranded RNA and shRNA.

4. Use according to claim 3, further comprising one or more of the following features:

1) the shRNA or double-stranded RNA target sequence is shown as SEQ ID NO:1 is shown in the specification;

2) the double-stranded RNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is shown as SEQ ID NO:2 is shown in the specification;

3) the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.