CN113917144A - Application of human SH2D2A gene and related product - Google Patents

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of human SH2D2A gene as a target in preparation of a gastric cancer treatment drug or a gastric cancer diagnosis drug. Extensive and intensive research shows that the expression of human SH2D2A gene is down-regulated by RNAi method, so that the proliferation of gastric cancer cells can be effectively inhibited, the apoptosis can be promoted, and the growth process of 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 capacity of gastric cancer cells, promote the apoptosis of the gastric cancer cells, inhibit the cloning of the gastric cancer cells and inhibit the growth of the gastric cancer cells, thereby treating the gastric cancer and opening up a new direction for the treatment of the gastric cancer.

Description

Application of human SH2D2A gene and related product

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of a human SH2D2A gene and a related product.

Background

The SH2D2A gene is a protein-encoding gene having an SH3 domain binding site, and is involved in signal transduction of T cells. Plays an important role in normal and pathological revascularization. There are multiple transcription mutants of this gene.

Gastric cancer (gastric carcinoma) is a malignant tumor originating from the epithelium of the gastric mucosa and can occur in any part of the stomach, more than half of which occur in the antrum of the stomach, where the greater curvature, lesser curvature and the anterior and posterior walls are affected. Most of gastric cancers belong to adenocarcinoma, have no obvious symptoms in the early stage, or have nonspecific symptoms such as epigastric discomfort, eructation and the like, are similar to the symptoms of chronic stomach diseases such as gastritis, gastric ulcer and the like, are easy to ignore, and have poorer prognosis due to the rapid development and transfer of the disease condition. In order to improve the therapeutic effect of gastric cancer, understanding the potential molecular mechanism and determining new and effective therapeutic targets are urgently needed.

Few reports on SH2D2A in the tumor-related field exist, and no reports on SH2D2A gene for gastric cancer treatment exist 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 SH2D2A gene and a related product.

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 application of the human SH2D2A gene as a target in preparing a gastric cancer treatment drug or a gastric cancer diagnosis drug is provided.

The human SH2D2A gene as a target for preparing the gastric cancer treatment drug specifically comprises the following steps: the SH2D2A gene is used as an action object, and the medicine or the preparation is screened to find the medicine which can inhibit the expression of the human SH2D2A gene and is used as a candidate medicine for treating the gastric cancer. The SH2D2A gene small interfering RNA (siRNA) is obtained by screening human SH2D2A gene serving as an action object and can be used as a medicament with the effect of inhibiting gastric cancer cell proliferation. In addition, SH2D2A gene can be used as an active substance such as an antibody drug, a small molecule drug, or the like.

The application of the human SH2D2A gene as a target in preparing gastric cancer diagnosis medicines specifically comprises the following steps: the SH2D2A gene expression product is used as a gastric cancer diagnosis index to be applied to the preparation of gastric cancer diagnosis medicaments.

The expression level of the SH2D2A gene in tumor tissues, normal tissues and normal tissues around the tumor is detected by an immunohistochemical method. The research finds that: the expression level of SH2D2A in gastric cancer tissues is obviously higher than that of normal tissues and normal tissues around tumors. It is suggested that the expression level of SH2D2A gene may be a marker for tumor diagnosis.

The gastric cancer treatment drug is a molecule which can specifically inhibit the transcription or translation of an SH2D2A gene or specifically inhibit the expression or activity of an SH2D2A protein, so that the expression level of the SH2D2A gene in gastric cancer cells is reduced, and the purposes of inhibiting the proliferation, growth, differentiation and/or survival of the gastric cancer cells are achieved.

The gastric cancer therapeutic drug or gastric cancer diagnostic drug prepared from the SH2D2A gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

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 amount of the gastric cancer therapeutic agent to be administered is a dose sufficient to reduce transcription or translation of the human SH2D2A gene, or to reduce expression or activity of the human SH2D2A protein. Such that the expression of the human SH2D2A 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 aim of treatment mainly by reducing the expression level of human SH2D2A gene to inhibit the proliferation of gastric cancer cells. Specifically, in the treatment, a substance effective in reducing the expression level of human SH2D2A gene is administered to the patient.

In one embodiment, the target sequence of the SH2D2A gene is set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-GGACCGAAGAATCAAACTT-3' are provided.

In a second aspect of the invention, there is provided the use of an SH2D2A inhibitor in the manufacture of a product having at least one of the following effects:

treating gastric cancer;

inhibiting the proliferation ability of gastric cancer cells;

promoting the apoptosis of gastric cancer cells;

inhibiting the cloning of gastric cancer cells;

inhibiting the growth of gastric cancer.

The product necessarily comprises an SH2D2A inhibitor and has the SH2D2A inhibitor as an effective component of the aforementioned effects.

In the product, the active ingredient for playing the aforementioned roles may be only an SH2D2A inhibitor, and may also include other molecules for playing the aforementioned roles.

That is, the SH2D2A 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 SH2D2A inhibitor can be a nucleic acid molecule, an antibody or a small molecule compound.

As exemplified in the examples of the present invention, the SH2D2A inhibitor may be a nucleic acid molecule that reduces SH2D2A gene expression 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 to a subject an SH2D2A inhibitor.

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 SH2D2A inhibitor may be administered to a subject before, during, or after treatment for gastric cancer.

The fourth aspect of the invention discloses a nucleic acid molecule for reducing SH2D2A gene expression 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 an SH2D2A gene;

the shRNA contains a nucleotide sequence capable of hybridizing with an SH2D2A 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 SH2D2A gene.

The target sequence in the SH2D2A gene is a segment in the SH2D2A gene corresponding to an mRNA segment which is recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the SH2D2A 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'-GGACCGAAGAATCAAACTT-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'-GGACCGAAGAAUCAAACUU-3'.

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

SEQ ID NO:2 is one strand of small interfering RNA designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at human SH2D2A gene, and 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 SH2D2A 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 SH2D2A 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 SH2D2A 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'-GGACCGAAGAAUCAAACUUCUCGAGAAGUUUGAUUCUUCGGUCC-3'.

Further, the SH2D2A gene is derived from human.

In the fifth aspect of the invention, the SH2D2A gene interfering nucleic acid construct contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.

The SH2D2A gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the human SH2D2A gene shRNA into a known vector.

Further, the SH2D2A gene interference nucleic acid construct is an SH2D2A gene interference lentiviral vector.

The SH2D2A gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the SH2D2A gene shRNA into a known vector, wherein most of the known vectors are lentiviral vectors, the SH2D2A gene interference lentiviral vector is packaged into infectious viral particles by virus, gastric cancer cells are infected, the shRNA is transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the SH2D2A gene.

Further, the SH2D2A gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding 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 discloses a human SH2D2A gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-SH2D 2A-siRNA.

The SH2D2A 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. SH2D2A gene interference lentiviral vector can be used for preparing the SH2D2A 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 SH2D2A gene interference lentivirus, which is formed by virus packaging of the SH2D2A 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 SH2D2A gene, thereby inhibiting the proliferation of the gastric cancer cells. The SH2D2A gene interference lentivirus can be used for preparing medicines for preventing or treating gastric cancer.

The seventh aspect of the present invention provides the use of the above nucleic acid molecule, or the above SH2D2A gene interfering nucleic acid construct, or the above SH2D2A gene interfering lentivirus, wherein: is used for preparing a medicine for preventing or treating gastric cancer or a kit for reducing SH2D2A gene expression 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 SH2D2A gene interfering nucleic acid construct; and/or the SH2D2A 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 SH2D2A gene is designed, and the corresponding SH2D2A RNAi vector is constructed, wherein the RNAi vector pGCSIL-GFP-SH2D2A-siRNA can remarkably reduce the expression of the SH2D2A gene at the mRNA level and the protein level. An RNAi vector pGCSIL-GFP-SH2D2A-siRNA carried by lentivirus (Lv) serving as a gene manipulation tool can be used for efficiently introducing an RNAi sequence aiming at the SH2D2A gene into gastric cancer AGS cells in a targeted manner, so that the expression level of the SH2D2A gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated SH2D2A 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:

extensive and intensive research shows that the expression of human SH2D2A gene is down-regulated by RNAi method, so that the proliferation of gastric cancer cells can be effectively inhibited, the apoptosis can be promoted, and the growth process of 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 capacity of gastric cancer cells, promote the apoptosis of the gastric cancer cells, inhibit the cloning of the gastric cancer cells and inhibit the growth of the gastric cancer cells, thereby treating the gastric cancer and opening up a new direction for the treatment of the gastric cancer.

Drawings

FIG. 1: and RT-PCR is used for detecting the target gene reduction efficiency of AGS cell mRNA level.

FIG. 2: the results of automatic analysis of Celigo cells revealed that the depletion of SH2D2A gene inhibited the proliferation of gastric cancer cells. (cell lines are AGS cells and cell numbers were counted 1, 2, 3, 4 and 5 days after viral infection)

FIG. 3: the influence of the SH2D2A gene on the proliferation capacity of the AGS cells is detected by a cell clone formation method, the AGS cells are infected by shRNA lentivirus, the clone number is observed after the AGS cells are cultured for 14 days, the left side is a digital camera record chart, and the right side bar result is displayed by the average value +/-standard deviation of the cell clone number.

FIG. 4: annexin V-APC flow apoptosis test SH SH2D2A influences the AGS apoptosis, the left graph is a schematic diagram of flow apoptosis, and the bar results of the right graph are shown by the cell percentage mean value plus or minus standard deviation.

In the drawings, there is shown in the drawings,

bar graphs represent the mean of three experiments and error bars represent 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 inventor of the invention has found through extensive and intensive research that SH2D2A gene is significantly highly expressed in gastric cancer tumor tissues; the inventor finds that after the expression of human SH2D2A gene is down-regulated by RNAi method, the proliferation of tumor cell can be effectively inhibited, the apoptosis can be promoted, and the growth process of tumor can be effectively controlled, and the research result shows that SH2D2A gene is protooncogene and can be used as the target point of tumor therapy. The inventors further synthesized and tested various siRNAs against SH2D2A gene, and screened out siRNAs capable of effectively inhibiting the expression of SH2D2A and further inhibiting the proliferation and growth of human gastric cancer AGS cells, thereby completing the invention.

SH2D2A inhibitors

Refers to a molecule having an inhibitory effect on SH2D 2A. Having an inhibitory effect on SH2D2A includes, but is not limited to: inhibiting the expression or activity of SH2D 2A.

Inhibition of SH2D2A activity refers to a decrease in SH2D2A activity. Preferably, the activity of SH2D2A 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 its activity prior to inhibition.

The inhibition of the expression of SH2D2A specifically can be the inhibition of the transcription or translation of SH2D2A gene, and specifically can be the inhibition of the expression of SH2D2A gene: the gene of SH2D2A is not transcribed, or the transcriptional activity of the gene of SH2D2A is reduced, or the gene of SH2D2A is not translated, or the translation level of the gene of SH2D2A is reduced.

The regulation of gene expression of SH2D2A can be accomplished by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.

The inhibition of SH2D2A gene expression was confirmed by PCR and Western Blot detection of the expression level.

Preferably, SH2D2A gene expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, still more preferably by at least 90%, most preferably by no expression of the SH2D2A gene, 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 SH2D2A gene expression in gastric cancer cells can be used; and/or, an SH2D2A gene interfering nucleic acid construct; and/or SH2D2A gene interferes lentivirus, and is 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 human SH2D2A Gene

1. Screening of effective siRNA target against human SH2D2A gene

SH2D2A (NM-003975) gene information was retrieved from Genbank; effective siRNA targets were designed against the SH2D2A gene. Table 1-1 lists the effective siRNA target sequences screened against the SH2D2A gene.

TABLE 1-1 siRNA target sequences targeting human SH2D2A gene

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

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 medicine science and technology Co., Ltd.), so that the vector is linearized, and the enzyme-cut fragment is identified by agarose gel electrophoresis.

TABLE 1-2 double-stranded DNAoligo 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 DNAoligo were ligated by T4 DNA ligase, and ligated overnight at 16 ℃ 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-SH2D 2A-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 plasmids are constructed, double-stranded DNAoligo sequences (tables 1-3) containing Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at Scr siRNA targets, and other construction methods, identification methods and conditions are the same as pGCSIL-GFP-SH2D 2A-siRNA.

TABLE 1-3 double-stranded DNAoligo 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 DNAoligo

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 strandDNAOligo(100ng/μl)	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

TABLE 1-7 PCR reaction System Programming

3. Packaging SH2D2A-siRNA lentivirus

The DNA of RNAi plasmid pGCSIL-GFP-SH2D2A-siRNA was extracted using a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l of stock solution.

24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were trypsinized and cell density was adjusted to 1.5X 10 in 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: (1) 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 process of the control lentivirus was the same as SH2D2A-siRNA lentivirus, and pGCSIL-GFP-Scr-siRNA vector was used only instead of pGCSIL-GFP-SH2D2A-siRNA vector.

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

Human gastric cancer AGS cells in logarithmic growth phase are 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 complex infection value (MOI, AGS: 10), an appropriate amount of the lentivirus prepared in example 1 is added, the medium is changed after 24h of culture, and cells are collected after the infection time reaches 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 SH2D2A gene were as follows: an upstream primer 5'-CATCAAACAGGGGCAAGC-3' (SEQ ID NO: 11) and a downstream primer 5'-GGTGTCGTGGAACAGGGA-3' (SEQ ID NO: 12). The housekeeping gene GAPDH is used as an internal reference, and the primer sequences are as follows: an upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 13) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 14). 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	0.5
M-MLV-RTase	1.0
		DEPC H2O	3.5
Total	11.0

TABLE 2-2 Real-time PCR reaction System

Reagent	Volume (μ l)
		SYBR premix ex taq	10.0
Upstream primer (2.5. mu.M)	0.5
		Downstream primer (2.5. mu.M)	0.5
cDNA	1.0
		ddH2O	8.0
Total	20.0

The program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 15 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 45 cycles were performed. Each time reading the absorbance value during the extension phase. After the PCR was completed, the DNA was denatured at 95 ℃ for 1min, and then cooled to 55 ℃ to allow the DNA double strands to be sufficiently bound. Melting curves were prepared by increasing the temperature from 55 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By adopting 2-^ΔΔCtThe assay calculates the abundance of expression of SH2D 2A-infected mRNA. Cells infected with the control virus served as controls. The results of the experiment are shown in FIG. 1, which indicates that the expression level of SH2D2A mRNA in human gastric cancer AGS cells is down-regulated by 65.1%.

Example 3 examination of the proliferative Capacity of tumor cells infected with SH2D2A-siRNA lentivirus

Human gastric cancer AGS cells in logarithmic growth phase are 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: 10), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. Complete medium resuspension into cell suspension (2X 10)⁴Per ml) at a cell density of about 3000 cells 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 readings were performed once a day with Celigo instrument (Thermo Fisher) starting the second day after plating, and were performed continuously for 5 days. By adjusting the input parameters of Celigo, the number of green fluorescent cells in the well plate for each scan was accurately calculated, and the data were statistically plotted to obtain a cell proliferation curve (the result is shown in FIG. 2). The results show that after each tumor of the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of the tumor cells of the control group, the number of the active cells is reduced by 56.7 percent, and the results show that the SH2D2A gene silencing causes human gastric cancerAGS cell proliferation ability is inhibited.

Example 4 examination of the clonogenic Capacity of tumor cells infected with SH2D2A-siRNA lentivirus

Human gastric cancer AGS cells are digested by trypsin and then inoculated into a 12-well plate, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. SH2D2A-siRNA lentiviruses were treated according to the multiplicity of infection MOI AGS: 10 were added to the plates and the medium was changed fresh 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

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 (200 cells/well), continuously culturing the inoculated cells in an incubator until the number of the cells in 14 days or most of single clones is more than 50, changing the liquid at intervals of 3day, 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 a result, as shown in fig. 3, the number of clones formed by AGS cells of human gastric cancer was significantly reduced and the volume of clones was significantly reduced after RNA interference reduced the expression of genes (KD group) compared to non-interference (control) and control interference (NC group); it is shown that SH2D2A gene silencing results in the reduction of human gastric cancer AGS cell cloning capacity. The plate cloning experiment detects that the cloning capacity of the gastric cancer cells is reduced after the expression of the genes is reduced.

Example 5 detection of apoptosis levels in tumor cells infected with SH2D2A-siRNA lentivirus

Human gastric cancer AGS cells are inoculated in a 6-well plate after being digested by trypsin, and the cell density is 20-30%. The next day was changed to fresh medium. SH2D2A-siRNA lentivirus was administered according to MOI, AGS: 10 were added to the plates and the medium was changed fresh 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

After trypsinizing the cells in logarithmic growth phase, resuspending the complete medium into a cell suspension; inoculating to 96-well plate, each100ul of holes; placing at 37 ℃ with 5% CO₂Culturing in an incubator; after culturing for 36h, removing the culture solution, and fixing the cells by using 85% ethanol precooled at 4 ℃; washing the plate twice with PBS; after RNase treatment, cells were PI stained and protected from light for 15 min. Scanning and analyzing the 96-well plate on a TTP instrument by using a preset template of analysis subG1 stage to obtain a result.

As shown in fig. 4, the experimental group showed a significant increase in apoptotic cells after RNA interference decreased SH2D2A gene expression (KD group) compared to the control interference (NC group); indicating that the gene silencing leads to the apoptosis of gastric cancer cells.

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.

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Claims (10)

1. The human SH2D2A gene is used as a target in the preparation of gastric cancer treatment medicines or gastric cancer diagnosis medicines.

Use of an SH2D2A inhibitor for the manufacture of a product having at least one of the following effects:

treating gastric cancer;

inhibiting the proliferation ability of gastric cancer cells;

promoting the apoptosis of gastric cancer cells;

inhibiting the cloning 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 SH2D2A inhibitor refers to a molecule having an inhibitory effect on SH2D 2A;

2) the SH2D2A inhibitor is the only effective component or one of the effective components of the product;

3) the SH2D2A inhibitor is selected from double-stranded RNA, shRNA, an antibody or a small molecule compound.

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.

5. A nucleic acid molecule for reducing SH2D2A gene expression in gastric cancer cells, the nucleic acid molecule comprising:

a. a double-stranded RNA containing a nucleotide sequence capable of hybridizing with the SH2D2A gene; or

shRNA containing a nucleotide sequence capable of hybridizing with an SH2D2A gene;

wherein 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 SH2D2A gene; 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 SH2D2A gene.

6. The nucleic acid molecule for reducing SH2D2A gene expression in a gastric cancer cell of claim 5, further comprising one or more of the following characteristics:

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 is siRNA, and the sequence of the first strand of the siRNA 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.

7. An SH2D2A gene interfering nucleic acid construct containing a gene segment encoding shRNA in the nucleic acid molecule according to any one of claims 5 to 6, capable of expressing the shRNA.

8. An SH2D2A gene interference lentivirus, which is formed by virus packaging of the interference nucleic acid construct of claim 7 with the help of lentivirus packaging plasmid and cell line.

9. The nucleic acid molecule of any one of claims 5 to 6, or the SH2D2A gene-interfering nucleic acid construct of claim 7, or the SH2D2A gene-interfering lentivirus of claim 8, for use in: is used for preparing a medicine for preventing or treating gastric cancer or a kit for reducing SH2D2A gene expression in gastric cancer cells.

10. A composition for preventing or treating gastric cancer, which comprises the following effective substances:

the nucleic acid molecule of any one of claims 5-6; and/or, the SH2D2A gene interfering nucleic acid construct of claim 7; and/or, the SH2D2A gene interfering lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.

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