CN113913513A - Application of human DSN1 gene and related product - Google Patents

Application of human DSN1 gene and related product Download PDF

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CN113913513A
CN113913513A CN202010653667.7A CN202010653667A CN113913513A CN 113913513 A CN113913513 A CN 113913513A CN 202010653667 A CN202010653667 A CN 202010653667A CN 113913513 A CN113913513 A CN 113913513A
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dsn1
gene
gastric cancer
strand
nucleic acid
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黄娴
曹跃琼
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Shanghai Jikai Gene Medical Technology Co ltd
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Shanghai Jikai Gene Medical Technology Co ltd
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Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human DSN1 gene as a target in preparation of a gastric cancer treatment drug or a gastric cancer diagnosis 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 human DSN1 gene is reduced by adopting an RNAi method, 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 and inhibit the growth of gastric cancer, thereby treating gastric cancer and opening up a new direction for treating gastric cancer.

Description

Application of human DSN1 gene and related product
Technical Field
The invention belongs to the field of biomedical research, and particularly relates to application of a human DSN1 gene and a related product.
Background
The research finds that the DSN1 gene is related to colorectal cancer and liver cancer. In one study, 68 genes with different copy number changes and gradually deregulated expression in colorectal cancer were discovered by genome-wide SNP genotyping and RNA sequence analysis of colon tissue from individual patients. Wherein Aurora A, SKA3 and DSN1 protein levels are up-regulated in sequence, and deletion of SKA3 or DSN1 results in G2/M block and reduction of migration and invasion. Another study finds that DSN1 is also expressed in liver cancer, and through Qpcr and immunohistochemistry, the result shows that DSN1 is up-regulated in liver cancer tissues and closely related to sex, alpha fetoprotein, tumor size, tumor node number, cancer thrombus and differentiation degree. In addition, high expression of DSN1 was associated with low survival rates in liver cancer patients.
At present, no report about the application of the DSN1 gene in treating gastric cancer exists.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide the application of the human DSN1 gene and related products.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
in the first aspect of the invention, the application of the human DSN1 gene as a target in preparing a gastric cancer treatment drug or a gastric cancer diagnosis drug is provided.
The human DSN1 gene as a target for preparing the gastric cancer treatment drug specifically comprises the following steps: the DSN1 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 DSN1 gene and is used as a candidate medicine for treating the gastric cancer. The DSN1 gene small interfering RNA (siRNA) is obtained by screening human DSN1 gene serving as an action object and can be used as a medicine for inhibiting gastric cancer cell proliferation. In addition, DSN1 gene can be used as an object of action, for example, antibody drugs, small molecule drugs, and the like.
The application of the human DSN1 gene as a target in preparing gastric cancer diagnosis medicines specifically comprises the following steps: the DSN1 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 DSN1 gene in tumor tissue, normal tissue and normal tissue around the tumor is detected by an immunohistochemical method. The research finds that: the expression level of DSN1 in gastric cancer tissue is significantly higher than that in normal tissue and normal tissue around tumor. It is suggested that the expression level of DSN1 gene may be a marker for tumor diagnosis.
The gastric cancer treatment drug is a molecule capable of specifically inhibiting the transcription or translation of a DSN1 gene or specifically inhibiting the expression or activity of a DSN1 protein, so that the expression level of the DSN1 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.
The gastric cancer therapeutic drug or gastric cancer diagnostic drug prepared from the DSN1 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 gastric cancer therapeutic agent is administered in an amount sufficient to reduce transcription or translation of human DSN1 gene, or to reduce expression or activity of human DSN1 protein. Such that the expression of the human DSN1 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 human DSN1 gene to inhibit the proliferation of gastric cancer cells. In particular, in therapy, a substance effective in reducing the expression level of human DSN1 gene is administered to a patient.
In one embodiment, the DSN1 gene has a target sequence as set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-TGAGATGAAGGAATACATA-3' are provided.
In a second aspect of the invention, there is provided the use of an inhibitor of DSN1 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;
inhibiting the growth of gastric cancer.
The product necessarily comprises a DSN1 inhibitor, and a DSN1 inhibitor as an effective ingredient for the aforementioned effects.
In the product, the active ingredient for playing the aforementioned function may be only the DSN1 inhibitor, and may also comprise other molecules for playing the aforementioned function.
That is, the DSN1 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 DSN1 inhibitor can be nucleic acid molecule, antibody, small molecule compound.
As exemplified in the examples herein, the DSN1 inhibitor can be a nucleic acid molecule that reduces DSN1 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 inhibitor of DSN 1.
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 DSN1 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 the DSN1 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 DSN1 gene;
the shRNA contains a nucleotide sequence capable of hybridizing with the DSN1 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 DSN1 gene.
The target sequence in the DSN1 gene is a segment in the DSN1 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 DSN1 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'-TGAGATGAAGGAATACATA-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'-UGAGAUGAAGGAAUACAUA-3'.
Further, the double-stranded RNA is small interfering RNA (siRNA).
SEQ ID NO:2 is one strand of small interfering RNA designed by using the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at the human DSN1 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 DSN1 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 a DSN1 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 DSN1 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'-GCUGAGAUGAAGGAAUACAUA UAUGUAUUCCUUCAUCUCAGC-3'.
Further, the DSN1 gene is derived from human.
In the fifth aspect of the invention, the DSN1 gene interference nucleic acid construct contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.
The DSN1 gene interfering nucleic acid construct can be obtained by cloning a gene segment for coding the shRNA of the human DSN1 gene into a known vector.
Further, the DSN1 gene interference nucleic acid construct is a DSN1 gene interference lentiviral vector.
The DSN1 gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the DSN1 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the DSN1 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 DSN1 gene.
Further, the DSN1 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 DSN1 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-DSN 1-siRNA.
The DSN1 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 DSN1 gene interference lentiviral vector can be used for preparing the DSN1 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 invention also discloses a DSN1 gene interference lentivirus, which is formed by virus packaging of the DSN1 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 the DSN1 gene, thereby inhibiting the proliferation of the gastric cancer cells. The DSN1 gene interference lentivirus can be used for preparing medicines for preventing or treating gastric cancer.
In a seventh aspect of the present invention, there is provided a use of the aforementioned nucleic acid molecule, or the aforementioned DSN1 gene interfering nucleic acid construct, or the aforementioned DSN1 gene interfering lentivirus, wherein: is used for preparing a medicine for preventing or treating gastric cancer or a kit for reducing the expression of the DSN1 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 DSN1 gene interfering nucleic acid construct; and/or the aforementioned DSN1 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 DSN1 gene is designed, and the corresponding DSN1RNAi vector is constructed, wherein the RNAi vector pGCSIL-GFP-DSN1-siRNA can obviously reduce the expression of the DSN1 gene at the mRNA level and the protein level. The use of lentivirus (Lv) as a gene manipulation tool to carry an RNAi vector pGCSIL-GFP-DSN1-siRNA can efficiently introduce the RNAi sequence aiming at the DSN1 gene into gastric cancer AGS cells in a targeted manner, reduce the expression level of the DSN1 gene and obviously inhibit the proliferation capacity of the tumor cells. Lentivirus-mediated DSN1 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 human DSN1 gene is reduced by adopting an RNAi method, 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 and inhibit the growth of gastric cancer, thereby treating gastric cancer and opening up a new direction for treating gastric cancer.
Drawings
FIG. 1: and (3) carrying out enzyme digestion electrophoresis picture on the carrier.
FIG. 2: and (3) an electrophoresis identification picture of the positive clone connected into the shRNA fragment.
FIG. 3: and RT-PCR is used for detecting the target gene reduction efficiency of AGS cell mRNA level.
FIG. 4: the results of automatic analysis of Celigo cells revealed that depletion of DSN1 gene inhibited 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)
In the drawings, there is shown in the drawings,
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
In some tumor cells, DSN1 gene is significantly highly expressed; the inventor of the invention finds that the proliferation of tumor cells can be effectively inhibited after the expression of the human DSN1 gene is down regulated by adopting an RNAi method, and the growth process of tumors can be effectively controlled, and the research result shows that the DSN1 gene is protooncogene and can be used as a target point for tumor treatment. The inventor further synthesizes and tests a plurality of siRNAs aiming at the DSN1 gene, screens out the siRNA which can effectively inhibit the expression of the DSN1 and further inhibit the proliferation and growth of AGS cells of human gastric cancer, and completes the invention on the basis.
DSN1 inhibitor
Refers to a molecule having an inhibitory effect on DSN 1. Having inhibitory effects on DSN1 include, but are not limited to: inhibiting expression or activity of DSN 1.
Inhibiting DSN1 activity refers to a decrease in DSN1 activity. Preferably, DSN1 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 prior to inhibition.
The inhibition of the expression of DSN1 may specifically be the inhibition of the transcription or translation of DSN1 gene, and specifically may be: the method comprises the steps of preventing the gene of DSN1 from being transcribed, reducing the transcription activity of the gene of DSN1, preventing the gene of DSN1 from being translated, or reducing the translation level of the gene of DSN 1.
The regulation of DSN1 gene expression 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 DSN1 gene expression was confirmed by PCR and Western Blot detection of expression level.
Preferably, DSN1 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 no DSN1 gene is expressed at all, compared to 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 DSN1 gene expression in gastric cancer cells can be utilized; and/or, a DSN1 gene interfering nucleic acid construct; and/or DSN1 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 DSN1 Gene
1. Screening effective siRNA target point aiming at human DSN1 gene
Calling DSN1 (NM-024918) gene information from Genbank; designing effective siRNA target point aiming at DSN1 gene. Table 1-1 lists the effective siRNA target sequences screened against the DSN1 gene.
TABLE 1-1 siRNA target sequences targeting the human DSN1 gene
SEQ ID NO TargetSeq(5’-3’)
1 TGAGATGAAGGAATACATA
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 DNA Oligo with Age I and EcoR I cleavage sites at both ends
Figure BDA0002575933640000081
The vector DNA linearized by double digestion (digestion system shown in tables 1-4, 37 ℃, reaction 1h) and the purified double-stranded DNA Oligo were ligated by T4DNA ligase at 16 ℃ 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-DSN 1-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 DNA Oligo sequences (shown in tables 1-3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at Scr siRNA targets, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-DSN 1-siRNA.
TABLE 1-3 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends
Figure BDA0002575933640000091
TABLE 1-4pGCSIL-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 1-6-1PCR 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 1-7PCR reaction System Programming
Figure BDA0002575933640000101
3. Packaging of DSN1-siRNA lentivirus
The DNA of RNAi plasmid pGCSIL-GFP-DSN1-siRNA was extracted with a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l 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. Concentrating the virusAnd subpackaging the 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 DSN1-siRNA lentivirus except that the pGCSIL-GFP-DSN1-siRNA vector was replaced with the pGCSIL-GFP-Scr-siRNA vector.
The result of the vector enzyme digestion electrophoresis is shown in figure 1, and the result of the positive clone electrophoresis identification of the segment connected with shRNA is shown in figure 2. The above results indicate that pGCSIL-GFP-DSN1-siRNA was successfully constructed.
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). The primers for the DSN1 gene were as follows: an upstream primer 5'-CTTTCCTAAGGGACACTAAGGGC-3' (SEQ ID NO: 11) and a downstream primer 5'-TTCCAGTCCGTCTGCAAAATG-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
Figure BDA0002575933640000111
Figure BDA0002575933640000121
TABLE 2-2Real-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 analytical method calculates the infection of DSN1Abundance of mRNA expression. Cells infected with the control virus served as controls. The results of the experiment are shown in FIG. 3, which indicates that the expression level of DSN1mRNA in human gastric cancer AGS cells is down-regulated by 53.7%.
Example 3 examination of the proliferative Capacity of tumor cells infected with DSN1-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. 4). 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 viable cells is reduced by 57.8 percent, and the result shows that the AGS cell proliferation capacity of the human gastric cancer caused by the silencing of the DSN1 gene is inhibited.
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
<110> Shanghai Jikai Gene medicine science and technology Co., Ltd
Application of <120> human DSN1 gene and related product
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Claims (10)

1. The application of the human DSN1 gene as a target in preparing gastric cancer treatment medicines or gastric cancer diagnosis medicines.
Use of an inhibitor of DSN1 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;
inhibiting the growth of gastric cancer.
3. Use according to claim 2, further comprising one or more of the following features:
1) the DSN1 inhibitor is a molecule having an inhibitory effect on DSN 1;
2) the DSN1 inhibitor is the only effective component or one of the effective components of the product;
3) the DSN1 inhibitor is selected from double-stranded RNA, shRNA, antibody or 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 that reduces DSN1 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 DSN1 gene; or
shRNA containing a nucleotide sequence capable of hybridizing with the DSN1 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 a DSN1 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 a DSN1 gene.
6. The nucleic acid molecule for reducing expression of DSN1 gene 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. A DSN1 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. A DSN1 gene interfering lentivirus, which is prepared by virus packaging the interfering 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 5-6, or the DSN1 gene interfering nucleic acid construct of claim 7, or the DSN1 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 the expression of the DSN1 gene 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 DSN1 gene interfering nucleic acid construct of claim 7; and/or the DSN1 gene interfering lentivirus of claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.
CN202010653667.7A 2020-07-08 2020-07-08 Application of human DSN1 gene and related product Pending CN113913513A (en)

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