CN111154723A - Research method for siRNA targeted inhibition of Zwilch and application thereof - Google Patents

Research method for siRNA targeted inhibition of Zwilch and application thereof Download PDF

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CN111154723A
CN111154723A CN202010039759.6A CN202010039759A CN111154723A CN 111154723 A CN111154723 A CN 111154723A CN 202010039759 A CN202010039759 A CN 202010039759A CN 111154723 A CN111154723 A CN 111154723A
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zwilch
u87mg
cells
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lentivirus
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顾金海
牛建国
文玉军
孙亚敏
王鹏
和祯泉
杨勇
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Ningxia Medical University
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention discloses a research method for siRNA targeting inhibition of Zwilch and application thereof, constructing siRNA targeting Zwilch gene to infect U87MG cells, and observing infection efficiency and inhibition effect of Zwilch protein expression by adopting a fluorescence microscope and Western blotting. After the siRNA effectively inhibits Zwilch expression, CCK-8 and a plate cloning experiment are used for detecting the change of the proliferation and the clone forming capacity of the U87MG cell. Research results show that the proliferation of U87MG cells can be obviously inhibited by the down-regulation of Zwilch expression, and the role of Zwilch in the pathogenesis of human brain glioma and the potential value of gene therapy are proved.

Description

Research method for siRNA targeted inhibition of Zwilch and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, relates to a research method for inhibiting Zwilch by siRNA targeting and application thereof, and particularly relates to a research method for inhibiting the influence of Zwilch expression on the proliferation of human brain glioma U87MG cells by siRNA targeting and application thereof.
Background
Glioma is the most common primary tumor of the central nervous system and has the characteristics of high incidence rate, high recurrence rate, high death rate, low cure rate and the like. Despite the continual exploration and innovation of surgical resection and chemotherapy and radiotherapy protocols in recent years, conventional treatment approaches have been less effective in improving prognosis and prolonging patient survival. According to statistics, the median survival time of patients with malignant glioma is only 12-15 months, while patients with recurrent glioma can only survive for 3-9 months. The main reason for this is that abnormal proliferation of tumor cells is not effectively inhibited. The Zwilch gene is a basic component of a mitotic checkpoint of an metazoan, plays a role through RZZ (ZW10/Rod/Zwilch) complex, can prevent cells from exiting mitosis prematurely, and plays an essential role in establishing kinetochore-microtubule junction devices in the mitosis process. The gene is expressed in dependence on the cell cycle and plays an important physiological role in the mitosis of cells.
Two key factors of poor prognosis and low survival rate of human brain glioma are abnormal proliferation and migration/invasion of tumor cells, which are very prominent biological characteristics of tumors, the process is complex, but the abnormal mitosis of tumor cells of a root line results from the abnormal mitosis of metazoan cells, the mitosis of metazoan cells is a complex and exquisite regulation process, in order to ensure the average distribution of genetic information, centromere on each sister chromosome pair needs to be connected with microtubules from two poles respectively, the process is monitored in real time by spindle assembly check points (acting), the signaling pathway of SAC mainly comprises four components, ① main components comprise Mad1/2/3, BubR1, Bub1/3 and the like, ② kinetochore protein comprises RZZ complex, Ndc80 complex and the like, ③ microtubule motor comprises dynein, CENP-E and the like, ④ effector comprises cdc20, APC/C and the like, the major mitotic protein in the study focus on the mitotic complex of kinetochore protein 2 in the study on the mitotic complex of malignant tumor cells, the recruitment of multiple mitotic receptors of the victimid ligand 3527, the mitotic receptor has been closely combined with the mitotic gene of the mitotic receptor, the victimid ligand of the victimid ligand, the mitotic receptor, the mitotic ligand of the receptor, which promotes the mitotic ligand of the tumor cell proliferation and the mitotic receptor of the tumor cell proliferation of the receptor, which is closely related to promote the tumor cell proliferation of the tumor cell growth of the receptor of the tumor cell type CD 20-9, the tumor cell, the receptor of the tumor cell, the receptor of the tumor cell type, the tumor cell.
Disclosure of Invention
The invention aims to provide a research method for inhibiting Zwilch by siRNA targeting and application thereof, wherein Zwilch-siRNA lentivirus is used to infect a human brain glioma cell U87MG, and the change of cell proliferation and clone forming capability of the cell is observed so as to discuss the effect of Zwilch in glioma occurrence and development.
The technical scheme is as follows:
a research method for siRNA targeted inhibition Zwilch comprises the following steps:
step 1 glioma cell culture
Human glioma cell line U87MG was cultured in DMED high-sugar medium containing 10% FBS at 37 deg.C and 5% CO2And (4) concentration.
Step 2 Lentiviral infection
Selecting a glioma cell line U87MG with a good growth state, and inoculating U87MG into a six-hole plate to ensure that the cell fusion degree is between 30 and 50 percent; infecting lentivirus under the condition of MOI of 10, firstly, properly diluting the lentivirus, calculating the required virus volume and the dose of a HitransG virus infecting reagent according to the virus titer and the number of cells in a six-well plate, then discarding a culture medium in the six-well plate, adding the lentivirus infecting mixed solution, and continuing culturing; after 12h, the lentivirus infection mixed liquor is discarded, and normal complete culture medium is added for continuous culture.
Step 3Zwilch Gene protein expression level analysis
After the Zwilch-siRNA expresses lentivirus and infects U87MG cells, glioma cells U87MG with good growth state and Zwilch knock-down and negative control cells infected by the lentivirus are collected for conventional Western blotting detection, β -actin is used as an internal reference, the change of protein expression quantity of the Zwilch gene is analyzed, GraphPad Prism 6 is used for analyzing the gray value of a strip, and the protein expression is quantified.
Step 4 cell proliferation Capacity analysis
Collecting glioma cells U87MG with good growth state and Zwilch knockdown and negative control cells infected by lentivirus, adding 100 ul (about 2000 cells) of cell suspension to a 96-well plate, filling edge wells with PBS, repeating 3 wells for each cell, inoculating 6 identical 96-well plates, taking out one plate every 24 hours, adding 10 ul CCK-8 solution to each well, incubating at 37 ℃ for 4 hours, determining OD value of each 450nm well, monitoring for 6 days, sorting the obtained OD values, drawing a broken line graph, and analyzing the change of the cell proliferation capacity of each group.
Step 5 plate cloning experiment
Observing the growth state of glioma cells U87MG, selecting U87MG with good growth state and Zwilch silent and negative control cells infected by lentivirus, inoculating the cells into 35mm cell culture dishes, inoculating 200 cells into each dish, changing the liquid every 3-4 days for each group of 3 dishes, and continuously culturing for two weeks; discarding the culture medium, washing with PBS for 3 times, fixing with methanol, staining with 1% crystal violet for 15min, discarding the staining solution of crystal violet, washing crystal violet with PBS, photographing, and counting the number of cell clone formation standard, wherein the diameter of cell clone formation standard is greater than 100 μm or the number of cells is greater than 50.
Step 6, SPSS 22.0 software is adopted for experimental data analysis of the statistical analysis method, the measured data in the experiment are expressed by means of the mean plus or minus standard deviation, variance analysis is adopted for overall comparison of four groups, LSD-t test is adopted for comparison between every two groups, and the difference is considered to have statistical significance when P is less than 0.05.
Further, in step 2, the dilution conditions are as follows: the virus was diluted with serum-free DMED high-sugar medium to 1/100 titer.
Further, in step 2, the degree of cell fusion was 40%.
The method disclosed by the invention is applied to the process of preparing the medicament for treating the glioma.
The invention has the beneficial effects that:
the invention uses human brain glioma cell U87MG as a research object, and researches and analyzes the influence of down-regulated Zwilch expression on the proliferation and clone forming capacity of U87MG cells after the high-efficiency transfection of Zwilch-siRNA mediated by lentivirus. The results show that Zwilch protein expression is significantly inhibited after the lentivirus with two target spots Zwilch-siRNA (59456-1 and 59460-1) for Knockdown (KD) is infected with U87 MG. After Zwilch is inhibited, the result of CCK-8 experiment shows that the proliferation capacity of U87MG is obviously weakened, and the clone forming capacity of U87MG is further proved to be obviously reduced by a plate clone experiment. These results indicate that down-regulation of Zwilch expression can significantly inhibit proliferation of U87MG cells, confirming the role of Zwilch in the pathogenesis of human brain glioma and the potential value of gene therapy.
Drawings
FIG. 1 is a microscopic observation of the infection efficiency of Zwilch-siRNA lentivirus in human brain glioma U87MG cells, wherein A1: observing the infection efficiency of Zwilch-siRNA (59456-1) under an optical microscope; a2: observing the infection efficiency of Zwilch-siRNA (59456-1) under a fluorescence microscope; b1: observing the infection efficiency of Zwilch-siRNA (59460-1) under an optical microscope; b2: the infection efficiency of Zwilch-siRNA (59460-1) was observed under a fluorescence microscope.
FIG. 2 is a Zwilch-siRNA lentivirus down-regulating the expression level of Zwilch protein in U87MG cells, wherein A: Zwilch-siRNA lentivirus down-regulates the expression level of Zwilch protein in U87MG cells; b: statistics of Zwilch-siRNA lentivirus downregulation of Zwilch protein expression levels in U87MG cells.
FIG. 3 shows that down-regulation of Zwilch expression significantly reduces the proliferative capacity of U87MG cells;
FIG. 4 is that down-regulation of Zwilch expression significantly attenuated clonogenic capacity of U87MG cells; wherein, A: down-regulation of Zwilch expression significantly reduces the clonogenic capacity of U87MG cells; b: statistical results of the down-regulation of Zwilch expression significantly attenuated the clonogenic capacity of U87MG cells.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific embodiments.
1 materials and methods
1.1 cell, animal and major reagent human glioma cell line U87MG were purchased from Kjeka Genenco chemical technology, Inc., Shanghai; DMEM high-glucose medium and PBS were purchased from Hyclone, usa; fetal bovine serum, 0.25% + EDTA pancreatin and penicillin-streptomycin were purchased from Thermo Fisher, USA; the slow virus mediated siRNA targeting Zwilch is designed and synthesized by Shanghai Jikai biotechnology limited; SDS-PAGE gel preparation kit, whole protein extraction kit and BCA protein quantification kit are all purchased from Jiangsu Kai-based biotechnology GmbH; the CCK-8 kit was purchased from Shanghai Bibo Bio Inc.
1.2 Experimental methods
1.2.1 glioma cell culture human brain glioma cell line U87MG was cultured in DMED high-sugar medium containing 10% FBS at 37 deg.C and 5% CO2And (4) concentration.
1.2.2 infecting by lentivirus, selecting a glioma cell line U87MG with good growth state, and inoculating U87MG into a six-well plate to ensure that the cell fusion degree is between 30 and 50 percent; infecting lentivirus under the condition of MOI 10, firstly diluting the lentivirus appropriately (in the experiment, the dilution condition is that the virus is diluted by serum-free DMED high-sugar culture medium, the diluted titer is 1/100, calculating the required virus volume and the dose of a HitransG virus infecting reagent according to the virus titer and the cell number in a six-well plate, then discarding the culture medium in the six-well plate, adding the lentivirus infecting mixed solution, and continuing culturing; after 12h, the lentivirus infection mixed liquor is discarded, and normal complete culture medium is added for continuous culture.
1.2.3 analyzing protein expression level of Zwilch gene, after Zwilch-siRNA expressing lentivirus infected U87MG cell, collecting glioma cell U87MG with good growth state and Zwilch knock-down and negative control cell infected by lentivirus, carrying out conventional Western blotting detection, using β -actin as internal reference, analyzing change of protein expression level of Zwilch gene, and analyzing gray value of band by GraphPad Prism 6 to quantify protein expression.
1.2.4 analysis of cell proliferation Capacity glioma cells U87MG with good growth status and lentivirus-infected Zwilch knockdown and negative control cells were collected, 100. mu.l (about 2000 cells) of cell suspension was added to a 96-well plate (the edge wells were filled with PBS), 3 wells were repeated for each cell, 6 identical 96-well plates were inoculated, one plate was removed every 24 hours, 10. mu.l of CCK-8 solution was added to each well, OD values of 450nm wells were measured after incubation at 37 ℃ for 4 hours, OD values obtained after monitoring for 6 days were collated and plotted as a line graph to analyze the change in proliferation capacity of each group of cells.
1.2.5 plate clone experiment observation glioma cell U87MG growth state, selecting good growth state U87MG and slow virus infected Zwilch silent and negative control cell, inoculating to 35mm cell culture dish, inoculating 200 cells per dish, each group 3 dishes, changing liquid every 3-4 days, continuously culturing for two weeks; discarding the culture medium, washing with PBS for 3 times, fixing with methanol, staining with 1% crystal violet for 15min, discarding the staining solution of crystal violet, washing crystal violet with PBS, photographing, and counting the number of cell clone formation standard, wherein the diameter of cell clone formation standard is greater than 100 μm or the number of cells is greater than 50.
1.3 statistical analysis method the experimental data analysis adopts SPSS 22.0 software, the measured data in the experiment is expressed by mean plus or minus standard deviation, the four groups of overall comparison is analyzed by variance, the comparison between every two groups adopts LSD-t test, and the difference is considered to have statistical significance when P is less than 0.05.
2 results
2.1Zwilch-siRNA Lentiviral highly infects human brain glioma cell U87MG
After the lentivirus is infected for 96 hours, the expression conditions of GFP fluorescence of two targets Zwilch-siRNA (59456-1) and Zwilch-siRNA (59460-1) in U87MG cells are observed under a fluorescence microscope, and the result shows that the infection efficiency of the two groups reaches 90 percent, which indicates that Zwilch-siRNA lentivirus efficiently infects human brain glioma cells U87MG (figure 1).
2.2Zwilch-siRNA Lentiviral downregulation of expression of Zwilch in U87MG cells
After lentivirus infection, Western blotting detects gene silencing effect, and the experimental result shows that after two target spots Zwilch-siRNA (59456-1 and 59460-1) lentivirus infects U87MG, Zwilch protein expression is obviously reduced, and the protein expression amounts of a blank control group, a negative control group and two experimental groups are respectively: 0.6400 + -0.0351, 0.6528 + -0.0351, 0.0150 + -0.0010, 0.0168 + -0.0025, the difference is statistically significant (P <0.05) (FIG. 2).
2.3 Down-regulation of Zwilch expression significantly reduced the proliferative capacity of human brain glioma cell U87MG
CCK-8 experimental results show that the proliferation capacity of cells in two Zwilch-siRNA lentivirus-infected groups is obviously reduced compared with a blank control group and a negative control group after lentivirus infection is continuously monitored for 6 days, wherein the inhibition effect on an interference target point is 59456-1 group at the 4 th day is most obvious, the inhibition effect on an interference target point is 59460-1 group at the 5 th day is most obvious, and the difference has statistical significance (P <0.05) (figure 3).
2.4 Down-regulation of Zwilch expression significantly reduced the clonogenic capacity of human brain glioma cell U87MG
After lentivirus infection, each group of cells are continuously cultured for two weeks under the same condition, and the plate cloning experiment result shows that the cloning formation numbers of the blank control group, the negative control group and the two Zwilch-siRNA lentivirus infected group cells are respectively as follows: 82.00 +/-4.00, 79.00 +/-2.65, 46.67 +/-3.512 and 49.00 +/-3.61. Compared with the blank control group and the negative control group, the clone forming capability of the cells of the two Zwilch-siRNA lentivirus infected groups is obviously reduced, and the difference has statistical significance (P <0.05) (figure 4).
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Claims (4)

1. A research method for siRNA targeted inhibition Zwilch is characterized by comprising the following steps:
step 1 glioma cell culture
Human glioma cell line U87MG was cultured in DMED high-sugar medium containing 10% FBS at 37 deg.C and 5% CO2Concentration;
step 2 Lentiviral infection
Selecting a glioma cell line U87MG with a good growth state, and inoculating U87MG into a six-hole plate to ensure that the cell fusion degree is between 30 and 50 percent; infecting lentivirus under the condition of MOI of 10, firstly, properly diluting the lentivirus, calculating the required virus volume and the dose of a HitransG virus infecting reagent according to the virus titer and the number of cells in a six-well plate, then discarding a culture medium in the six-well plate, adding the lentivirus infecting mixed solution, and continuing culturing; after 12h, discarding the slow virus infection mixed solution, and adding a normal complete culture medium to continue culturing;
step 3Zwilch Gene protein expression level analysis
After the Zwilch-siRNA expresses lentivirus and infects U87MG cells, collecting glioma cells U87MG with good growth state and Zwilch knockdown and negative control cells infected by the lentivirus, carrying out conventional Western blotting detection, analyzing the change of protein expression quantity of Zwilch genes by taking β -actin as an internal reference, analyzing the gray value of a strip by using GraphPad Prism 6, and quantifying protein expression;
step 4 cell proliferation Capacity analysis
Collecting glioma cells U87MG with good growth state and Zwilch knockdown and negative control cells infected by lentivirus, adding 100 mu l of cell suspension to a 96-well plate, filling edge holes with PBS, repeating 3 holes for each cell, inoculating 6 identical 96-well plates, taking out one plate every 24 hours, adding 10 mu l of CCK-8 solution into each hole, incubating for 4 hours at 37 ℃, determining the OD value of each hole at 450nm, monitoring for 6 days, sorting the obtained OD values, drawing a line graph, and analyzing the change of the cell proliferation capacity of each group;
step 5 plate cloning experiment
Observing the growth state of glioma cells U87MG, selecting U87MG with good growth state and Zwilch silent and negative control cells infected by lentivirus, inoculating the cells into 35mm cell culture dishes, inoculating 200 cells into each dish, changing the liquid every 3-4 days for each group of 3 dishes, and continuously culturing for two weeks; discarding the culture medium, washing with PBS for 3 times, fixing with methanol, staining with 1% crystal violet for 15min, discarding the staining solution of crystal violet, washing crystal violet with PBS, taking a picture, and counting the number of cell clone formation, wherein the standard of cell clone formation is that the diameter is more than 100 μm or the number of cells is more than 50;
step 6 statistical analysis
SPSS 22.0 software is adopted for experimental data analysis, metering data in the experiment are represented by means of +/-standard deviation, variance analysis is adopted for four groups of overall comparison, LSD-t test is adopted for two-to-two group comparison, and the difference is considered to have statistical significance when P is less than 0.05.
2. The method for studying targeted inhibition Zwilch of siRNA according to claim 1, wherein in step 2, the dilution conditions are: the virus was diluted with serum-free DMED high-sugar medium to 1/100 titer.
3. The method for studying targeted inhibition of Zwilch by siRNA according to claim 1, wherein in step 2, the degree of cell fusion is 40%.
4. Use of the method of claim 1 in the manufacture of a medicament for the treatment of glioma.
CN202010039759.6A 2020-01-15 2020-01-15 Research method for siRNA targeted inhibition of Zwilch and application thereof Pending CN111154723A (en)

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Application publication date: 20200515