| Objective: Glioma is the most common intracranial primary malignancy in adults.Although comprehensive treatments such as surgery,radiotherapy and radiotherapy are currently used,the prognosis of patients is still poor.According to the World Health Organization(WHO)classification,glioblastoma(GBM,glioblastoma)is the most malignant glioma,and the main cause of poor prognosis of GBM is its high recurrence rate.Despite the combination of temozolomide and standard radiotherapy in recent years,the mean survival of primary GBM patients was only 14.6 months,and the 5-year survival rate was only 9.8%.Changes in the phenotype of GBM genes based on individual patients may be a breakthrough in individualized treatment.Micro RNAs(mi RNAs)are short,non-coding RNA molecules involved in a range of important biological processes such as cell proliferation,apoptosis,metabolism and differentiation.Recent studies have confirmed that mi RNAs play an important role in the production,angiogenesis,invasion and apoptosis of different human tumors.A considerable number of studies have shown that the expression of certain specific mi RNAs in malignant gliomas is dysregulated.In our recent study,we found that mi R-196 a is significantly higher in high-grade glioblastoma than non-tumor brain tissue,but the specific role and mechanism of mi R-196 a in glioma is unclear.In this experiment,we cloned the sequence of mature micro RNA-196 a and studied its biological function in glioma cells.In this study,we infected a glioma cell line with a lentiviral vector and established a stable cell line that overexpresses mi R-196 a and a stable cell line that inhibits mi R-196 a expression;using CCK-8 method,PI detects cells Cyclic and Trans-well assays for the effects of mi R-196 a on glioma cell growth,migration,invasion and apoptosis;mi R-196 a effects on glioma growth in nude mice subcutaneous xenograft models;bioinformatics software prediction In combination with the microarray analysis method,the downstream target gene of mi R-196 a was searched and verified by a dual luciferase reporter system and Western blot.Finally,we verified whether mi R-196 a exerts a biological role through its target gene by overexpressing the target gene and simulating a rescue experiment. Our results show that inhibition of mi R-196 a expression in glioblastoma cell lines U87 and U373 inhibits glioma cell proliferation,cell cycle progression and activity,while inhibiting the cell cycle of glioma cells.In molecular mechanism studies,we found that mi R-196 a overexpression promotes the expression of the cycle-associated proteins cyclin D1 and p RB,while inhibiting the expression of the cycle-associated protein p27Kip1.In glioma cells,we found that overexpression of mi R-196 a down-regulated the expression of GATA6 and confirmed that the effect of mi R-196 a on the biological behavior of glioma was achieved to some extent by inhibition of GATA6.Taken together,our results provide sufficient evidence to demonstrate the important role of mi R-196 a in the development of gliomas.Methods: 1.Cell culture: Human glioma cells U87 and U373 cell lines were cultured in DMEM medium containing 10% fetal bovine serum,and the two cells were cultured in a37 °C,5% CO 2 incubator..U87-derived glioma stem cells GSC U87 and GSC 1295 were cultured in stem cell culture medium.2.LV-mi R-196a-GFP lentivirus-infected cells:cell lines U87 and U373 were chronic virus vectors containing mature mi R-196 a sequence(Shanghai Jikai Biotech Co.,Ltd.)was transfected and labeled with green fluorescent protein,and the green fluorescence intensity was observed under a fluorescence microscope to evaluate the transfection efficiency.A cell line overexpressing mi R-196 a and inhibiting expression of mi R-196 a was stably cultured,and the cells were infected with a viral vector containing mi R-NC as a control group.3.CCK-8 method for measuring cell proliferation activity: constructing mi R-196 a overexpressing cells,cells inhibiting mi R-196 a expression,mi R-196 a overexpression,and GATA6 overexpressing cells and mi R-NC cells were seeded in 96-well plates(A total of 5 plates were plated in 2000 cells per well.Each well was incubated with CCK-8for 2 hours in the dark,and the absorbance at 450 nm was measured with a microplate reader for 5 days.Draw a cell proliferation curve.4.Flow cytometry analysis of cell cycle: Prior to testing,the cell culture medium was changed to serum-free DMEM for synchronization.After collecting the cells,the cells were fixed in 4% 70% ice ethanol for2 hours,containing 25 mg/ml PI(propidium iodide)and 10 mg/ml RNA.The enzyme inhibitor was stained with PBS and incubated for 30 minutes at 37 ° C in the dark;DNA content was detected by flow cytometry,and red fluorescence at 488 nm was recorded and analyzed by Modifit software.5.Glioma stem cell glomeration test: Solid tumors grow in a three-dimensional(3D)spatial conformation,resulting in uneven exposure of oxygen and nutrients as well as other physical and chemical stresses.To mimic the 3D spatial conformation,a 3D in vitro culture model has been used for cancer research.Cancer stem cells(CSCs)are defined as a small fraction of cells within a tumor that have the ability to self-renew and often drive tumor progression and relapse after chemotherapy.Traditionally,cancer stem cells have been isolated from cancer cell lines and tumor biopsies and grown in 3D tumor sphere suspension cultures.In order to promote cell globular growth and prevent cell adherence,a serum-free medium(with B27 and a suitable concentration of growth factor)is used in combination.Cell aggregation was inhibited using a 1% concentration of methyl cellulose.After 10-14 days of culture using the above method,the number of cell spheres having a diameter greater than 75 um was counted.Cell spheres after 10-14 days of culture were collected over 70μm cell sieves,digested into single cells by trypsinization,and appropriate cells were plated into 96-well plates for further culture for 10-14 days.The number of cell spheres was counted and SFE was calculated.(SFE = number of cell spheres larger than 75 um in diameter per well / total number of cells inoculated in each well).6.Invasion ability test:(1)Pre-cool the gun head,Transwell chamber,Matrigel gel and Eppendorf tube at 4 ° C for 1 hour;(2)Resuspend the cells in PBS for 1 wash and in serum-free medium Dilute the medium;(3)dilute the Matrigel gel to 1 ng / n L;add 50 jx L to the upper end of the Transwell chamber and add 1 hour to the incubator;Transwell small chamber to add 800^ 10% FBS complete medium,then in the chamber Add 100 μL of cell suspension to the upper end,each set of three replicate wells,placed in a 5% CO 2 incubator for 24 hours;(4)remove the chamber,the cotton swab erases the cells at the upper end of the chamber;(5)at room temperature Fix with a mixed solution of 1:1 methanol and acetone for 10minutes;(6)rinse 3 times with tap water,and stain with crystal violet for 30 minutes;(7)rinse the tap water 3 times,dry,cut the film with a spatula,seal the gel;8)Under the microscope observation,three fields were randomly selected from each group.7.Tumor formation experiment in nude mice: 12 BALB / C-null nude mice(Beijing Weitong Lihua Biotechnology Co.,Ltd.)were used.The nude mice were placed in a sterile laminar flow-free specific pathogen(SPF)environment at the Animal Experimental Center of China Medical University,and the autoclaved water and feed were freely consumed.Twelve mice were equally divided into an experimental group(mi R-196a)control group(mi R-NC),and 100 μl(1 × 10 7 cells)of the tumor cell suspension was subcutaneously injected into the side of each nude mouse.The long diameter(D)and short diameter(d)of the tumor were measured weekly using a vernier caliper.According to the formula tumor volume(V)=(D × d2)/ 2 mm3,the mice were sacrificed after 6weeks,and the tumor was weighed.8.m RNA chip analysis: After m RNA expression analysis of mi R-196 a overexpression,m RNA expression in U87 cells was changed,and m RNA with more than 2-fold decrease in expression and statistical significance(P < 0.05)was selected as mi R-196 a.Potential target genes.9.Bioinformatics analysis: The target genes of mi R-196 a were predicted by target gene prediction software Targetscan,mi Randa and PITA,respectively,and the results were summarized to obtain intersections.The results of this crossover and the results of the m RNA chip analysis were combined to determine the most likely mi R-196 a target gene.10.Dual luciferase reporter assay: The wild-type or mutant GATA6-3’-UTR luciferase reporter vector was designed and synthesized by Guangzhou Ruibo Biotechnology Co.,Ltd.HEK293 cells were seeded in96-well plates for 24 hours and reached 80% confluence prior to transfection.100 ng of wild-type or mutant GATA6-3’-UTR luciferase reporter vector,total concentration of 100 nmol mi R-196 a mimic or mi R-NC mimic,was co-transfected into HEK293 cells.Firefly and Renilla luciferase activities were detected 24 hours after transfection using the dual luciferase reporter system(Promega).Using Renilla luciferase activity as a standard,firefly luciferase activity was used as a reference for standardized transfection efficiency.11.Immunofluorescence: mi R-196 a cells and mi R-NC cells were evenly plated in24-well plates with cell fusion of approximately 40%.The cells were fixed with 4%paraformaldehyde the next day,washed 3 times with PBS,blocked with 5% BSA working solution for 1 hour,and incubated overnight at 1 °C.The next day,1 hour was added at room temperature for 1 hour,and the nuclei were stained with DAPI for 15 minutes and then observed under an inverted fluorescence microscope.The first antibody was GATA6(1:200,Abcam)and the second antibody was TRITC(1:200,Proteintech).12.Transient transfection: The construction of GATA6-pc DNA3.1 and Empty-Vector vector was completed by Guangzhou Ruibo Biotechnology Co.,Ltd.,and the transfection reagent was Limpofectamine 2000 of Invitrogen.Cells were harvested 48 hours after transfection for subsequent experiments.13.Western Blot: RIPA cell protein lysate containing 1% PMSF cleaves proteins extracted from glioma cells and transplanted tumor tissues and is electrophoresed according to molecular weight using an SDS-PAGE gel at a concentration of 8%-15%.Protein is separated.Transfer to a PVDF membrane,block with 5% skim milk for 2 hours,and incubate overnight by adding a primary antibody to a 4 °C shaker.Antibody concentrations were GATA6(1:5000,Abcam),cyclin D1(1:2000,CST),cyclin E1(1:2000,CST),PARP,cleaved PARP(1:1000,Santa Cruz),Caspase-3,cleaved-caspase-3(1:1000,Santa Cruz),p RB(1:1000,Ser780,CST),internal reference is β-actin(1:2000),the next day Incubate with secondary antibodies of the same source,illuminate and photograph using the ECL method.14.Statistical analysis: Statistical analysis was performed on the differences between groups by t test.The analytical tools were SPSS and R.P < 0.05 was considered statistically significant.Results: 1.Overexpression of mi R-196 a promotes proliferation of glioma cells: Stable expression of cells stably expressing and overexpressing glioma cell lines U87 and U373 and inhibiting expression of mi R-196 a and GFP by lentiviral-mediated stable infection system.The results of CCK-8 showed that mi R-196 a overexpression promoted the growth of glioma cells U87 and U373;cell cycle experiments showed that mi R-196 a overexpression promoted cell cycle progression from G1 / G0 to S phase.Western blot analysis showed that mi R-196 a overexpression promoted the expression of the cycle-associated proteins cyclin D1 and p RB and inhibited the expression of the cycle-associated protein p27Kip1.2.Overexpression of mi R-196 a promotes invasion of glioma cells: Trans-well experiments have shown that overexpression of mi R-196 a promotes invasion of glioma cells.In addition,Western blot analysis showed that overexpression of mi R-196 a promoted the activation of the mesenchymal-related proteins ZEB1 and MMP2 and inhibited the expression of the epithelial-associated protein E-cadherin.3.GATA6 is a target gene downstream of mi R-196a: combined with the results of microarray analysis and target gene software prediction,GATA6 is a potential target gene of mi R-196 a.The mi R-196 a mimic and the wild-type GATA63’-UTR were co-transfected by the dual luciferase reporter assay.Luciferase activity was reduced when the gene vector was reported,and there was no change in luciferase activity when co-transfected with the reporter vector of the mi R-196 a mimetic and the mutant GATA6 3’-UTR.Therefore,we believe that mi R-196 a can bind to the GATA63’-UTR site.GATA6 was confirmed to be a target gene of mi R-196 a in glioma by subsequent cellular immunofluorescence and Western blotting.4.mi R-196 a promotes the proliferation of glioma cells and promotes the transition to the interstitial phenotype by inhibiting the expression of GATA6: overexpression of the expression level of GATA6 and analysis of biological behavior in cell lines overexpressing mi R-196 a.The results showed that due to mi R-196 a,overexpression of GATA6 could reduce the ability of tumor cells to proliferate to some extent,and inhibit the ability of mi R-196 a to promote the transformation of glioma cells into mesenchymal phenotype.Conclusion: 1.mi R-196 a promotes the growth of glioma cells and promotes their invasion.2.GATA6 is a downstream target gene of mi R-196 a.3.mi R-196 a inhibits the proliferation of glioma cells and promotes their transformation to mesenchymal phenotype by inhibiting the expression of GATA6. |
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