Effect Of MiR-136 On The Biological Functions Of Proliferation,Invasion And Migration Of Glioma Cell Line U251

Objective:MiR-136 plays as a tumor suppressor gene in the development of a variety of tumors and is involved in the process of differentiation,growth,proliferation and apoptosis of various tumor cells.By studying the target gene of miR-136,to investigate the mechanism of miR-136 inhibiting the proliferation,invasion and migration of glioma U251 cells.In the future,miR-136 and FZD4 may be used as potential markers for the treatment of glioma.Methods:The U251 cell line in glioma was selected and cultured with high glucose DMEM medium containing 10%fetal bovine serum,1%penicillin and streptomycin,and then incubate in a incubator,at constant temperature of 37?and 5%CO₂.Glioma cell line U251 were divided in to two groups,miR-136 mimic?mimic group?and miR-136 mimic negative control?control group?were transfected respectively by lipofectamine2000.qRT-PCR was used to detedct the expression of miR-136 in the two groups cell.24 h after transfection,the cell density of the two groups was observed under microscope.CCK-8 assay was applied to evaluate the proliferative ability of the glioma cells.The invasion and migration of glioma U251 cells was evaluated by Transwell assay and scratch wound assay.The binding sites of miR-136were predicted by miRanda.The expression of FZD4 mRNA and FZD4 protein was detected by qRT-PCR and western blotting,respectively.Result:1.The result of qRT-PCR shows the expression of miR-136 in miR-136 mimic group was significantly higher than control group.It indicates that the transfection model is successful.2.24h after transfection,the cells of miR-136 mimic group were less than the control group;CCK-8 showed that the proliferative activity of U251cells in the miR-136 mimic group was significantly lower than the control group.These two experiments showed that miR-136 could inhibit the proliferation of glioma U251cells.3.Transwell assays found that the number of U251 cells in the miR-136 mimic group was significantly less than that of the control group after 24 h transfection,indicating that miR-136 could inhibit the invasion of U251 cells.4.Scratch wound assays revealed that the migration distance of U251 cells in the miR-136 mimic group was shorter than that of the control group after 24 h transfection,indicating that miR-136 could inhibit the migration of U251 cells.5.According to the miRanda,we analyzed the target gene of miR-136,and found that there was a binding area between miR-136 and FZD4-mRNA 3'-UTR,suggesting that FZD4 might be the downstream target gene of miR-136.6.qRT-PCR found that the expression of FZD4 mRNA in the miR-136 mimic group was significantly lower than the control group,indicating that miR-136inhibited the expression of FZD4 mRNA.7.The results of western blot showed that the expression of FZD4 protein in the miR-136 mimic group was significantly lower than that of the control group,which showed that miR-136 inhibited the expression of FZD4 protein.Conclusion:1.Mi R-136 might inhibit the proliferation,invasion and migration of U251 cells by regulating FZD4.2.Mi R-136 and FZD4 may be used as potential markers for the treatment of glioma in future.