MiR-200a Targeting Inhibited MAGED4 And Its Effect On Migration And Invasion Of Glioma

Objective:To investigate whether there is a target regulatory relationship between miR-200a and MAGED4,and the effect of miR-200a on migration and invasion of glioma.Methods:A variety of bioinformatics online software was used to obtain3’UTR downstream sequence of MAGED4 gene and predict potential microRNA regulatory factors and binding sites of MAGED4.Using bioinformatics online software to obtain the relevant information of microRNA gene;（2）qRT-PCR detection of normal brain tissue and glioma cell line miR-200a expression;（3）Glioma cell lines transferred to different states of exogenous miR-200a,upregulated or downregulated miR-200a expression;（4）MAGED4 3’UTR wild type（WT）and mutant type（MUT）vector were constructed respectively,and the target regulation effect of miR-200a on MAGED4 was verified by the double luciferase reporter gene test;（5）The expression of MAGED4 mRNA and protein in glioma cells with up regulation or down regulation of miR-200a expression was detected by qRT-PCR and Western Bloting;（6）The migration ability of glioma cells with up regulation or down regulation of miR-200a expression was detected by Wound-healing assay,Transwell migration assay;The invasion ability of glioma cells with up regulation or down regulation of miR-200a expression was detected by Transwell Martrigel assay.Results:（1）The results of multiple bioinformatics software analysis showed that MAGED4 3’UTR had miR-200a binding sites,and the seed region was completely matched,and miR-200a was a potential regulator of MAGED4;（2）Compared with normal brain tissue,miR-200a showed low expression in A172,U87 and U251,with significant difference（P<0.01）;（3）The glioma cell lines（A172,U87 and U251）were transferred to miR-200a Minics,Inhibitors and Ngative control（NC）respectively.The detection results showed that there was no significant difference in the relative expression between the NC group and the Mock group.However,relative expression level of miR-200a in group Minics was significantly higher than that of group NC and group Mock（P<0.01）,and the relative expression level of miR-200a in group Inhibitors decreased significantly（P<0.01）;（4）Luciferase activity assay showed that:In 293T,A172 and U87 cells,compared with the WT^{MAGED4-3′UTR} group,the luciferase activity of the WTMAGED4-3′UTR group transfected with miR-200a NC and the MUT^MAGED4-3′UTRAGED4-3′UTR experiment group had no significant effect,but the luciferase activity of WT^{MAGED4-3′UTR} group transfected with miR-200a Mimics decreased significantly（P<0.05）,and the luciferase activity of MUT^{MAGED4-3′UTR} group was not changed（P>0.05）;（5）The glioma cells were transfected to miR-200a Minics,Inhibitors and Ngative control（NC）respectively.qRT-PCR was used to detect the mRNA and protein expression of MAGED4.The results showed that there was no difference between the NC group and Mock group.After transfection of miR-200a Minics,the expression of MAGED4 mRNA and protein decreased,the difference was statistically significant（P<0.01）.After transfection of miR-200a Inhibitors,and the mRNA and protein expression of MAGED4 increased significantly（P<0.01）;（6）The Wound-healing data showed that compared with the Mock group,there was no significant difference in the wound sealing rate in the NC group（P>0.05）.However,compared with the Mock and NC group,wound sealing rate in the Minics group was significantly lower（P<0.01）while the wound sealing rate of the Inhibitors group was significantly increased（P<0.01）.The Transwell migration test showed that there was no significant difference between the NC group and the Mock group（P>0.05）.Compared to the control NC group and the Mock group,the number of membrane cells in the Minics group decreased（P<0.01）,but the number of membrane cells in the Inhibitors group increased（P<0.01）.There was no significant difference in the difference between the two groups（P>0.05）.Compared with the control group NC and group Mock,the number of penetrating cells in group Minics was significantly reduced（P<0.01）,and the number of penetrating cells in Inhibitors group increased significantly（P<0.01）.Conclusion:There is a targeting regulation relationship between miR-200a and MAGED4,and miR-200a inhibits the expression of MAGED4 mRNA and protein in glioma cells,and miR-200a may weaken the migration and invasion of glioma cells by mediating the expression of MAGED4.This study will lay a foundation for in vivo experiments and provide new directions and ideas for further revealing the regulation mechanism of MAGED4 expression.