MiR-211 Regulates The Expression Of Pluripotency Factors In F9 Cells By Targeting Meis1

CHIR99021 can effectively activate the Wnt/β-catenin signaling pathway in J1 and F9 cells and promote the expression of the pluripotency gene Nanog.While miR-211 is a miRNA that responds to the Wnt/β-catenin signaling pathway,CHIR induces miR-211 expression by activating Wnt/β-catenin.MiR-211 is less reported in regulating cellular pluripotency.Therefore,this project mainly studies the role of miR-211 in the regulation of pluripotency factors in F9 cells.We treated the mouse F9 cells with CHIR according to the literature,and found that the expression of miR-211 was significantly up-regulated in cells at a CHIR concentration of 10 μmol.Next,miR-211 mimic and its NC,miR-211 inhibitor and its NC were transfected,qPCR and western detection were used to detect the expression of several pluripotency factors.The results showed that after the transfection of mimic,Nanog,Klf4 and Oct4 did not show significant changes at the m RNA level,whereas the expression at the protein level showed an upward trend.After the transfection of inhibitor,they were no significant change in the m RNA level,but the expression at the protein level showed a downward trend.Immunofluorescence showed that after the transfection of mimic,the fluorescence intensity of Nanog,Klf4,and Oct4 was enhanced compared to the control.It indicates that miR-211 has some effects on the maintenance of pluripotency of F9 cells.To further explore how miR-211 exerts the funtion,we start with its targets.The target genes of miR-211 were predicted by software and crossover was performed.After cluster analysis,the target genes on the pathway(which was related to cell pluripotency)were selected for verification.Transfection of mimic in F9 cells,qPCR initially detect the four targets of selected,the results show that the Meis1 had the most significant downward trend.To verify whether Meis1 is a target gene of miR-211,the wild type and mutant reporter vectors of Meis1 were first constructed based on the predicted binding site of miR-211 and Meis1.The dual luciferase reporter assay results showed that Meis1 is inhibited by miR-211 mimic,after mutating the binding sites,the inhibitory effect will be released,indicating that the binding site is the mutation site.QPCR and western blot results showed that after transfection of miR-211 mimic,the expression of Meis1 was significantly down-regulated both at mRNA and protein levels.After disturbing miR-211,the expression of Meis1 was up-regulated both at mRNA and protein levels.It can be determined that Meis1 is the target of miR-211.To study the function of Meis1,we first constructed the expression vector of Meis1.Treatment of F9 cells with CHIR showed that Meis1 was significantly down-regulated in the m RNA level at a concentration of 10 μmol,indicating that CHIR inhibits the expression of Meis1 probably by promoting the expression of miR-211.Next,the effect of Meis1 on the regulation of pluripotency factors in F9 cells was examined by qPCR and western.The results showed that after overexpression of Meis1,Nanog was significantly down-regulated both in m RNA and protein levels,while Klf4 and Oct4 had a downward trend at the mRNA level but not significant,both of them were significantly down-regulated at the protein level.After interference with Meis1,Nanog and Oct4 were significantly upregulated both in mRNA and protein levels.Klf4 was not up-regulated at the mRNA level,but was significantly up-regulated at the protein level.This shows that the regulation of miR-211 to Nanog,Klf4 and Oct4 in F9 cells is likely to be accomplished by targeting Meis1.This study mainly explored that after treating the F9 cells will CHIR,Promoting the expression of miR-211.MiR-211 regulates the expression of Nanog,Klf4 and Oct4 in cells by targeting Meis1.This shows that miR-211 has some effects on the maintenance of pluripotency in F9 cells.