Effects Of MiR-21-5p On Proliferation,Migration And Invasion Of Endometrial Carcinoma Cells

Ⅰ.Objective This study is aimed to investigate the expression of mi R-21-5p in endometrial carcinoma cell lines,Ishikawa and HEC-1A,and the effect of cell proliferation,migration and invasion by up-regulating or down-regulating the mi R-21-5p gene.Exploration of the direct target molecule of mi R-21-5p provides a new target for diagnosis and treatment of endometrial carcinoma.Ⅱ.Methods1.Real-time PCR method was used to detect and analyze the difference of mi R-21-5p expression in endometrial carcinoma cell lines Ishikawa and HEC-1A;2.By using lipofectamine transfection comparing mi R-21-5p mimics(up-regulated mi R-21-5p)and control(Negative control)or mi R-21-5p inhibitor(reduced mi R-21-5p)and control(Inhibitor NC)into Ishikawa and HEC-1A cells by Real-time PCR method to detect and analyze the expression of mi R-21-5p;3.The effect of up-regulated or down-regulated mi R-21-5p on the proliferation of Ishikawa and HEC-1A cells was detected by CCK8 assay;4.The effect of up-regulation or down-regulation of mi R-21-5p on migration and invasion of Ishikawa and HEC-1A cells were detected by Transwell assay and cell wound assay;5.By using Target Scan,mi RDB and other target genes prediction software to search for mi R-21-5p gene related to invasion and metastasis;6.The m RNA expression of target genes(PITX2,VCL,JAG1 and FBXO11)after transfecting with mi R-21-5p mimics in HEC-1A cells;The protein expression of PITX2 and VCL transfected up-regulation or down-regulation of mi R-21-5p into Ishikawa and HEC-1A cell lines by western blot;7.The construction of mi R-21-5p target genes(PITX2,VCL,JAG1 and FBXO11)3 ’UTR wild type clone and dual luciferase recombinant vector;lipofectamine transfection by dual luciferase assay to detect the luciferase activity of each group respectively.Ⅲ.Results1.The expression of mi R-21-5p in HEC-1A cells was significantly higher than Ishikawa cells;2.mi R-21-5p mimics(group Negative control)transfected into Ishikawa and HEC-1A cells,the expression of mi R-21-5p increased(Ishikawa cells increased 140 times,HEC-1A 15 times higher);mi R-21-5p inhibitor(Inhibitor group NC)transfected into Ishikawa and HEC-1A cells,the expression of mi R-21-5p decreased in Ishikawa cells(2 times lower,HEC-1A decreased by2.5 times);3.Up-regulation of mi R-21-5p can promote the proliferation ability of Ishikawa and HEC-1A cells,the difference was statistically significant(P <0.05);The down-regulation of mi R-21-5p had no significant effect on the proliferation of Ishikawa and HEC-1A;4.Up-regulation of mi R-21-5p can promote the migration and invasion ability of Ishikawa and HEC-1A cells;Down-regulation of mi R-21-5p inhibited the migration and invasion ability of Ishikawa and HEC-1A cells;The difference is statistically significant(P <0.05);5.Targetscan Human7.1 predicted that the "seed" region of mi R-21-5p was "UAUUCGA",and the target genes PITX2,VCL,JAG1 and FBXO11 related to invasion and metastasis were screened out by mi R-21-5p;6.The m RNA of target genes PITX2,VCL,JAG1 and FBXO11 were significantly decreased compared with the control group after transfected with mi R-21-5p mimics in HEC-1A cells by Real-time method;The protein expression of PITX2 and VCL was decreased by up-regulation the expression of mi R-21-5p into Ishikawa and HEC-1A cells;The protein expression of PITX2 and VCL was increased by down-regulation of the expression of mi R-21-5p into Ishikawa and HEC-1A cells;7.Comparison result from the target gene ’mi R-21-5p’ 3 ’UTR region cloning vector,wild type and mutant recombinant vector sequencing comparied are consistent with the expected results;double luciferase results showed that PITX2,VCL,JAG1 and FBXO11 are the direct target genes.Ⅳ.Conclusion1.The expression of mi R-21-5p in HEC-1A was significantly higher than that in Ishikawa cells.2.Up-regulation of mi R-21-5p can promote the proliferation,migration and invasion of Ishikawa cells and HEC-1A cells.3.This study proved that JAG1,FBX011,VCL and PITX2 are the target genes of mi R-21-5p.