Tumor Suppressive MicroRNA-34a Inhibits Cancer Cell Migration By Targeting Transcription Factor Yin Yang1(YY1) In Esophageal Squamous Cell Carcinoma

PartⅠThe effect of miR-34a on the biological function of TE-1cellsObjective: To investigate the effect of miR-34a on the biological function of TE-1cells.Methods: The mature type human miR-34a was synthesized chemically. Thesynthesized miR-34a was transfected into TE-1cells via Lipofectamine-2000. Theexpression of mature type miR-34a was detected by real-time PCR. MTT assay and fociformation assay were used to study the function of miR-34a on the proliferation of TE-1cells. Flow cytometry was conducted for the detection of cell apoptosis. The invation ofTE-1cells investigated by Transwell Chamber assay. The metastasis of TE-1cells wasdetected by cell scratch assay. The expression levels of MMPs or bal-x were detected byWestern blot.Results： In order to confirm the biological effects of miR-34a during thedevelopment of ESCC, we synthesis human miR-34a mimic and miR-34a inhibitor,which was transfected into esophageal cancer cell lines. Increased miR-34a expressionsuppressed cell growth, focus formation, while overexpression of miR-34a significiantlypromoted apoptosis by flow cytometry. miR-34a treatment resulted in significantinduction of Bcl-2and PARP-1expression in TE-1cells, but the expression of Bax wasincreased. Through wound healing test and Transwell migration assay, we observed thattransfection of human miR-34a mimic, reduced the migration ability compared with thecontrol group. On the contrary, TE-1cells treated with miR-34a inhibitor, the functionwas opposive. The expression of MMP-2and MMP-9were inhibited byWestern blot.Conclusions：Synthesized mature type human miR-34a can effectively enhancemiR-34a expression after transfection. The successful transfection of miR-34a couldinhibits the proliferation, promote the apoptosis of TE-1cells and inhibit the ablity of migration and invasion.Part ⅡStudy on the mechanism of miR-34a regulatory effect on the TE-1cellmigration and invasion by targeting YY1Objective: To investigate the invasion and migration of YY1on TE-1cellsMethods: we initially determines the relationship between miR-34a and YY1through dual-luciferase reporter system. In TE-1cell lines, the miR-34a mimic treatedcells reduced protein expression level of YY1by Western blot. The invation of TE-1cells investigated by Transwell Chamber assay. The metastasis of TE-1cells wasdetected by cell scratch assay. The expression levels of MMPs were detected by Westernblot.Results: Through dual-luciferase reporter system, co-transfected with miR-34a andYY13’UTR, significantly attenuated the activity of luciferase. In TE-1cell lines, themiR-34a mimic treated cells reduced protein expression level of YY1by Western blot.To above experimental results, we hypothesized that YY1may be one of miR-34a targetgenes. Overexpression of YY1dramatically increased tumor cell mobility in TE-1cellcompared with corresponding control, In contrast, the wound healing and invasion ofTE-1cells was deceased when endogenous YY1was inhibited with shRNA-YY1. InWestern blot, the expression levels of MMP2and MMP9were corresponding changedin YY1overexpression and silencing vectors treated cells. Cells were co-transfectedwith miR-34a mimic and shRNA-YY1, Western blot and Transwell migration assaydiscovered that shRNA-YY1could reverse the migrate inhibition of miR-34a to TE-1cells.Conclusions：YY1is one of the miR-34a target genes, miR-34a in ESCC cells caninhibit the ability of the invasion and migration, may be associated with the target geneYY1. Down-expression of YY1could reverse the migrate inhibition of miR-34a to TE-1cells.Part ⅢThe expression of miR-34a and YY1of TE-1cell to ionizing radiationObjective: To investigate the expression of miR-34a and YY1of TE-1cell toionizing radiation.Methods: The expression of miR-34a was detected by real-time PCR. The expression of YY1was detected by Western blot.Results：The expression of miR-34a was reduced and the protein level of YY1wasincreased by a time and dose-depentend manner after ionizing radiation.Conclusions：YY1is a target gene of miR-34a, the expression of YY1wasincreased, the level of miR-34a was decreased after IR.