Effects Of MiR-4306 On The Malignant Phenotype Of Esophageal Cancer Cells And Its Mechanism

Objective:1.To investigate the effects of miR-4306 on the proliferation,cell colony formation,invasion,migration and apoptosis of ESCC cells.2.To investigate the molecular mechanism of miR-4306 suppressing the malignant phenotype of ESCC cells.Methods:1.Expression levels of miR-4306 in esophageal cancer tissues,adjacent tissues and cells were detected by qRT-PCR.2.Proliferatiom,clone formation,migration,invasion,and apoptosis of esophageal cancer cells with miR-4306 up-regulation or down-regulation were detected by RTCA-MP,transwell,colony-forming assay and flow cytometry,respectively.3.Western blotting was used to detect the alter of the EMT molecules(N-cadherin and E-cadherin),and metastasis-related molecular MMP-2 expression after ESCC cells transfected with a miR-4306 mimic or inhibitor for 48 h.4.miR-4306 target genes predicted by miRDB,Targetscan,TargetMiner and miRWalk,luciferase reporter assay verified that miR-4306 function by targeting SIX3.5.The mRNA and protein levels of SIX3 were determined by qRT-PCR and Western blotting after ESCC cells transfected with a miR-4306 mimic or inhibitor for 48 h.6.The levels of SIX3 protein expression in ESCC cell-lines were determined by Western blotting.Overexpression SIX3 expressin effects proliferation,invasion and migration of ESCC cell lines were detected by RTCA-MP system and transwell assay,respectively.7.Proliferatiom was measured by RTCA-MP system in ESCC cells co-transfected with SIX3 plasmids and miR-4306 mimic.Results:1.qRT-PCR demonstrated that expression levels of miR-4306 in esophageal cancer tissues were lower than those in the adjacent normal tissues(P<0.05).The expression levels of miR-4306 in esophageal cancer cell were lower than those in normal esophageal epithelial cells.2.After esophageal cancer cells transfected with a miR-4306 mimic or inhibitor for 48 h,RTCA-MP system and colony formation assay demonstrated that over-expression of miR-4306 inhibited proliferation and clone formation abilities of KYSE410 and KYSE450(P < 0.05,P < 0.01,P < 0.001),while low-expression of miR-4306 promoted proliferation and clone formation abilities of KYSE140 and KYSE150(P<0.01).Transwell assays showed that over-expression of miR-4306 significantly suppressed cell migration and invasion of KYSE410 and KYSE450 as compared with the negative control(P<0.05,P<0.01),while silenced expression of miR-4306 increased cell migration and invasion of KYSE140 and KYSE150 as compared with the negative control(P<0.05).Using the flow cytometry,we founded that the apoptosis of KYSE450 cell transfected with miR-4306 mimic was significantly attenuated;By contrast,the apoptosis of KYSE150 cell transfected with miR-4306 mimic was significantly promoted.3.After KYSE450 and KYSE150 cells transfected with miR-4306 mimic or inhibitor for 48 h,Western blotting showed overexpression of miR-4306 down-regulated the protein level of MMP-2,Simultaneously,we found that the expression of N-cadherin was decreased,while E-cadherin showed a higher level of expression.However,the result was the opposite of that found in KYSE150 cells,which were transfected with the miR-4306 inhibitor.4.86 potential target genes for miR-4306 predicted by miRDB,Targetscan,TargetMiner and miRWalk.we selected the target gene SIX3 eventually.After KYSE410 and KYSE450 cells co-transfected with WT or Mut reporter plasmids SIX3 and miR-4306 mimic for 48 h,dual-luciferase reporter assay showed overexpression miR-4306 significantly reduced the luciferase activity of WT plasmid(P<0.001,P<0.05),wheas not changed the luciferase activity of MUT plasmid.5.After transfection of the miR-4306 mimic or inhibitor in KYSE450 and KYSE150 cells for 48 h,we detected the mRNA and protein levels of SIX3 by qRT-PCR and Western blotting,then founded that overexpression miR-4306 markedly decreased the mRNA and protein levels of SIX3.The result was the opposite of that found in KYSE150 cells,which were transfected with the miR-4306 inhibitor.6.Western blotting showed that the expression levels of SIX3 in esophageal cancer cell were higher than those in normal esophageal epithelial cells.RTCA-MP system demonstrated that over-expression of SIX3 increased proliferation abilities of KYSE410 and KYSE450(P < 0.05,P < 0.01,P < 0.001).Transwell assays showed that over-expression of SIX3 not affected cell migration and invasion of KYSE410 and KYSE450.7.RTCA-MP system demonstrated that co-transfected with SIX3 plasmids and miR-4306 mimic rescued proliferation abilities of KYSE410 and KYSE450 as compared with the only transfected miR-4306 mimic.Conclusions:1.miR-4306 was down-regulated in ESCC tissue samples and ESCC cells,which indicates that miR-4306 is a tumor suppressor in ESCC.2.miR-4306 inhibited proliferatiom,clone formation,migration and invasion,simultaneously it promoted apoptosis of ESCC.It relied on influencing the MMP-2 expression and EMT process regulate ESCC cellular metastasis.3.miR-4306 inhibited the expression of SIX3 by binding its 3'-UTR,and then regulated cell proliferation.