MiR-205Regulates EMT In Non Small Cell Lung Cancer Cells

Background and Objective: Around the world, the incidence and mortality of lungcancer is increasing every year. Clarify the pathogenesis of lung cancer, especiallynon-small cell lung cancer, is vitally important for the individualized treatment. Epithelial-mesenchymal transformation (EMT) allows tumor cells access to the ability of invasionand metastasis. This constitutes the first step of metastasis for tumor from the primarylesion. Researches have shown that miR-205involved in the regulation of EMT process fortumor cells in breast cancer by targeting ZEB1and ZEB2. In this study, we intend toinvestigate:1.Whether miR-205is involved in the regulation of EMT process in non-smallcell lung cancer cells;2.The mechanism for miR-205participates in the EMT process innon small cell lung cancer cells.Methods:(1).With5ng/ml TGF-β1stimulating non-small cell lung cancer cell linesA549, H226, first we observe the changes in cells morphology,and then we extract theRNAs and proteins in cells before and after stimulation with5ng/ml TGF-β1.In furtherstudy, we will use Real-time PCR、immunohistochemistry, Western blot to detect thechanges of EMT molecular marker proteins (E-cadherin, N-cadherin, Vimentin).(2)Wethen use Real-time PCR to detect the expression of miR-205and transcription factor,ZEB1and ZEB2before and after stimulated with TGF-β1;(3) The miR-205mimics andnegative control will be transfected into A549, H226cells, after48h,we will again useReal-time PCR to detect the expression of these proteins (E-cadherin, N-cadherin,Vimentin)and transcription factors ZEB1and ZEB2in these cells;(4) The two kinds ofcells will be stimulated with5ng/ml TGF-β1after transfecting with miR-205mimics andnegative control for48h, then waiting another48h,we will measure the expression of theproteins of (E-cadherin, N-cadherin, Vimentin) using Western blot.Results:(1) After stimulated with TGF-β1at5ng/ml,the A549and H226cellsshowed morphological changes in spindle and the expression of E-cadherin,molecularproteins of epithelial cells characteristics,was significantly decreased, while the expression of N-cadherin,Vimentin,molecular protein of mesenchymal cells characteristics, increased,suggested that the two cells had occurred EMT.(2) The expression for miR-205in A549and H226cells decreased,while the expression of transcription factors ZEB1,ZEB2increased remarkablely in both after stimulation.(3)When transiently transfected withmiR-205mimics and negative control into A549cells,the expression of E-cadherinrised,while the ZEB1, and ZEB2decreased, The change of N-cadherin, Vimentin was notobvious. In H226cells, the expression of E-cadherin rised, N-cadherin, Vimentin, ZEB1,ZEB2decreased.(4)While the cells which have been transiently transfected with miR-205mimics(mimics group) and negative control(NC group) for48h were stimulated withTGF-β1another48h,then we found that in A549cells,the expression of E-cadherin inmimics group decreased unremarkablely than in NC group,while the N-cadherin andVimentin slightly changed;in H226cells,the expression of N-cadherin and Vimentinincreased slightly in NC group,while changed unobviously in mimics group.Conclusions: Our results suggest that the A549and H226cells can undergo theprogress of EMT after stimulated with TGF-β1at5ng/ml.MiR-205may through upregulating the expression of E-cadherin to inhibition EMT by down regulating the ZEB1and ZEB2in A549cells. But in H226cells, ectopic expression of miR-205may throughdown regulating the expression of N-cadherin and Vimentin to inhibition EMT byinhibiting the expression of ZEB1and ZEB2.