Effect Of Over-expression Of E-cadherin On Epithelial-mesenchymal-transition That Induced By TGF-βin Peritoneal Mesothelial Cells

Objective:To transfect with E-cadherin plasmid(pIRES2-EGFP-ECAD)into human peritoneal mesothelium cells immortalized cell lines, and to investigate the effects of E-cad over-express on the epithelial-mesenchymal transition of peritoneal mesothelium cells.Methods:(1) Using E. coli DH5a to amplify the pIRES2-EGFP-ECAD plasmid and blank plasmid, the plasmid was identified by enzyme digestion and RT-PCR.(2) HMrSV5were cultured and divided into3groups:normal control group,pIRES2-EGFP-ECAD transfection group and blank plasmid transfection group, then the pIRES2-EGFP-ECAD and blank plasmids were transfected into HMrSV5using Lipofectamine2000. The morphological changes of cells were observed by inverted microscope; Flow cytometry evaluate were used to assess transfection efficiency. The level of mRNA and protein expression in12h,24h,36h,48h,72h about E-cad were detected by RT-PCR and Western blot.(3) HMrSV5were divided into normal control group and TGF-β stimulation group, Western blot and immunofluorescence were used to investigate the protein expression in12h,24h,36h and48h after TGF-β stimulating.(4) HMrSV5were divided into normal control group and TGF-β stimulation group,pIRES2-EGFP-ECAD transfection+TGF-β stimulation group. The level of mRNA expression in12h,24h,36h,48h about E-cad were detected by RT-PCR. The protein expression of α-SMA、Vimentin were detected by Western blot. Immunofluorescence were used to investigate the E-cad、α-SMA protein expression in12h、24h、36h and48h after transfected+TOF-β stimulating and stimulated alone. To investigate the effect of E-cad over-expression on the EMT of HMrSV5after TGF-β stimulation.Results:(1) The recombinant plasmids are verified by sequencing analysis, identifying with the sequence in genebank.(2) The HMrSV5intracellular E-cad expression is increased after transfected with E-cad for12hours, and peaked on36hours, then declined regularly, there is significant difference compared with the normal control group(p<0.01).(3) The HMrSV5intracellular expression of a-SMA is increased regularly after TGF-Pstimulation in a time-dependent manner and there is significant difference compared with the normal control group(p<0.01).(4) The intracellular expression of a-SMA and Vimentin is decreased,and the expression of E-cad is increased regularly after transfection+TGF-β stimulation compared with stimulation alone, there is significant difference between them(p<0.01).Conclusion:(1) The protein expression of E-cad in HMrSV5cells is increased after transfected with pIRES2-EGFP-ECAD plasmid.(2) Over-expression of E-cad can inhibit the phenotype transition of HMrSV5after stimulated with TGF-β.