The Affect Of E-cadherin Expression And Tumor Growth By Ecombinant Has-mir-373Eukaryotic Exprcssion Plasmid Vector

Objective: The aims of this study was to elucidate whether theexpression of E-cadherin can be affected by the recombinant has-mir-373eukaryotic exprcssion plasmid vector through tests in vitro and in vivo, andto analyze the relationship between the expression of E-cadherin andtumor growth.Methods: According to the has-mir-373sequence in miRBasedatabase, two template DNA sequences were designed. The has-mir-373sequence and a control sequence were synthesized and inserted intopGenesil-1eukaryotic exprcssion plasmid vector. The recombinantedexprcssion plasmids were indentified by double endonuclease digestion andsequencing. The recombinant plasmids were transfected into human lungcancer A549cells by liposome mediated method. and the G418was used toscreene the positive transfected cell clones. The mir-373expression inA549cells was detected by using real-time quantitative polymerase chainreaction(real-time PCR)．The MTT (methyl thiazolyl tetrazolium) and theflow cytometry were used to analyze the cancer cell cycle. RT-PCR andWestern blotting were used to evaluate the levels of E-cadherin mRNA and protein expression，respectively．The expression of E-cadherin in cellswas determined by immunocytochemistry. The mobility capability oftransfected cells were evaluated by using wound healing assay in vitro.The stablely expressing cell lines and cells without trnsfeetion weresubcutaneous1y injected into nude mice．At the end of the experiment，themice were euthanized and tumor weights were recorded．The expressionlevels of mRNA and proteins of E-cadherin in the tumor mass weredetermined by RT-PCR, Western Blotting and immunohistochemicaltechnique．Results: The recombinant vectors were confirmed by doubleendonuclease digestion and sequencing. The fluorescent light was observedunder fluorescence microscope．RT-PCR indicated that the mRNA ofE-cadherin increased, and the Western blotting results also displayed thatmir-373promoted the expression of the E-cadherin protein．Compared withthe control groups, MTT method and wound healing assay demonstratedthat both the growth rate and migration of A549cells transfected with therecombinant has-mir-373eukaryotic exprcssion plasmid was alsodecreased significantly (p<0.001). The differences between the other twocontrol groups were not significant(p>0.05). While flow cytometry showedthat the of cell cycle in three groups was no difference significantly amongthe three groups，respectively. The immunocytochemistry demonstrated asignificant increase of E-cadherin protein levels in the cells transfected with mir-373, but not in the cells of control group cells. The experimentshowed that the expression levels of E-cadherin mRNA and protein wereincreased in experiment group in vivo. The growth of lung cancer A549cells in vivo can be significantly suppressed by the increasing expression ofE-cadherin．Conclusion: Mir-373could increase the expression levels of theE-cadherin and decrease the migration ability of human lung cancer A549cells in in vitro and in vivo.