摘要
目的探讨膜-细胞骨架联接蛋白Ezrin对肾癌细胞株786-0细胞增殖、侵袭和迁移能力的影响。方法以ezrin为靶基因,以Pgenesil-1质粒为载体设计和构建重组体,设计2条发夹式RNA(shRNA),合成后克隆入载体Pgenesil-1,脂质体Lipofectamine2000转染进786-0细胞,观察RNA干扰Ezrin后肾癌细胞株786-0细胞增殖及侵袭能力的改变。结果稳定转染细胞荧光定量PCR结果显示shRNA-ezrin1对ezrin mRNA的表达量抑制率66.33%,稳定转染细胞Ezrin下调率达93%,shRNA干扰后786-0细胞增殖活性减弱,GO/G1时段明显延长(P<0.01),PI缩短(P<0.01),细胞凋亡率增加(P<0.01);细胞黏附力减弱,细胞运动、穿孔能力减弱(P<0.01)。结论 RNA干扰Ezrin可有效抑制786-0细胞增殖活性、降低细胞的侵袭力,Ezrin在人肾癌细胞增殖、侵袭中发挥重要作用。
Objective To determine the effect of membrane-cytoskelet on linker Ezrin in the invasion and migration of human renal carcinoma cells line 786-0.Methods With ezrin as the target gene,2 pairs of shRNA was designed and chemically synthesized,and then inserted into plasmid pGenesil-shRNA.The recombinant plasmid was transformed into DH5α.The recombinant plasmid was extracted in middle quantity and transfected into 786-0 cells line by Lipofectamine 2000.Ezrin expression in 786-0 cells transfected by shRNA recombinant plasmid was measured by qRT-PCR and Western blotting.786-0 cells were transfected by effective shRNA plasmid and cultured in 1640 media containing G418（800 μg/ml）.qRT-PCR and Western blotting were applied respectively to detect the Ezrin mRNA and protein expression to evaluate the blocking effect of shRNA-Ezrin.MTT assay and flow cytometry was used to further assess cell proliferation and apoptosis.Migration and invasion was determined by cell adhesion analysis and chemotactic motion assay.Results Our designed shRNA knocked down 93% Ezrin in 786-0 cells which had a stable expression of Ezrin small hairpin RNAs.After transfected with p-GeneSil-l-ezrin,the capability of 786-0 cells of proliferation,invasion and adhesion were decreased dramatically.Compared with the cells treated with shRNA-ezrin1,the percentage of G0/G1 cells was significantly higher than those of untransfected cells and 786-0cells transfected with shRNA-hk（P0.01）,and proliferation index（PI） was decreased in the cells treated with shRNA-ezrin1 compared with those of untransfected cells and 786-0cells transfected with shRNA-hk（P0.01）.Heterotypic cell adhesion analysis showed that the numbers of adhesive cell in 786-0 cells transfected with shRNA-ezrin1 were less than the 786-0 cells untransfected and transfected with shRNA-hk（P0.01）.Chemotactic motion was decreased in 786-0 cells transfected with shRNA-ezrin1.Conclusion Ezrin plays an important role in cell migration and invasion of human renal carcinoma cells,which may be regarded as a promising target for tumor gene therapy.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第20期2136-2140,共5页
Journal of Third Military Medical University
基金
兰州军区2010年A类医药卫生课题(Acwsloja14)~~
