人源MRTF-A重组腺病毒构建及其对平滑肌细胞小窝蛋白表达的影响

Construction of MRTF-A recombinant adenovirus vector and its effect on the expression of caveolae protein of smooth muscle cells

目的探讨心肌素相关转录因子(MRTF)-A对膀胱平滑肌细胞小窝蛋白(caveolin)及小窝关联蛋白(cavin)表达的影响,有望为膀胱出口梗阻所致膀胱壁病理改变提供新的治疗靶点。方法首先从人源MRTF-A表达质粒pcDNA3.1-MRTF-A-3flag扩增MRTF-A基因序列并构建重组穿梭质粒AdEasy-h-MRTF-A,将成功构建的MRTF-A穿梭质粒与骨架质粒pHBAd-BHG共转染HEK293细胞通过AdEasy系统实现重组,获得HBAd-h-MRTF-A毒种后继续扩增、纯化及滴度测定。然后将正常构建的HBAd-h-MRTF-A重组腺病毒(MRTF-A组)与空载腺病毒(空载组)分别感染人膀胱平滑肌细胞,RT-qPCR检测两组细胞caveolin-1(CAV1)及cavin-1(CAVIN1)的mRNA表达水平,Western blot检测两组细胞MRTF-A、caveolin-1及cavin-1的蛋白表达水平。结果经测序分析验证重组腺病毒HBAd-h-MRTF-A含有正确的MRTF-A基因序列,其在人膀胱平滑肌细胞中过表达的蛋白在大约145 kDa水平可被MRTF-A特异性抗体所识别。与空载组相比,MRTF-A组CAV1的mRNA表达水平上调1.90±0.26倍;CAVIN1的mRNA表达水平上调1.77±0.12倍,差异均具有统计学意义(P<0.05)。同样,与空载组相比,MRTF-A组的caveolin-1蛋白表达水平上调1.84±0.31倍;cavin-1的蛋白表达水平上调2.14±0.28倍,差异均具有统计学意义(P<0.05)。结论成功构建人源MRTF-A重组腺病毒载体HBAd-h-MRTF-A;且过表达MRTF-A可促进膀胱平滑肌细胞caveolin-1及cavin-1的表达;MRTF-A可能成为调控平滑肌细胞小窝结构的重要靶点。

Objective To investigate the regulatory effect of myocardin-related transcription factor A(MRTF-A)on the expression of caveolin and cavin in bladder smooth muscle cells.Methods Human MRTF-A gene sequence was amplified from MRTF-A expressing plasmid pcDNA3.1-MRTF-A-3flag and was inserted to the polyclonal sites of adenovirus shuttle plasmid to generate a recombinant shuttle plasmid AdEasy-h-MRTF-A.HEK293 cells were co-transfected with the AdEasy-h-MRTF-A plasmid and the framework plasmids,and the original generation of recombinant adenovirus HBAd-h-MRTF-A was generated using the AdEasy recombinant system,followed by virus amplification,purification,and titer-measurement.Human bladder smooth muscle cells(HBSMCs)were transduced with HBAd-h-MRTF-A(MRTF-A group)or empty vectorsas a control(empty-vector group).mRNA expression levels of caveolin-1(CAV1)and cavin-1(CAVIN1)were detected by RT-qPCR and the protein expression of MRTF-A,caveolin-1 and cavin-1 were measured by western blot.Results The sequencing analysis showed that the recombinant adenovirus vector HBAd-h-MRTF-A contains a correct DNA sequence of MRTF-A.The protein overexpressed by the recombinant vector in human bladder smooth muscle cells can be regulated by MRTF-A specific antibody at the molecular weight of about 145 kDa.Compared to the empty-vector group,the mRNA level of CAV1 raised up to 1.90±0.26 folds(P<0.05),the mRNA level of CAVIN1 raised up to 1.77±0.12 folds(P<0.05),For the protein level,caveolin-1 of MRTF-A group increased to 1.84±0.31 folds(P<0.05)and cavin-1 climbed to 2.14±0.28 folds(P<0.05),compared tothe empty-vector group respectively.Conclusions The recombinant adenovirus vector HBAd-h-MRTF-A is successfully constructed,and up-regulation of caveolin-1 and cavin-1 can be induced by overexpression of MRTF-A,which might be a potential target to regulate the caveolae ultrastructure of smooth muscle cells.