miR-129-5p在腹膜间皮细胞转分化中的作用与机制  

Role of miR-129-5p in regulation of epithelial-mesenchymal transition of peritoneal mesothelial cells

作　　者：周循[1] 刘伏友[1] 罗盈[1] 唐丹[1] 阳石坤[1] 孙林[1] 肖力[1] 

机构地区：[1]中南大学湘雅二医院肾内科中南大学肾脏病研究所,长沙410011

出　　处：《中华肾脏病杂志》2015年第4期269-276,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金青年基金（81100541）;国家自然科学基金面上项目（81370832）;中央高校基本科研业务费项目（2012QNZT155）

摘　　要：目的探讨miR-129—5p（microRNA-129—5p）对腹膜间皮细胞上皮．间充质转分化（EMT）的调节作用及可能的分子机制。方法通过新开管腹膜透析（PD）和PD6个月以上两组患者腹透流出液分离培养腹膜间皮细胞，采用Taqman探针实时荧光定量PCR法检测其中miR-129-5p的表达差异。5μg/L TGF—β1刺激腹膜间皮细胞株（HPMCs）0～72h，观察TGF—β1对HPMCs细胞miR-129—5p表达的影响；预转染miR-129-5p前体（pre-miR-129—5p）使HPMCs中miR-129—5p过表达，再给予5μg/L TGF—β1刺激48h，采用实时荧光定量PCR、Western印迹和细胞免疫荧光等方法，观察过表达miR-129-5p对TGF—β1诱导的HPMCs EMT相关蛋白和基因表达的影响。同时，观察5μg/L TGF-β1作用不同时间（0—72h）对HPMCsSmad作用蛋白1（SIP1，miR-129-5p生物预测下游因子）表达的影响以及过表达miR-129—5p对其mRNA和蛋白水平的调节作用。结果PD6个月以上组患者透出液分离培养的腹膜间皮细胞中miR-129—5p的表达显著低于新开管组（P〈0．01）；5μg／L外源性TGF-β1刺激，可使HPMCs的miR-129-5p表达明显降低，呈时间依赖性，pre—miR-129—5p预转染可明显恢复TGF-β1诱导的上皮标志物E—cadherin mRNA和蛋白的表达，并抑制TGF-β1引起的间充质标志物Vimentin mRNA和蛋白的高表达（均P〈0．01）。另外，TGF-β1可呈时间依赖性增加SIP1的mRNA和蛋白表达（均P〈0．01），而pre-miR-129—5p可明显抑制TGF-β1诱导的SIP1蛋白表达（P〈0．01），对其mRNA表达无明显影响（P〉0．05）。结论miR-129—5p可能参与PD状态下腹膜间皮细胞EMT，并可能通过转录后调控SIP1参与该过程，以miR-129-5p／SIP1为靶点，可望为PD腹膜纤维化防治提供一种新途径。Objective To investigate the role of microRNA-129- 5p （miR-129- 5p） in the regulation of epithelial-mesenchymal transition （EMT） of human peritoneal mesothelial cells （HPMCs） isolated from peritoneal dialysate effluents and TGF- β1 induced HPMCs line. Methods The isolated cells were cultured from peritoneal dialysate effluents overnight of 10 patients just started PD and 12 patients with PD over 6 months. Taqman PCR assay was used to determine the expression of miR- 129-5p in the HPMCs. Moreover, the expression of miR- 129-5p in HPMCs induced by 5 μg/L TGF-151 for 0-72 h was also detected by Taqman PCR. HPMCs were pre-transfected with miR-129-5p precursor （pre- mir- 129- 5p） to overexpress miR- 129- 5p, then incubated with TGF- β1 for 48 h, and the expression of EMT associated gene and protein was detected by real-time PCR, Western blotting and immunofluorescence, respectively. Furthermore, the effect of TGF- 131 on the expression of Smad interacting protein-1 （SIP1） and the regulation of pre-miR-129-5p on the SIP1 expression also were investigated. Results MiR-129-5p expression significantly down-regulated in the HPMCs isolated from PD patients over 6 months than from PD start patients（P 〈 0.01）. Similarly, TGF-151 remarkably decreased miR- 129- 5p in HPMCs lines on time- dependent manner （P 〈 0.01）. Pre- mir-129- 5p dramatically restored the expression of epithelial marker E-cadherin, while inhibited the expression of Vimentin, a mesenchymal marker, in HPMCs induced by TGF-β1 （all P 〈 0.01）. In addition, TGF-β1 increased SIP1 expression in HPMCs time dependently, while the high level of SIP1 protein was obviously repressed after transfected of pre-miR-129-5p （P 〈 0.01）, but there was no obvious change of its mRNA expression. Conclusion MiR- 129- 5p modulates EMT formation of HPMCs in PD process, possibly by posttranscriptional inhibition of SIP1. Targeting miR - 129 - 5p/SIP1 may provide a new approach for the prevention and treatment of peritoneal fibrosis durin

关 键 词：腹膜透析 细胞转分化 miR-129-5p Smad作用蛋白-1 

分 类 号：R459.5[医药卫生—治疗学]