高糖诱导腹膜间皮细胞发生上皮间质转化中microRNA的异常变化

目的:定腹膜间皮细胞在高糖引起上皮间质转化(Epithelial-mesenchymal transition,EMT)过程中microRNA的表达差异。方法:常规培养PMC细胞,利用高糖培养液刺激诱导腹膜间皮细胞发生EMT,倒置显微镜观察各组细胞形态学变化,实时定量PCR检测EMT标志基因变化,以此确定高糖诱导EMT的发生。利用特异茎环结构的引物合成microRNA的cDNA,实时定量PCR检测重要microRNA的表达变化。结果:高糖培养液培养腹膜间皮细胞48hr后,细胞形态呈梭形改变,同时EMT标记基因E-cadherin表达明显减低(P0.01),Vimentin表达显著升高(P0.01),说明高糖诱导腹膜间皮细胞发生了EMT。利用microRNA特异的茎环结构引物,实时定量PCR检测结果发现高糖刺激后miR-193a的表达明显上调(P0.01);miR-15a和let-7e的表达明显降低(P0.01);miR-16和miR-21的表达无明显变化(P0.05),同时检测发现高糖刺激后miR-193a的表达水平随刺激时间表达升高。结论:异常变化的microRNA可能对高糖诱导的腹膜间皮细胞EMT具有重要调控作用。

The Abnormal Expression Level of MicroRNA in Rpithelial-MesenchymalTransition of Eritoneal Mesothelial Cells Induced by High Glucose

Objective:To determine the expression level of microRNA in the process of epithelial-mesenchymal transition (EMT)of peritoneal mesothelial cells induced by high glucose.Methods:The peritoneal mesothelial cells were cultured routinely by M199medium with 10%fetal bovine serum, and the EMT was induced by D-glucose stimulation. The occurrence of EMT was determined bythe change of cell morphology and EMT marker genes expression levels. Cell morphology change was observed by inverted microscope,and expression levels of EMT marker genes were determined by real-time PCR. The expression levels of microRNA were detected byreal-time PCR with microRNA-specific stem-loop structure primer.Results:The peritoneal mesothelial cells were changed to fusiformcellmorphology following high-glucose medium stimulated 48 hr, and the EMT marker genes were changed significantly, such as thedecreased of E-cadherin and increased of Vimentin (P<0.01 respectively). These result proved the EMT had been induced byhigh-glucose. Applied real-time PCR with microRNA -specific stem-loop structure primer, miR-193a was detected increased notably(P<0.01), and miR-15a and let-7e was increased (P<0.01), while miR-16 and miR-21 had no significant change (P>0.05). Importantly theiincrease of miR-193a was positive correlation with stimulus duration.Conclusion:It is indicated that the abnormal expression ofMicroRNA in rpithelial-Mesenchymal could influence the transition of eritoneal mesothelial cells which was induced by high glucose