lnc-MGC对高糖诱导的人腹膜间皮细胞转分化的影响

Effect of lnc-MGC on the trans-differentiation of peritoneal mesothelial cells induced by high glucose

目的探索长链非编码RNA-MGC(lnc-MGC)在高糖诱导的人腹膜间皮细胞(HPMCs)转分化中的作用。方法使用永生化的HPMCs,设置对照组及高糖组,对照组给予常规培养基培养,高糖组给予含60mmol/L葡萄糖的常规培养液培养72h。采用Real-time PCR检测两组lnc-MGC的表达变化,并采用Real-time PCR及Western bloting检测两组上皮细胞E-钙黏蛋白(E-Cadherin)、α-平滑肌肌动蛋白(α-SMA)、结缔组织生长因子(CTGF)、Ⅰ型胶原蛋白(COL-1)和Ⅲ型胶原蛋白(COL-3)mRNA及蛋白表达的变化。采用慢病毒转染HPMCs,上调和下调lnc-MGC后,分别观察上述指标的变化。结果高糖刺激HPMCs 72h后,Real-time PCR检测结果显示:与对照组相比,高糖组lnc-MGC及α-SMA、CTGF、COL-1、COL-3 mRNA表达水平均明显增加(P<0.05),E-Cadherin mRNA表达水平明显降低(P<0.05);Western blotting检测结果显示其蛋白表达与mRNA表达变化趋势一致,差异均有统计学意义(P<0.05)。此外,Real-time PCR检测结果还显示,与对照组相比,慢病毒转染下调lnc-MGC后,lnc-MGC、α-SMA、CTGF、COL-1、COL-3的表达明显降低(P<0.05),E-Cadherin的表达明显升高(P<0.05);Western blotting检测结果显示其蛋白表达与mRNA表达变化趋势一致,差异均有统计学意义(P<0.05)。而慢病毒转染上调lnc-MGC后,Real-time PCR检测结果显示,与对照组相比,lncMGC、α-SMA、CTGF、COL-1、COL-3的表达增加(P<0.05),E-Cadherin的表达降低(P<0.05);Western blotting检测结果显示其蛋白表达与mRNA表达变化趋势一致,差异均有统计学意义(P<0.05)。采用mi RBase数据库预测所得lncRNA下游靶分子为miRNA126-3p,与对照组相比,高糖刺激后miRNA126-3p表达明显升高(P<0.05),转染下调lnc-MGC的慢病毒后miRNA126-3p表达明显降低(P<0.05),转染上调lnc-MGC的慢病毒后miRNA126-3p表达明显升高(P<0.05)。结论lnc-MGC通过调控miRNA126-3p参与HPMCs的转分化过程,调控lnc-MGC的表达可以改变HPMCs的表型转换,并有可能延缓腹膜纤维化的发展。

Objective To explore the role of long noncoding RNA-MGC（lnc-MGC） on the trans-differentiation of human peritoneal mesothelial cells（HPMCs） induced by high glucose. Methods The immortalized HPMCs were used to establish control group and high glucose group（60 mmol/L） respectively. Cells in control group were cultured with ordinary cell medium, and in high glucose group were stimulated with high glucose medium for 72 h. Changes of lnc-MGC expression in the both groups were measured by RT-PCR, and the changes of mRNA and protein expression of E-Cadherin, α smooth muscle actin（α-SMA）, connective tissue growth factor（CTGF）, type Ⅰ collagen（COL-1） and type Ⅲ collagen（COL-3） in epithelial cells of both groups were measured by RT-PCR and Western blotting. The HPMCs were transfected with lentivirus, and then the changes of the above indexes were observed after up-and down-regulation of lnc-MGC. Results By high glucose stimulation of HPMCs for 72 h, RTPCR results showed that the expressions of lnc-MGC, α-SMA, CTGF, COL-1 and COL-3 mRNA increased obviously（P0.05）, and the expression of E-Cadherin mRNA decreased markedly（P0.05） in high glucose group than in control group; Western-blotting results indicated that the expression of protein was consistent with that of mRNA, and the differences were statistically significant（P0.05）. After lentivirus transfection and down-regulation of lnc-MGC, RT-PCR results showed that the expressions of lnc-MGC, α-SMA, CTGF, COL-1 and COL-3 mRNA decreased obviously（P0.05）, and the expression of E-Cadherin mRNA increased markedly（P0.05） in high glucose group than in control group; Western-blotting results showed that the expression of protein was consistent with that of mRNA, and the differences were statistically significant（P0.05）. After lentivirus transfection and upregulation of lnc-MGC, RT-PCR results showed that the expressions of lnc-MGC, α-SMA, CTGF, COL-1 and COL-3 mRNA increased significantly（P0.05）, and the expression of E-Cadherin mRNA decreased obviously（P0.05） in high glucose group than in control group; Western blotting results showed that the expression of protein was consistent with that of mRNA, and the differences were statistically significant（P0.05）. The downstream target was predicted as miRNA126-3 p, and compared with the control group, the expression of miRNA126-3 p increased（P0.05） after high glucose stimulation, and after transfection with down regulated lnc-MGC lentivirus, the expression of miRNA126-3 p decreased obviously（P0.05）, and transfection with up regulated lnc-MGC lentivirus, the expression of miRNA126-3 p increased obviously（P0.05）. Conclusions lnc-MGC participates in the process of HPMCs transdifferentiation through regulating miRNA126-3 p. Regulation of lnc-MGC expression level may control the phenotype transition of HPMCs, and delay the development of peritoneal fibrosis.