miR-155、miR-15a和miR-181c在糖尿病环境下对人阴茎海绵体血管内皮细胞功能的影响

The expressions of miR-155,miR-15a and miR-181c in human cavernous endothelial cells grown in diabetic-like medium

目的：晚期糖基化终产物AGEs联合高糖（AGE-BSA＋高糖）刺激人阴茎海绵体血管内皮细胞（HCECs）诱导糖尿病环境，观察其对内皮功能相关的miR-155、miR-15a和miR-181c及其靶基因的表达变化影响。方法利用人阴茎海绵体分离鉴定的HCECs，随机分为两组：正常＋BSA组（NC组）、AGE-BSA＋高糖（DM组）。采用蛋白印迹实验检测RAGE蛋白表达确定诱导体外糖尿病环境；采用实时定量PCR来检测miR-155、miR-15a和miR-181c及对应靶基因eNOS、TXNIP、VEGFA、IRS1和KLF6的mRNA表达变化；采用蛋白印迹实验验证靶基因的蛋白表达变化。结果与NC组相比，DM组在AGE-BSA＋高糖刺激下，RAGE基因的mRNA水平和蛋白水平均明显升高；同时观察到miR-155表达明显升高而miR-15a和miR-181c的表达明显降低。miR-155对应的靶基因eNOS的mRNA水平和蛋白水平明显降低；miR-15a对应的靶基因TXNIP的mRNA水平和蛋白水平明显升高，靶基因IRS1的mRNA水平明显升高但蛋白水平变化不明显，靶基因VEGFA的mRNA水平没有显著变化；miR-181c对应的靶基因KLF6的mRNA水平和蛋白表达水平明显升高，而靶基因IRS1的mRNA水平明显升高但蛋白水平变化不明显。结论 HCECs在AGE-BSA＋高糖刺激的糖尿病环境下，miR-155、miR-15a和miR-181c均有显著的表达差异，并负调控它们对应的靶基因eNOS、TXNIP及KLF6发生不同程度的表达变化。结果提示糖尿病环境下的HCECs中多种miRNAs可能参与了糖尿病内皮功能障碍的发生发展过程，其具体的机制有待进一步深入研究。

Objective To investigate the expressions of miR-155, miR-15a and miR-181c and their targeting genes in human cavernous endothelial cells （HCECs） grown in diabetic-like medium.Methods Purified HCECs were divided into two groups：normal group ＋ BSA （NC group）, AGE-BSA ＋ Glucose group （DM group）. The expressions of miR-155, miR-15a and miR-181c and their target genes were detected by real-time PCR. The protein levels of relevant target genes were further validated by western blot.Results Compared with that of NC group, mRNA and protein expression of RAGE were all significantly increased in DM group. The expression of miR-155 in DM group was obviously higher than that in NC group, whereas the expressions of miR-15a and miR-181 were remarkably lower than those in DM group. Compared with that of NC group, mRNA expression of eNOS （miR-155） were decreased significantly in DM group, but mRNA expressions of TXNIP （miR-15a）, IRS1 （miR-15a, miR-181c）, KLF6 （miR-181c） were increased significantly in DM group; No obvious change in VEGFA （miR-15a） expression was found in DM group. Protein expressions of eNOS, TXNIP and KLF6 except IRS1 were all consistent with their mRNA expression.Conclusion Under stimulation of AGE-BSA combined with glucose, miR-155, miR-15a and miR-181c and their target genes in HCECs are differentially expressed, suggesting that a complexity of multiple miRNAs may be involved in regulating the process of endothelial dysfunction. The detailed mechanism needs further more investigation.