长链非编码RNA H 19调控小鼠睾丸间质细胞功能的机制

The Study of Mechanism of Mouse Leydig Cell's Function Regulated by Long Noncoding RNA H 19

目的探讨长链非编码RNA H 19对小鼠睾丸间质细胞(LCs)合成分泌睾酮功能的影响及其可能的机制。方法采用原代小鼠LCs和TM3细胞为研究对象,细胞分为二甲基亚砜(DMSO)组、2-硝基丙烷(2-NP)处理组、si control组、si H19组、si Creb1组、Mimic control组、miR-181c-5p mimic组、Inhibitor control组、miR-181c-5p inhibitor组、si H19+Inhibitor control组和si H19+miR-181c-5p inhibitor组共11组。干扰H 19表达后,ELISA法检测其睾酮分泌水平的变化,CCK8法检测LCs增殖变化;生物信息学分析预测各基因的结合位点。RT-qPCR法和Western-blot检测各组小鼠LCs转染后各相关基因及蛋白表达的变化。结果(1)与si control组比较,si H19组LCs培养液睾酮水平及细胞增殖水平均显著下降(72 h后,P<0.01)。(2)各相关基因之间存在结合位点。(3)与DMSO组比较,2-NP组LCs的p-STAT1表达增加、H 19表达明显升高,而miR-181c-5p表达明显降低(P<0.01)。(4)与si control组比较,si H19处理可显著下调LCs的H 19、Creb1、HSD3B1和HSD3B6 RNA表达水平并显著上调miR-181c-5p表达水平(P<0.01),si Creb1处理可显著下调Creb1、HSD3B1和HSD3B6 mRNA表达水平(P<0.01);si H19组和si Creb1组Creb1、3β-HSD蛋白表达均明显降低(P<0.01);与Mimic control组比较,miR-181c-5p mimic组H 19、Creb1、HSD3B1和HSD3B6 mRNA表达明显降低,miR-181c-5p表达水平明显升高且Creb1、3β-HSD蛋白表达显著降低(P<0.01);与Inhibitor control组比较,miR-181c-5p inhibitor组的各相关基因及蛋白表达呈现与miR-181c-5p mimic组作用相反的效应;与si H19+Inhibitor control组比较,si H19+miR-181c-5p inhibitor组H 19表达显著升高,miR-181c-5p表达水平明显降低,Creb1、HSD的mRNA和蛋白表达均明显升高(P<0.01)。结论H 19低表达可通过抑制细胞增殖、miR-181c-5p表达升高、Creb1蛋白表达降低和3β-HSD蛋白表达降低导致LCs合成分泌睾酮能力降低。STAT1/H 19/miR-181c-5p/Creb1/3β-HSD通路可能在LCs功能调控中发挥重要作用。

Objective To explore the effects of long noncoding RNA H 19 on the function of the Leydig cells(LCs)and it's mechanism.Methods Primary mouse LCs and TM3 cells were used as study materials.Cells were divided into 11 groups:Dimethyl sulfoxide(DMSO)group,2-nitropropane(2-NP)treated group,si control group,si H19 group,si Creb1 group,Mimic control group,miR-181c-5p mimic group,Inhibitor control group,miR-181c-5p inhibitor group,si H19+Inhibitor control group and si H19+miR-181c-5p inhibitor group.Testosterone level was measured by ELISA after manipulation of H 19 expression,the proliferation of LCs was measured by CCK8;bioinformatics analysis was performed to predict the binding site of related genes.The expression of related genes and their proteins of LCs in each group after transfection were determined by RT-qPCR and Western\|blot analysis.Results(1)Compared with the si control group,testosterone levels in the culture media and the level of cell proliferation of LCs in si H19 group were decreased significantly(after 72 h,P<0.01).(2)There are binding sites among the related genes.(3)Compared with the DMSO group,expression of H 19 and p-STAT1 were significantly higher and the expression of miR-181c-5p was obvious lower in the LCs of 2-NP group(P<0.01).(4)Compared with the si control group,treatment with si H19 significantly down-regulated the expression of H 19,Creb1,HSD3B1 and HSD3B6 of LCs and significantly up-regulated expression of the miR-181c-5p of LCs(P<0.01).Treatment with si Creb1 significantly down-regulated the expression of Creb1,HSD3B1 and HSD3B6 mRNA(P<0.01).The protein expression of Creb1 and 3β-HSD were significantly decreased in both the si H19 group and the si Creb1 group(P<0.01).Compared with the Mimic control group,The expression of H 19,Creb1,HSD3B1,and HSD3B6 were significantly decreased,the expression of miR-181c-5p was significantly increased and the expression of Creb1 and 3β-HSD protein were significantly decreased in the miR-181c-5p mimic group(P<0.01).Compared with the inhibitor control group, the expression of the related genes and proteins in the miR-181c-5p inhibitor group showed the opposite effect to that of the miR-181c-5p mimic group. Compared with the si H19 + Inhibitor control group, the H 19 expression was significantly increased, the expression of miR-181c-5p was significantly reduced, the mRNA and protein expression of Creb1 and HSD were significantly increased in the si H19 + miR-181c-5p inhibitor group ( P <0.01). Conclusion Low expression of H 19 can reduce the testosterone secretion ability of LCs by inhibiting cell proliferation and increased expression of miR-181c-5p , decreased expression of Creb1 protein, and decreased 3 β -HSD protein expression. The STAT1 / H 19/ miR-181c-5p /Creb1/3 β -HSD pathway may play an important role in the regulation of LCs function.