lncRNA-NAS对湖羊睾丸间质细胞睾酮分泌的影响及其靶基因鉴定

Effects of lncRNA-NAS on testosterone secretion and identification of target genes in Leydig cells of Hu sheep

[目的]本文旨在探究lncRNA-NAS对湖羊睾丸间质细胞(LC)睾酮分泌的影响。[方法]利用RT-qPCR对湖羊lncRNA-NAS进行组织表达谱分析,针对lncRNA-NAS设计siRNA并将其转染睾丸间质细胞;利用ELISA、Western blot、细胞计数(CCK-8)、RT-qPCR等技术分析干扰lncRNA-NAS对睾丸间质细胞睾酮分泌、细胞增殖和凋亡的影响;在高通量测序基础上对lncRNA-NAS可能作用的靶基因进行分析。[结果]lncRNA-NAS在湖羊心脏、肝、脾、肾、空肠、睾丸组织中均有表达,但其在睾丸中的表达量显著高于其他组织(P<0.05);lncRNA-NAS在睾丸中的表达量随着湖羊年龄增长而呈上升趋势,2周岁(2 Y)表达量显著高于5日龄(5 D)、3月龄(3 M)和9月龄(9 M)(P<0.05),9 M表达量显著高于5 D和3 M(P<0.05),5 D和3 M表达量差异不显著(P>0.05);相比对照组,干扰lncRNA-NAS后,睾丸间质细胞中睾酮分泌水平显著升高(P<0.05),睾酮分泌相关基因和蛋白表达量显著升高(P<0.05),细胞增殖活力增强(P<0.05),细胞凋亡相关基因Bax/Bcl-2 mRNA表达量和蛋白比值均显著降低(P<0.05);分析lncRNA-NAS潜在的9个靶基因,2个为未知基因,针对7个已知基因进行研究,当lncRNA-NAS干扰后,发现只有生殖细胞发育相关基因Nanos3表达水平发生变化,表现为显著升高(P<0.05),提示Nanos3可能为lncRNA-NAS作用的靶基因。[结论]lncRNA-NAS可能通过调控靶基因Nanos3影响湖羊睾丸间质细胞睾酮的分泌。

[Objectives]The paper aimed to explore the effect of lncRNA-NAS on testosterone secretion of Hu sheep Leydig cells(LC).[Methods]The tissue expression of lncRNA-NAS in Hu sheep was detected by RT-qPCR;siRNA transfection was performed in sheep LC,siRNA was designed against lncRNA-NAS and transfected into LC with Lipofectamine 3000,its testosterone secretion was tested by ELISA,Western blot,cell counts(CCK-8),the LC proliferation and apoptosis was examined by RT-qPCR;and the target genes were predicted by lncRNA-NAS on the previous sequencing.[Results]lncRNA-NAS was expressed in the heart,liver,spleen,lung,jejunum and testis of Hu sheep,and the expression level in testis was significantly higher than that in other tissues(P<0.05).The expression level of lncRNA-NAS was an upward trend with the increasing days.The expression level of 2-year-old(2 Y)was significantly higher than 5-day-old(5 D),3-month-old(3 M)and 9-month-old(9 M)(P<0.05).The expression levels of lncRNA-NAS in 9 M were significantly higher than 5 D and 3 M(P<0.05),and there was no significantly difference between 5 D and 3 M(P>0.05).Compared with the control group,the level of testosterone secretion in LC significantly increased(P<0.05),the expression levels of testosterone-related genes and proteins significantly increased(P<0.05),the cell proliferation showed an upward trend(P>0.05),and the mRNA and protein levels of apoptosis-related genes Bax/Bcl-2 significantly reduced(P<0.05).By analyzing 9 target genes of lncRNA-NAS,two of them were unknown genes and subsequent research was on 7 known genes.When lncRNA-NAS interfered,it was found that only the target gene Nanos3 expression level significantly increased(P<0.05),which showed that lncRNA-NAS may regulate the target genes Nanos3.[Conclusions]lncRNA-NAS may regulate target gene Nanos3 to affect testosterone secretion in LC of Hu sheep.