miR-660在牛骨骼肌生长发育过程中表达谱分析及相关功能研究

发布时间：2018-02-12 13:33

本文关键词： mi R-660 ARHGEF12 骨骼肌细胞增殖分化　出处：《江苏师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：microRNA是当今生命科学中研究最为普遍的一类非编码RNA,现有相关文献资料揭示,microRNA在动物骨骼肌发育阶段扮演着重要的调控角色。在本研究中,借助实验室先前有关秦川牛骨骼肌microRNA高通量测序结果,初步筛选出时空差异表达的microRNA,采用RT-qPCR技术获取秦川牛不同发育时期(胎牛期、犊牛期),不同组织(腿肌、心脏、肝脏、脾脏,肺、肾脏、胃、肠)的microRNA表达谱,并加以整理分析,最终选择miR-660为实验对象,研究其对骨骼肌发育的影响。本研究通过miR-660 mimics或miR-660 inhibitor在小鼠成肌细胞系C2C12中过表达或干扰miR-660,并诱导C2C12增殖及分化,利用RT-qPCR,Westerrn Blot在mRNA及蛋白水平上测定相关增殖分化标志基因的表达,探究miR-660对骨骼肌增殖分化的影响。双荧光素酶报告基因系统确认了ARHGEF12是miR-660的靶基因,此后通过RT-qPCR,Westerrn Blot检测,在mRNA及蛋白水平上进一步证实miR-660与ARHGEF12间的靶向关系。此外,本实验还对靶基因ARHGEF12参与Rho A/Rho A kinase信号通路的可能性进行了探讨。本研究取得的主要结果如下:(1)相关序列保守性分析显示成熟miR-660以及其靶基因ARHGEF12 3’UTR上的microRNA结合位点在人、牛等不同物种间具有高度保守性。(2)miR-660在秦川牛不同发育阶段(胎牛期、犊牛期),不同组织(腿肌、心脏、肝脏、脾脏、肺、肾脏、胃、肠)内普遍表达,并且miR-660在胎牛及成年牛骨骼肌中的表达量呈现出显著的差异(P0.01),表明miR-660属于非特异性肌肉相关microRNA。(3)将miR-660 mimics,negative control(NC),miR-660 inhibitors或anti-negative control(anti-NC)转染至C2C12细胞,过表达或抑制miR-660。利用RT-qPCR,Westerrn Blot技术验证过表达或抑制miR-660对C2C12细胞增殖分化的影响。结果表明miR-660促进成肌细胞的增殖,抑制分化。(4)成功构建了ARHGEF12、CDH13、ETV1、GNAS、HIF1A、SDC1、TPM4这7个靶基因及其突变体荧光素酶报告基因载体。双荧光素酶报告基因系统确认了ARHGEF12是miR-660的靶基因。RT-qPCR、Westerrn Blot检测过表达或干扰miR-660对ARHGEF12表达量的影响,结果进一步证实ARHGEF12是miR-660的靶基因。(5)通过研究miR-660与Rho A/Rho A kinase信号通路的联系,推测miR-660可能通过靶向ARHGEF12影响骨骼肌的分化。
[Abstract]:MicroRNA is one of the most common noncoding RNAs in the life sciences. The current literature shows that microRNAs play an important role in the development of animal skeletal muscle. Based on the results of high throughput sequencing of microRNA in the skeletal muscle of Qinchuan cattle, the spatio-temporal differentially expressed microRNAs were preliminarily screened. The RT-qPCR technique was used to obtain different tissues (leg muscle, heart, liver) of Qinchuan cattle at different developmental stages (fetal cattle stage, calf stage, calf stage), and different tissues (leg muscle, heart, liver). MicroRNA expression profiles of spleen, lung, kidney, stomach and intestine were analyzed, and miR-660 was selected as experimental object. In this study, miR-660 mimics or miR-660 inhibitor were used to overexpression or interfere with miR-660 in mouse myoblast cell line C2C12, and to induce proliferation and differentiation of C2C12. In order to explore the effect of miR-660 on skeletal muscle proliferation and differentiation, RT-qPCR Westerrn Blot was used to detect the expression of related proliferative and differentiation marker genes at the mRNA and protein levels. The double luciferase reporter gene system confirmed that ARHGEF12 was the target gene of miR-660, and then detected by RT-qPCRG Westerrn Blot. The targeting relationship between miR-660 and ARHGEF12 was further confirmed at the level of mRNA and protein. The possibility of the target gene ARHGEF12 participating in the Rho A / Rho A kinase signaling pathway was also investigated. The main results obtained in this study are as follows: 1) the analysis of the conserved sequence shows that the mature miR-660 and the microRNA binding site on its target gene ARHGEF12 3 UTR are human. Among different species such as cattle, there is a high degree of conserved expression in Qinchuan cattle at different developmental stages (fetal cattle, calves, leg muscle, heart, liver, spleen, lung, kidney, stomach, intestine), and in different tissues (leg muscle, heart, liver, spleen, lung, kidney, stomach, intestine). There was a significant difference in the expression of miR-660 between fetal and adult bovine skeletal muscle, indicating that miR-660 belongs to non-specific muscle-related microRNA.3. miR-660 mimicsnegative control inhibitors was transfected into C2C12 cells by transfection of miR-660 mimicsnegative control inhibitors or anti-negative control-anti-NC12 cells. Overexpression or inhibition of miR-660. the effects of overexpression or inhibition of miR-660 on the proliferation and differentiation of C2C12 cells were examined by RT-qPCR- Westerrn Blot technique. The results showed that miR-660 promoted the proliferation of myoblasts. The seven target genes and their mutant luciferase reporter gene vectors, ARHGEF12, CDH13, ETV1, GNASHIF1, TPM4, and its mutant luciferase reporter gene, were successfully constructed. The double luciferase reporter gene system confirmed that ARHGEF12 is the target gene of miR-660. RT-qPCRWesterrn Blot was used to detect the effect of overexpression or interference miR-660 on the expression of ARHGEF12. Results further confirmed that ARHGEF12 is the target gene of miR-660. By studying the relationship between miR-660 and Rho A / Rho A kinase signaling pathway, we speculated that miR-660 might affect skeletal muscle differentiation by targeting ARHGEF12.
【学位授予单位】：江苏师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823;Q78

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