miRNA-432在成肌细胞增殖分化中的作用及机制研究

发布时间：2019-02-18 22:49

【摘要】：骨骼肌是机体运动的主要执行者及代谢活动的主要调控因子,其生长发育主要受一系列转录因子及表观遗传调控因子的调控。mircoRNA长度为18-24个核苷酸,通过与靶基因mRNA的3'非翻译区(3'UTR)结合,在转录后水平调控靶基因的表达。研究表明,mircoRNA在骨骼肌的发育过程中发挥着重要的作用。通过mircoRNA测序分析,我们发现miR-432-5p在35日龄仔猪的背最长肌中的表达水平显著高于在287日龄成年荣昌猪的表达水平,差异高达7倍;且miR-432在小鼠、人和猪等不同物种中高度保守。基于此,本研究以C2C12成肌细胞系为研究材料,旨在探索miR-432在骨骼肌成肌细胞增殖与分化中的作用及调控机制。本实验通过对成肌细胞转染miR-432模拟物(mimics)或miR-432抑制物(inhibitor),研究了miR-432对成肌细胞增殖和分化的作用;运用生物信息学和双荧光素酶报告试验,预测并验证miR-432的靶基因,进一步探索了miR-432调控成肌细胞增殖与分化的分子机制。获得如下主要结果:1.miR-432抑制成肌细胞的增殖在成肌细胞中过表达miR-432可以显著下调增殖相关基因Cyclin E,PCNA和CDK2mRNA与蛋白表达水平,并显著下调S期的细胞数及总的细胞数目;相反,抑制miR-432后促进了成肌细胞的增殖过程。2.miR-432抑制成肌细胞的分化过表达miR-432后可以显著抑制分化关键转录因子MyoG和标志基因MyHC mRNA和蛋白表达水平;通过免疫荧光染色技术,发现miR-432能显著抑制肌管的形成并下调分化指数。3.miR-432直接靶定E2F3和P55PIK构建野生型及突变型双荧光素酶报告载体后,miR-432能显著抑制E2F3和P55PIK野生型荧光素酶活性,而不影响其突变型荧光活性。不同浓度的mimics处理成肌细胞后,E2F3和P55PIK的mRNA表达呈现浓度依赖式下降。4.miR-432通过抑制P55PIK介导的PI3K/Akt/mTOR通路抑制成肌细胞的分化过表达miR-432能显著下调分化期的p-Akt/t-Akt及p-mTOR/t-mTOR比值,但不影响增殖期的p-Akt/t-Akt及p-mTOR/t-mTOR比值。当加入低浓度的胰岛素(insulin)后,miR-432对分化的抑制作用被削弱,而加入高浓度的insulin后能基本消除miR-432对分化的抑制作用。本研究基于miRNA测序,解析了我国西部地区肉质优良地方品种荣昌猪的肌肉发育特性,发现miRNA-432-5p在35日龄荣昌猪背最长肌的表达显著高于287日龄,进一步研究表明该miRNA靶向E2F3和P55PIK,并通过PI3K/AKT/mTOR信号通路抑制成肌过程。研究结果为探索我国地方猪品种肉质优良特性的非编码RNA调控提供了新的候选基因,也为猪优良肉品质性状选育提供了科学依据。
[Abstract]:Skeletal muscle is the main executor of body movement and the main regulator of metabolic activity. The growth and development of skeletal muscle are mainly regulated by a series of transcription factors and epigenetic regulators. The length of mircoRNA is 18-24 nucleotides. The expression of target gene was regulated at post-transcriptional level by binding to 3 'untranslated region (3'UTR) of target gene mRNA. MircoRNA plays an important role in skeletal muscle development. By mircoRNA sequencing, we found that the expression level of miR-432-5p in the longissimus dorsi muscle of 35-day-old piglets was significantly higher than that in 287-day-old Rongchang pigs, and the difference was as high as 7 times. MiR-432 is highly conserved in different species, such as mice, humans and pigs. Therefore, the purpose of this study was to explore the role of miR-432 in the proliferation and differentiation of skeletal myoblasts and its regulatory mechanism. The effects of miR-432 on the proliferation and differentiation of myoblasts were studied by transfection of miR-432 mimics (mimics) or miR-432 inhibitor (inhibitor), into myoblasts. The target genes of miR-432 were predicted and verified by bioinformatics and double luciferase report test. The molecular mechanism of miR-432 regulating the proliferation and differentiation of myoblasts was further explored. The main results were as follows: 1.miR-432 inhibited the proliferation of myoblasts and overexpression of miR-432 in myoblasts could significantly down-regulate the expression levels of proliferation-associated genes Cyclin, CDK2mRNA and protein. The number of S phase cells and the total number of cells were significantly down-regulated. On the contrary, inhibition of miR-432 promoted the proliferation of myoblasts. 2.miR-432 inhibited the over-expression of miR-432 in myoblasts and significantly inhibited the expression of MyoG, the marker gene MyHC mRNA and protein. Immunofluorescence staining showed that miR-432 could significantly inhibit the formation of myotubes and down-regulate the differentiation index. 3.miR-432 directly targeted E2F3 and P55PIK to construct wild-type and mutant double-luciferase report vectors. MiR-432 significantly inhibited the activity of E2F3 and P55PIK wild-type luciferase, but did not affect its mutant fluorescence activity. The myoblasts were treated with different concentrations of mimics. The expression of mRNA in E2F3 and P55PIK decreased in a concentration-dependent manner. 4.miR-432 could significantly down-regulate p-Akt/t-Akt and P in differentiation phase by inhibiting P55PIK mediated PI3K/Akt/mTOR pathway to inhibit myoblast differentiation and overexpression of miR-432. -mTOR/t-mTOR ratio, However, the ratio of p-Akt/t-Akt and p-mTOR/t-mTOR in proliferative stage was not affected. When low concentration of insulin (insulin) was added, the inhibitory effect of miR-432 on differentiation was weakened, but the inhibitory effect of miR-432 on differentiation was basically eliminated by adding high concentration of insulin. Based on miRNA sequencing, the muscle development characteristics of Rongchang pig with good meat quality in western China were analyzed. It was found that the expression of miRNA-432-5p in the longissimus dorsi muscle of 35 day old Rongchang pig was significantly higher than that of 287-day old Rongchang pig. Further studies showed that the miRNA targeted E2F3 and P55 Pi, and inhibited myogenesis through PI3K/AKT/mTOR signaling pathway. The results provided a new candidate gene for exploring the non-coding RNA regulation of the fine meat quality of local pig breeds in China, and provided scientific basis for the breeding of good meat quality traits of pigs.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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