miR-486对绵羊骨骼肌卫星细胞增殖及PI3k-Akt信号通路相关基因表达的影响

王世银 张伟 刘晓娜 杨力伟 邓双义

摘要：【目的】明確miR-486对绵羊骨骼肌卫星细胞增殖及PI3K-Akt信号通路相关基因表达的影响，为揭示miR-486对绵羊骨骼肌发育的调控机制打下基础。【方法】以巴什拜羊1日龄羔羊后肢骨骼肌卫星细胞为研究对象，通过转染miR-486 mimics和miR-486 inhibitor致使绵羊骨骼肌卫星细胞中miR-486水平上调或下调，探究miR-486对骨骼肌卫星细胞增殖速度及PI3K-Akt信号通路中PTEN、GSK3b、PRKCα、Raf-1、MAPK1、PKN1、Casp9和SGK1等8个相关基因表达变化的影响。【结果】空白对照及转染miR-486 mimics、miR-486 mimics negative control和miR-486 inhibitor negative control的绵羊骨骼肌卫星细胞均表现出典型的S形增殖曲线，但各处理间的细胞增殖速度存在明显差异。当绵羊骨骼肌卫星细胞中miR-486水平上调可促使细胞进入快速增殖状态，而miR-486水平下调可使细胞保持静止状态。实时荧光定量PCR检测结果表明，当绵羊骨骼肌卫星细胞中miR-486水平上调时，可引起PTEN、Raf-1和MAPK1基因相对表达量极显著下调（P<0.01，下同），PRKCα和PKN1基因相对表达量极显著上调，SGK1基因相对表达量显著上调（P<0.05）;而GSK3β和Casp9基因相对表达量在不同处理组绵羊骨骼肌卫星细胞间的差异均不显著（P>0.05），可能在维持绵羊骨骼肌卫星细胞基本生命活动中发挥作用。【结论】miR-486通过调控PI3K-Akt信号通路相关基因的表达而参与绵羊骨骼肌卫星细胞生长发育及增殖，为深入研究miR-486对绵羊骨骼肌发育的调控机理及优质肉羊品种培育打下了理论基础。

关键词： 绵羊;miR-486;骨骼肌卫星细胞;PI3K-Akt信号通路;增殖;基因表达

中图分类号：S826.8                             文献标志码： A 文章编号：2095-1191（2021）08-2276-08＼

Effects of miR-486 on proliferation of sheep （Ovis aries） skeletal muscle satellite cells and expression of genes related

to PI3K-Akt signal pathway

WANG Shi-yin， ZHANG Wei*， LIU Xiao-na， YANG Li-wei， DENG Shuang-yi

（Xinjiang Agricultural Professional Technological College， Changji， Xinjiang  831100， China）

Abstract：【Objective】To investigate the effects of miR-486 on proliferation of sheep （Ovis aries） skeletal muscle sa-tellite cell and expression level of genes related to PI3K-Akt signal pathway， and finally lay the foundation for illumina-ting the  regulatory mechanism of miR-486 on development of sheep skeletal muscle. 【Method】Here the skeletal muscle satellite cells of one day-old Bashby sheep were applied， and the proliferation of satellite cells and the expression levels of 8 key genes， PTEN， GSK3b， PRKCα， Raf-1， MAPK1， PKN1， Casp9 and SGK1 in PI3K-Akt signal pathway were detected when the level of miR-486 in cells were up- or down-regulated by transferring into miR-486 mimics and miR-486 inhibitor. 【Result】The result showed that the skeletal muscle satellite cells in blank control and treated by miR-486 mi-mics， miR-486 mimics negative control， and miR-486 inhibitor negative control all performed the typical S-shaped proli-feration curve， but thespeed of proliferation existed great differenceamong treatments. The proliferation of satellite cells was accelerated when the level of miR-486 was up-regulated， conversely when the level of miR-486 was down-regulated and the cells kept rest state. The results of real-time fluorescence quantitative PCR showed that the expression levels of PTEN， Raf-1， and MAPK1 were extremely significantly down-regulated （P<0.01， the same below）， levels of PRKCα and PKN1 were extremely significantly up-regulated， and level of SGK1gene was also significantly up-regulated（P<0.05）， when the level of miR-486 was up-regulated in satellite cells， but the relative expression levels of GSK3β and Casp9 gene did not show significantly different（P>0.05） no matter the level of miR-486 was up-regulated or down-regulated in satellite cells， maybe these two genes played important roles in essential activity of satellite cells. 【Conclusion】miR-486 can affect the proliferation of sheep skeletal muscle satellite cell by regulating the expression of genes in PI3K-Akt signal pathway， and provided a theoretical basis for further investigate the regulation mechanism of miR-486 on skeletal muscle development of sheep and breeding of high-quality mutton sheep breeds.

Key words： sheep; miR-486; skeletal muscle satellite cell; PI3K-Akt signal pathway; proliferation; gene expression

Foundation item： Xinjiang Natural Science Foundation （2017D01A51）

0 引言

【研究意義】提高产肉性能一直是绵羊育种的重要目标。在动物骨骼肌生长发育过程中，从胚胎期成肌细胞的迁移、增殖和分化，到出生后骨骼肌细胞的进一步生长发育及稳态维持，均受一个分子网络的精细调控（Puri and Sartorelli，2000），但目前对于这一网络调控的作用机制知之甚少。……