小鼠miR-487b-3p对C2C12增殖和分化调控作用的研究

doi:10.11843/j.issn.0366-6964.2018.11.008

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (11): 2371-2383.doi: 10.11843/j.issn.0366-6964.2018.11.008

小鼠miR-487b-3p对C2C12增殖和分化调控作用的研究

凌笑笑¹, 唐朋^1,2, 贾聪俊¹, 梁春年¹, 吴晓云¹, 褚敏¹, 阎萍^1*

1. 中国农业科学院兰州畜牧与兽药研究所, 甘肃省牦牛繁育重点实验室, 兰州 730050;
2. 甘肃农业大学动物科技学院, 兰州 730050

收稿日期:2018-04-02 出版日期:2018-11-23 发布日期:2018-11-23
通讯作者: 阎萍,研究员,主要从事动物遗传育种与繁殖研究,E-mail:pingyanlz@163.com
作者简介:凌笑笑(1991-),女,山西临汾人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:1391545241@qq.com
基金资助:
现代肉牛牦牛产业技术体系（CARS-37）；中国农业科学院创新工程项目（CAAS-ASTIP-2014-LIHPS-01）

Study on the Regulation of Mouse miR-487b-3p in C2C12 Proliferation and Differentiation

LING Xiao-xiao¹, TANG Peng^1,2, JIA Cong-jun¹, LIANG Chun-nian¹, WU Xiao-yun¹, CHU Min¹, YAN Ping^1*

1. Key Laboratory of Yak Breeding Engineering of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agricultural Sciences, Lanzhou 730050, China;
2. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730050, China

Received:2018-04-02 Online:2018-11-23 Published:2018-11-23

摘要/Abstract

摘要：

旨在研究miR-487b-3p对小鼠C2C12细胞增殖与分化的具体调控机制。本研究以小鼠成肌细胞系（C2C12）为研究对象，利用qRT-PCR检测miR-487b-3p在小鼠各组织和C2C12细胞增殖与分化过程中的表达情况；通过转染miR-487b-3p mimics和miR-487b-3p inhibitor后，利用qRT-PCR检测转染效率，并采用qRT-PCR和Western blotting检测增殖标记基因PCNA和分化标记基因MYOD、MYOG和MYHC的表达差异；利用生物信息学软件对miR-487b-3p靶基因进行预测，利用qRT-PCR检测小鼠C2C12细胞增殖与分化过程中PITX2的表达情况，并检测过表达（抑制） miR-487b-3p后PITX2的表达差异，通过双荧光素酶报告系统验证miR-487b-3p和PITX2的靶标关系。结果表明，miR-487b-3p在小鼠骨骼肌中相对表达最高，在C2C12细胞增殖与分化的过程中miR-487b-3p的表达量整体上呈现上升趋势，推测miR-487b-3p参与调控骨骼肌发育。过表达miR-487b-3p后，极显著上调miR-487b-3p的表达（P<0.01），在转录和蛋白水平均极显著上调PCNA的表达（P<0.01），显著下调MYHC、MYOD和MYOG基因的表达（P<0.05），同时显著下调MYHC （P<0.05）、MYOD （P<0.01）和MYOG蛋白（P<0.05）的表达；而抑制miR-487b-3p后，极显著下调miR-487b-3p的表达（P<0.01），显著下调PCNA基因的表达（P<0.05），极显著下调PCNA蛋白的表达（P<0.01），显著上调MYHC、MYOD和MYOG蛋白的表达（P<0.05），上调MYHC、MYOD和MYOG基因的表达，但未达显著水平（P>0.05）。在C2C12细胞增殖和分化过程中，PITX2与miR-487b-3p表达趋势相反；过表达miR-487b-3p能极显著下调PITX2 mRNA的表达（P<0.01），相反，抑制miR-487b-3p的表达则极显著上调PITX2 mRNA的表达（P<0.01）；双荧光素酶报告系统验证PITX2是miR-487b-3p的直接靶基因。综上表明，miR-487b-3p通过靶向PITX2促进C2C12细胞增殖而抑制其分化。

Abstract:

This study aimed to explore the regulatory mechanism of miR-487b-3p on proliferation and differentiation of mouse C2C12 cells. In this study,mouse myoblast cell line(C2C12) was used as the research material,qRT-PCR was used to detect the expression of miR-487b-3p in various tissues and in C2C12 cells during proliferation and differentiation in mouse. After transfection of miR-487b-3p mimics and miR-487b-3p inhibitor,the transfection efficiency was detected by qRT-PCR,and the expression of proliferation marker gene PCNA and differentiation marker genes MYOD, MYOG and MYHC were detected by qRT-PCR and Western blotting. The bioinformatics softwares were used to predict target genes of miR-487b-3p,and the expression differences of PITX2 were detected by qRT-PCR in the proliferation and differentiation of mouse C2C12 cells,and the expression of PITX2 after over-expression(inhibition-expression) of miR-487b-3p was detected,and target relationship between miR-487b-3p and PITX2 was verified via the Dual-luciferase reporter assay. The results showed that the expression of miR-487b-3p was the highest in mouse skeletal muscle, and its expression level increased during the proliferation and differentiation of C2C12 cells. It was speculated that miR-487b-3p participated in the regulation of skeletal muscle development. Over-expression of miR-487b-3p significantly up-regulated the expression of miR-487b-3p(P<0.01), and significantly up-regulated the expression of PCNA at transcription and protein levels(P<0.01),and significantly down-regulated MYHC, MYOD and MYOG genes expression(P<0.05), simultaneously, significantly down-regulated the expression of MYHC(P<0.05),MYOD(P<0.01) and MYOG(P<0.05) proteins; After inhibiting the expression of miR-487b-3p, the expression of miR-487b-3p was significantly down-regulated(P<0.01),and the expression of PCNA gene was significantly down-regulated(P<0.05),and the expression of PCNA protein was significantly down-regulated(P<0.01),MYHC,MYOD and MYOG proteins expression were significantly up-regulated(P<0.05). The expression levels of MYHC,MYOD and MYOG genes increased,but the change was not significant(P>0.05). In the process of proliferation and differentiation of C2C12 cells, the expression trends of miR-487b-3p and PITX2 was reversed;Over-expression of miR-487b-3p could significantly down-regulate PITX2 mRNA expression(P<0.01),on the contrary,inhibition of miR-487b-3p could significantly up-regulate PITX2 mRNA expression(P<0.01). Dual-luciferase reporter assay verified that PITX2 was a direct target gene of miR-487b-3p. In summary,miR-487b-3p promotes the proliferation and inhibits the differentiation of C2C12 cells by targeting PITX2 in mouse.

中图分类号:

S811.2

凌笑笑, 唐朋, 贾聪俊, 梁春年, 吴晓云, 褚敏, 阎萍. 小鼠miR-487b-3p对C2C12增殖和分化调控作用的研究[J]. 畜牧兽医学报, 2018, 49(11): 2371-2383.

LING Xiao-xiao, TANG Peng, JIA Cong-jun, LIANG Chun-nian, WU Xiao-yun, CHU Min, YAN Ping. Study on the Regulation of Mouse miR-487b-3p in C2C12 Proliferation and Differentiation[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2371-2383.

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小鼠miR-487b-3p对C2C12增殖和分化调控作用的研究

Study on the Regulation of Mouse miR-487b-3p in C2C12 Proliferation and Differentiation

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