miR-495-3p对山羊骨骼肌细胞增殖分化的影响

doi:10.16431/j.cnki.1671-7236.2022.04.008

摘要/Abstract

摘要： 【目的】研究miR-495-3p对山羊骨骼肌细胞增殖分化的影响及其在不同组织中的表达情况,为探究miRNA在肌肉发育中的调控机制提供理论基础。【方法】利用生物信息学方法预测miR-495-3p的靶基因;实时荧光定量PCR检测miR-495-3p及其靶基因在心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、腿肌等组织中的表达水平。构建miR-495-3p的过表达(miR-495-3p mimic、miR-495-3p mimic NC)及抑制物(miR-495-3p inhibitor、miR-495-3p inhibitor NC),在山羊骨骼肌细胞汇合度达60%~70%时进行转染,并用含2%马血清的培养基进行诱导分化,用CCK-8检测细胞增殖活力,实时荧光定量PCR检测过表达和干扰效率及增殖分化相关基因配对盒基因(paired-box 7,Pax7)、细胞蛋白周期E(Cyclin E)、肌细胞生成素(myogenin,myoG)、生肌因子5(recombinant myogenic factor 5,Myf5)的表达;构建miR-495-3p靶基因的野生型和突变型载体并转染至293T细胞,用双荧光素酶测定miR-495-3p与其靶基因的结合情况。【结果】生物信息学预测结果表明,miR-495-3p的靶基因为心肌肌动蛋白α1(alpha cardiacactin 1,ACTC1),且miR-495-3p和ACTC1在1和10月龄山羊背最长肌中表达量均差异极显著(P<0.01)。miR-495-3p和ACTC1在心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、腿肌6种组织中均有表达,在背最长肌中表达量较高。细胞转染结果表明,miR-495-3p mimic极显著促进miR-495-3p的表达(P<0.01),miR-495-3p inhibitor极显著抑制miR-495-3p的表达(P<0.01),说明miR-495-3p mimic和miR-495-3p inhibitor可用于后续试验。实时荧光定量PCR结果表明,过表达miR-495-3p促进骨骼肌细胞分化相关标志基因MyoG、Myf5的表达,抑制miR-495-3p则降低MyoG、Myf5基因的表达;过表达、干扰miR-495-3p对Pax7、Cyclin E基因的表达及细胞增殖活力均无显著影响(P>0.05)。双荧光素酶报告结果表明,miR-495-3p与ACTC1基因存在靶向关系。【结论】 miR-495-3p对山羊骨骼肌细胞的增殖无显著影响,但可通过靶向ACTC1基因促进山羊骨骼肌细胞分化。

关键词: 山羊; miR-495-3p; 骨骼肌细胞; 分化

Abstract: 【Objective】 The experiment was conducted to study the effects of miR-495-3p on the proliferation and differentiation of goat skeletal muscle cells and its expression in different tissues,in order to provide a theoretical basis for exploring the regulatory mechanism of miRNA in muscle development.【Method】 Target genes of miR-495-3p were predicted by bioinformatics methods.The expression levels of miR-495-3p and its target genes in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and leg muscle were detected by Real-time quantitative PCR.The overexpression (miR-495-3p mimic,miR-495-3p mimic NC) and inhibitor (miR-495-3p inhibitor,miR-495-3p inhibitor NC) of miR-495-3p were constructed,and when the confluence of goat skeletal muscle cells reached 60%-70%,they were transfected and induced to differentiate with the medium containing 2% horse serum,CCK-8 was used to detect cell proliferation activity,and Real-time quantitative PCR was used to detect the overexpression and interference efficiency,and the expression of proliferation and differentiation related genes paired box 7 (Pax7),Cyclin E,myogenin (MyoG) and myogenic factor 5 (Myf5).The wild-type and mutant vectors of miR-495-3p target gene were constructed and transfected into 293T cells,the double luciferase report was used to verify the binding of miR-495-3p and its target gene.【Result】 Bioinformatics prediction results showed that the target gene of miR-495-3p was alpha cardiocactin 1 (ACTC1),and there were extremely significant differences of miR-495-3p and ACTC1 in longissimus dorsi muscle at the age of 1 and 10 months goats (P<0.01).miR-495-3p and ACTC1 were expressed in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and leg muscle,and the expression was higher in longissimus dorsi muscle.The results of cell transfection showed that miR-495-3p mimic significantly promoted the expression of miR-495-3p (P<0.01),and miR-495-3p inhibitor significantly inhibited the expression of miR-495-3p (P<0.01),indicating that miR-495-3p mimic and miR-495-3p inhibitor could be used in subsequent trials.Real-time quantitative PCR results showed that overexpression of miR-495-3p promoted the expression of skeletal muscle cell differentiation related marker genes MyoG and Myf5,while inhibition of miR-495-3p decreased the expression of MyoG and Myf5 genes.Overexpression and interference of miR-495-3p had no significant effect on the expression of Pax7,Cyclin E genes and cell proliferation activity of skeletal muscle cells (P>0.05).The results of double luciferase report showed that miR-495-3p had a targeting relationship with ACTC1 gene.【Conclusion】 miR-495-3p had no significant effect on the proliferation of goat skeletal muscle cells,but could promote the differentiation of goat skeletal muscle cells by targeting ACTC1 gene.

Key words: goat; miR-495-3p; skeletal muscle cells; differentiation

中图分类号:

S826.8

杨丽丽, 张琼文, 张瑜, 刘笑笑, 陈婷, 江明生. miR-495-3p对山羊骨骼肌细胞增殖分化的影响[J]. 中国畜牧兽医, 2022, 49(4): 1271-1279.

YANG Lili, ZHANG Qiongwen, ZHANG Yu, LIU Xiaoxiao, CHEN Ting, JIANG Mingsheng. Effect of miR-495-3p on Proliferation and Differentiation of Goat Skeletal Muscle Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1271-1279.

参考文献

[1] BRODERSEN P,VOINNET O.Revisiting the principles of microRNA target recognition and mode of action[J].Nature Reviews Molecular Cell Biology,2009,10(2):141-148.
[2] 范永辉.miR-34b通过靶向SYISL调节成肌细胞增殖和分化[D].武汉:华中农业大学,2019. FAN Y H.miR-34b regulates myoblast proliferation and differentiation by targeting SYISL[D].Wuhan:Huazhong Agricultural University,2019.(in Chinese)
[3] GUO Z,MAKI M,DING R,et al.Genome-wide survey of tissue-specific microRNA and transcription factor regulatory networks in 12 tissues[J].Scientific Reports,2014,3(4):5150.
[4] SWEETMAN D,GOLJANEK K,RATHJEN T,et al.Specific requirements of MRFs for the expression of muscle specific microRNAs,miR-1,miR-206 and miR-133[J].Developmental Biology,2008,321(2):491-499.
[5] ZHANG D,LI X,CHEN C,et al.Attenuation of p38-mediated miR-1/133 expression facilitates myoblast proliferation during the early stage of muscle regeneration[J].PLoS One,2012,7(7):e41478.
[6] SAFA A,BAHROUDI Z,SHOOREI H,et al.miR-1:A comprehensive review of its role in normal development and diverse disorders[J].Biomedecine & Pharmacotherapie,2020,132:110903.
[7] 李丹丹.miRNA-101a对山羊骨骼肌卫星细胞增殖与分化调控作用研究[D].成都:四川农业大学,2015. LI D D.Regulation of miRNA-101a on proliferation and differentiation of goat skeletal muscle satellite cells[D].Chengdu:Sichuan Agricultural University,2015.(in Chinese)
[8] XIA Y,ZHOU Y,HAN H,et al.lncRNA NEAT1 facilitates melanoma cell proliferation,migration,and invasion via regulating miR-495-3p and E2F3[J].Journal of Cellular Physiology,2019,234(11):19592-19601.
[9] SUN L,LIU L,YANG J,et al.SATB1 3'-UTR and lncRNA-UCA1 competitively bind to miR-495-3p and together regulate the proliferation and invasion of gastric cancer[J].Journal of Cellular Biochemistry,2018,120(4):6671-6682.
[10] GOOSSENS E,VRIES M,JUKEMA J W,et al.Myostatin inhibits vascular smooth muscle cell proliferation and local 14q32 microRNA expression,but not systemic inflammation or restenosis[J].International Journal of Molecular Sciences,2020,21(10):3508.
[11] CESANA M,CACCHIARELLI D,LEGNINI I,et al.A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA[J].Cell,2011,147(2):358-369.
[12] MA X,WEI D,CHENG G,et al.Bta-miR-130a/b regulates preadipocyte differentiation by targeting PPARG and CYP2U1 in beef cattle[J].Molecular and Cellular Probes,2018,42:10-17.
[13] 孙加节.秦川牛肌肉与脂肪组织发育相关miRNA鉴定及miR-10020调控机制解析[D].杨凌:西北农林科技大学,2015. SUN J J.Identification of miRNA related to muscle and adipose tissue development and analysis of miR-10020 regulatory mechanism in Qinchuan cattle[D].Yangling:Northwest A&F University,2015.(in Chinese).
[14] 郑盼盼.miR-206和miR-486及其相关调节因子在卫星细胞增殖、分化中的变化[D].上海:上海体育学院,2015. ZHENG P P.Changes of miR-206 and miR-486 and their related regulatory factors in the proliferation and differentiation of satellite cells[D].Shanghai:Shanghai University of Physical Education,2015.(in Chinese).
[15] LEE B,SHIN Y J,LEE S H,et al.miR-3074-3p promotes myoblast differentiation by targeting Cav1[J].BMB Reports,2020,53(5):278-283.
[16] KEEL B N,ZAREK C M,KEELE J W,et al.RNA-Seq meta-analysis identifies genes in skeletal muscle associated with gain and intake across a multi-season study of crossbred beef steers[J].BMC Genomics,2018,19(1):430.
[17] BOUTILIER J K,TAYLOR R L,RAM R,et al.Variable cardiac alpha-actin (Actc1) expression in early adult skeletal muscle correlates with promoter methylation[J].Biochimica et Biophysica Acta. Gene Regulatory Mechanisms,2017,1860(10):1025-1036.
[18] LEE J H,KIM S W,HAN J S,et al.Functional analyses of miRNA-146b-5p during myogenic proliferation and differentiation in chicken myoblasts[J].BMC Molecular and Cell Biology,2020,21(1):40.
[19] YE Z J,SHI J,NING Z C,et al.miR-92b-3p inhibits proliferation and migration of C2C12 cells[J].Cell Cycle,2020,19(21):2906-2917.
[20] QIN J,SUN Y,LIU S,et al.microRNA-323-3p promotes myogenesis by targeting Smad2[J].Journal of Cellular Biochemistry,2019,120(11):18751-18761.