miR-665靶向BCL 2L11调控武安山羊成肌细胞增殖  

miR-665 Targeting BCL 2L11 Regulates the Myoblasts Proliferation of Wu’an Goat

作　　者：冯婧[1,2,3] 盛辉 张效生 郭晓飞[1,2,3] 姚大为 李玉鹏 陈龙宾[1,2,3] 张金龙 FENG Jing;SHENG Hui;ZHANG Xiaosheng;GUO Xiaofei;YAO Dawei;LI Yupeng;CHEN Longbin;ZHANG Jinlong(Institute of Animal Science and Veterinary,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China;Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology,Tianjin 300381,China;Tianjin Engineering Research Center of Animal Healthy Farming,Tianjin 300381,China)

机构地区：[1]天津市农业科学院畜牧兽医研究所,天津300381 [2]天津市畜禽分子育种与生物技术重点实验室,天津300381 [3]天津市畜禽健康养殖工程技术中心,天津300381

出　　处：《畜牧兽医学报》2025年第2期582-590,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：天津市科技计划项目(23YFZCSN00170,23ZYCGSN00090);天津市自然科学基金(23JCYBJC00260);天津市农业科学院种业创新项目(2024ZYCX012)。

摘　　要：旨在利用分子生物学方法探究miR-665靶向调控BCL 2L11对山羊成肌细胞增殖的影响。本研究选取1月龄和9月龄的健康山公羊各3只,各年龄段体重均接近且饲养环境相同。本试验通过RT-qPCR分析BCL 2L11在武安山羊不同组织中的表达情况;利用RT-qPCR检测了不同月龄武安山羊背最长肌组织中BCL 2L11和miRNA-665的表达情况;构建了BCL 2L113′UTR野生型和突变型载体,以及miR-665 mimics和对照组,将其共转染到HEK293T细胞,通过使用双荧光素酶报告基因检测系统鉴定了miR-665与BCL 2L11存在靶向性关系。在山羊成肌细胞转染miR-665 mimic及阴性对照,采用RT-qPCR检测了细胞凋亡标志因子XIAP和Fas表达水平,同时利用EdU试验分析miR-665过表达对山羊成肌细胞增殖的影响。结果表明,BCL 2L11在背最长肌组织中高表达;miR-665和BCL 2L11在9月龄和1月龄山羊背最长肌组织中表达情况存在差异,miR-665在1月龄的表达量高于9月龄,而BCL2L11则相反,两者存在负调控现象;通过双荧光素酶报告分析显示,当转染过表达miR-665 mimic后,显著抑制了BCL 2L11荧光素酶的活性,同时靶基因BCL 2L11的mRNA表达水平也明显降低。为进一步验证miR-665的功能,转染miR-665 mimic后发现细胞凋亡标志基因XIAP与Fas的表达量也显著降低;EdU试验分析显示,过表达miR-665后促进了成肌细胞的增殖。综上所述,在山羊背最长肌组织中miR-665作为BCL 2L11的调控元件,能够显著抑制山羊成肌细胞中BCL 2L11的表达水平,当miR-665过表达后,能够促进成肌细胞的增殖情况。This study aimed to use molecular biological methods to explore the effect of miR-665 targeting BCL 2L11 on the myoblasts proliferation of goat.Three healthy male Wu’an goats aged 1 month and 9 months each,with similar body weights and feeding environments for each age group was selected.The expression of BCL 2L11 and miRNA-665 in the longest dorsal muscle tissue of Wu′an goats at different ages was detected using RT-qPCR;The expression of BCL 2L11 in different tissue of Wu′an goats was also detected by RT-qPCR;Wild-type and mutant vectors of BCL 2L113′UTR,as well as miR-665 mimics and control groups,were constructed and cotransfected into HEK293T cells.The targeting relationship between miR-665 and BCL 2L11 was identified using a dual luciferase reporter gene detection system.The goat myoblasts were transfected with miR-665 mimic and negative control,and the expression levels of apoptosis markers factors XIAP and Fas were detected by RT-qPCR,and the effect of miR-665 overexpression on the proliferation of goat myoblasts was analyzed by EdU test simultaneously.The results showed that BCL 2L11 was highly expressed in the longissimus dorsi muscle tissue;There were differences in the expression of miR-665 and BCL 2L11 in the longissimus dorsi muscle tissue of goats aged 9-months and 1-months,and the expression of miR-665 at 1-month was higher than that at 9-month,while the expression of BCL 2L11 was opposite,and the two were negative regulated;The dual luciferase report analysis showed that,after transfecting overexpressed miR-665 mimic,the luciferase activity of BCL 2L11 was significantly inhibited,and the mRNA expression level of the target gene BCL 2L11 was also significantly reduced.In order to further verify the function of miR-665,the expression of apoptosis marker genes XIAP and Fas was also significantly reduced after transfection of miR-665 mimic;EdU test analysis showed that the proliferation of myoblasts increased significantly after overexpression of miR-665.To sum up,miR-665 as a regulatory elem

关 键 词：miR-665 BCL 2L11 肌肉 武安山羊 成肌细胞 

分 类 号：S827.2[农业科学—畜牧学]