miR-665靶向BCL2L11调控武安山羊成肌细胞增殖

doi:10.11843/j.issn.0366-6964.2025.02.010

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (2): 582-590.doi: 10.11843/j.issn.0366-6964.2025.02.010

miR-665靶向BCL2L11调控武安山羊成肌细胞增殖

冯婧¹^,²^,³(), 盛辉¹^,²^,³, 张效生¹^,²^,³, 郭晓飞¹^,²^,³, 姚大为¹^,²^,³, 李玉鹏¹^,²^,³, 陈龙宾¹^,²^,³, 张金龙¹^,²^,³^,*()

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

收稿日期:2024-08-09 出版日期:2025-02-23 发布日期:2025-02-26
通讯作者: 张金龙 E-mail:fengjing_2024@126.com;jlzhang1010@163.com
作者简介:冯婧(1986-)，女，黑龙江哈尔滨人，助理研究员，主要从事营养与繁殖调控研究，E-mail: fengjing_2024@126.com
基金资助:
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)

miR-665 Targeting BCL2L11 Regulates the Myoblasts Proliferation of Wu'an Goat

FENG Jing¹^,²^,³(), SHENG Hui¹^,²^,³, ZHANG Xiaosheng¹^,²^,³, GUO Xiaofei¹^,²^,³, YAO Dawei¹^,²^,³, LI Yupeng¹^,²^,³, CHEN Longbin¹^,²^,³, ZHANG Jinlong¹^,²^,³^,*()

1. Institute of Animal Science and Veterinary, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China
2. Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology, Tianjin 300381, China
3. Tianjin Engineering Research Center of Animal Healthy Farming, Tianjin 300381, China

Received:2024-08-09 Online:2025-02-23 Published:2025-02-26
Contact: ZHANG Jinlong E-mail:fengjing_2024@126.com;jlzhang1010@163.com

摘要/Abstract

摘要：

旨在利用分子生物学方法探究miR-665靶向调控BCL2L11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BCL2L11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BCL2L11和miRNA-665的表达情况；构建了BCL2L11 3'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BCL2L11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，BCL2L11在背最长肌组织中高表达；miR-665和BCL2L11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BCL2L11荧光素酶的活性，同时靶基因BCL2L11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BCL2L11的调控元件，能够显著抑制山羊成肌细胞中BCL2L11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。

关键词: miR-665, BCL2L11, 肌肉, 武安山羊, 成肌细胞

Abstract:

This study aimed to use molecular biological methods to explore the effect of miR-665 targeting BCL2L11 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 BCL2L11 and miRNA-665 in the longest dorsal muscle tissue of Wu'an goats at different ages was detected using RT-qPCR; The expression of BCL2L11 in different tissue of Wu'an goats was also detected by RT-qPCR; Wild-type and mutant vectors of BCL2L11 3'UTR, as well as miR-665 mimics and control groups, were constructed and cotransfected into HEK293T cells. The targeting relationship between miR-665 and BCL2L11 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 BCL2L11 was highly expressed in the longissimus dorsi muscle tissue; There were differences in the expression of miR-665 and BCL2L11 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 BCL2L11 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 BCL2L11 was significantly inhibited, and the mRNA expression level of the target gene BCL2L11 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 element of BCL2L11 in goat longissimus dorsi muscle tissue, could significantly inhibit the expression level of BCL2L11 in goat myoblasts, when miR-665 was overexpressed, it could promote the proliferation of myoblasts.

Key words: miR-665, BCL2L11, muscle, Wu'an goat, myoblast

中图分类号:

S827.2

冯婧, 盛辉, 张效生, 郭晓飞, 姚大为, 李玉鹏, 陈龙宾, 张金龙. miR-665靶向BCL2L11调控武安山羊成肌细胞增殖[J]. 畜牧兽医学报, 2025, 56(2): 582-590.

FENG Jing, SHENG Hui, ZHANG Xiaosheng, GUO Xiaofei, YAO Dawei, LI Yupeng, CHEN Longbin, ZHANG Jinlong. miR-665 Targeting BCL2L11 Regulates the Myoblasts Proliferation of Wu'an Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 582-590.

图/表 8

表 1

图 1

图 2

图 3

图 4

图 5

图 6

图 7

参考文献 31

1	HENKEL J , SAIF R , JAGANNATHAN V , et al. Selection signatures in goats reveal copy number variants underlying breed-defining coat color phenotypes[J]. PLoS Genet, 2019, 15 (12): e1008536. doi: 10.1371/journal.pgen.1008536
2	TORRES-HERNÁNDEZ G , MALDONADO-JÁQUEZ J A , GRANADOS-RIVERA L D , et al. Status quo of genetic improvement in local goats: a review[J]. Arch Anim Breed, 2022, 65 (2): 207- 221. doi: 10.5194/aab-65-207-2022
3	张乐超, 刘月琴, 段春辉, 等. 7个地方山羊品种遗传多样性及遗传结构分析[J]. 生物技术通报, 2020, 36 (6): 183- 190.
	ZHANG L C , LIU Y Q , DUAN C H , et al. Analysis of genetic diversity and genetic structure in 7 local goat breeds[J]. Biotechnology Bulletin, 2020, 36 (6): 183- 190.
4	ARIAS J E R , BUSSKAMP V . Challenges in microRNAs' targetome prediction and validation[J]. Neural Regener Res, 2019, 14 (10): 1672- 1677. doi: 10.4103/1673-5374.257514
5	YANG D D , WAN X P , DENNIS A T , et al. MicroRNA biophysically modulates cardiac action potential by direct binding to ion channel[J]. Circulation, 2021, 143 (16): 1597- 1613. doi: 10.1161/CIRCULATIONAHA.120.050098
6	屈文燕. 以miRNAs为中介基于深度学习预测lncRNA-蛋白质相互作用[D]. 北京: 北京工业大学, 2023.
	QU W Y. LPI-MAM: predicting lncRNA-protein interactions with miRNAs as mediators based on deep learning[D]. Beijing: Beijing University of Technology, 2023. (in Chinese)
7	李潇凡, 耿丹丹, 毕瑜林, 等. miRNA的非经典作用机制研究进展[J]. 生物技术通报, 2022, 38 (12): 1- 10.
	LI X F , GENG D D , BI Y L , et al. Research progress in unconventional miRNA functions[J]. Biotechnology Bulletin, 2022, 38 (12): 1- 10.
8	LAI L J , LI H Y , FENG Q , et al. Multi-factor mediated functional modules identify novel classification of ulcerative colitis and functional gene panel[J]. Sci Rep, 2021, 11 (1): 5669. doi: 10.1038/s41598-021-85000-3
9	RANI V , SENGAR R S . Biogenesis and Mechanisms of microRNA-mediated gene regulation[J]. Biotechnol Bioeng, 2022, 119 (3): 685- 692. doi: 10.1002/bit.28029
10	HU X Y , XU S N , CHEN Y B , et al. Depletion of Ars2 inhibits cell proliferation and leukemogenesis in acute myeloid leukemia by modulating the miR-6734-3p/p27 axis[J]. Leukemia, 2019, 33 (5): 1090- 1101. doi: 10.1038/s41375-018-0301-z
11	FU J J , LIU J , ZOU X , et al. Transcriptome analysis of mRNA and miRNA in the development of LeiZhou goat muscles[J]. Sci Rep, 2024, 14 (1): 9858. doi: 10.1038/s41598-024-60521-9
12	MARTINEZ B , PEPLOW P V . MicroRNA expression in animal models of amyotrophic lateral sclerosis and potential therapeutic approaches[J]. Neural Regener Res, 2022, 17 (4): 728- 740. doi: 10.4103/1673-5374.322431
13	MCCORMICK R , GOLJANEK-WHYSALL K . MicroRNA dysregulation in aging and pathologies of the skeletal muscle[J]. Int Rev Cell Mol Biol, 2017, 334, 265- 308.
14	HU Y C , YANG C , YANG S K , et al. RETRACTED ARTICLE: miR-665 promotes hepatocellular carcinoma cell migration, invasion, and proliferation by decreasing Hippo signaling through targeting PTPRB[J]. Cell Death Dis, 2018, 9 (10): 954. doi: 10.1038/s41419-018-0978-y
15	CONCANNON C G , TUFFY L P , WEISOVÁ P , et al. AMP kinase-mediated activation of the BH3-only protein Bim couples energy depletion to stress-induced apoptosis[J]. J Cell Biol, 2010, 189 (1): 83- 94. doi: 10.1083/jcb.200909166
16	刘玉芳, 陈玉林, 周祖阳, 等. miR-221-3p靶向BCL2L11调控小尾寒羊卵泡颗粒细胞凋亡[J]. 中国农业科学, 2022, 55 (9): 1868- 1876.
	LIU Y F , CHEN Y L , ZHOU Z Y , et al. miR-221-3p regulates ovarian granulosa cells apoptosis by targeting BCL2L11 in Small-Tail Han sheep[J]. Scientia Agricultura Sinica, 2022, 55 (9): 1868- 1876.
17	KULUS M , KRANC W , SUJKA-KORDOWSKA P , et al. The processes of cellular growth, aging, and programmed cell death are involved in lifespan of ovarian granulosa cells during short-term IVC- study based on animal model[J]. Theriogenology, 2020, 148, 76- 88. doi: 10.1016/j.theriogenology.2020.02.044
18	TRIVEDI P , STEELE C D , AU F K C , et al. Mitotic tethering enables inheritance of shattered micronuclear chromosomes[J]. Nature, 2023, 618 (7967): 1049- 1056. doi: 10.1038/s41586-023-06216-z
19	TAO J , ZENG Y P , DAI B , et al. Excess PrP^C inhibits muscle cell differentiation via miRNA-enhanced liquid-liquid phase separation implicated in myopathy[J]. Nat Commun, 2023, 14 (1): 8131. doi: 10.1038/s41467-023-43826-7
20	SONG Y , LI M M , LEI S J , et al. Silk sericin patches delivering miRNA-29-enriched extracellular vesicles-decorated myoblasts (SPEED) enhances regeneration and functional repair after severe skeletal muscle injury[J]. Biomaterials, 2022, 287, 121630. doi: 10.1016/j.biomaterials.2022.121630
21	ARÁNEGA A E , LOZANO-VELASCO E , RODRIGUEZ-OUTEIRIÑO L , et al. MiRNAs and muscle regeneration: therapeutic targets in Duchenne muscular dystrophy[J]. Int J Mol Sci, 2021, 22 (8): 4236. doi: 10.3390/ijms22084236
22	DMITRIEV P , BARAT A , POLESSKAYA A , et al. Simultaneous miRNA and mRNA transcriptome profiling of human myoblasts reveals a novel set of myogenic differentiation-associated miRNAs and their target genes[J]. BMC Genomics, 2013, 14, 265. doi: 10.1186/1471-2164-14-265
23	LAMON S , ZACHAREWICZ E , BUTCHART L C , et al. MicroRNA expression patterns in post-natal mouse skeletal muscle development[J]. BMC Genomics, 2017, 18 (1): 52. doi: 10.1186/s12864-016-3399-2
24	RAO P K , KUMAR R M , FARKHONDEH M , et al. Myogenic factors that regulate expression of muscle-specific microRNAs[J]. Proc Natl Acad Sci U S A, 2006, 103 (23): 8721- 8726. doi: 10.1073/pnas.0602831103
25	杨琛. miRNA-1对C2C12细胞衰老及肌肉特异性miRNA表达的影响研究[D]. 武汉: 华中师范大学, 2019.
	YANG C. Effects of MiRNA-1 on senescence and the expression of muscle-specific MicroRNAs of C2C12 myotubes[D]. Wuhan: Central China Normal University, 2019. (in Chinese)
26	CLOP A , MARCQ F , TAKEDA H , et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep[J]. Nat Genet, 2006, 38 (7): 813- 818. doi: 10.1038/ng1810
27	VAN ROOIJ E , QUIAT D , JOHNSON B A , et al. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance[J]. Dev Cell, 2009, 17 (5): 662- 673. doi: 10.1016/j.devcel.2009.10.013
28	SHEN J Y , HAO Z Y , LUO Y Z , et al. Deep small RNA sequencing reveals important miRNAs related to muscle development and intramuscular fat deposition in Longissimus dorsi muscle from different goat breeds[J]. Front Vet Sci, 2022, 9, 911166. doi: 10.3389/fvets.2022.911166
29	GUO Y C , PENG X P , CHENG R , et al. Long non-coding RNA-X-inactive specific transcript inhibits cell viability, and induces apoptosis through the microRNA-30c-5p/Bcl2-like protein 11 signaling axis in human granulosa-like tumor cells[J]. Bioengineered, 2022, 13 (6): 14107- 14117. doi: 10.1080/21655979.2022.2080366
30	KUDRYASHOVA T V , GONCHAROV D A , PENA A , et al. HIPPO-integrin-linked kinase cross-talk controls self-sustaining proliferation and survival in pulmonary hypertension[J]. Am J Respir Crit Care Med, 2016, 194 (7): 866- 877. doi: 10.1164/rccm.201510-2003OC
31	杨丽丽, 张琼文, 张瑜, 等. 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 L L , ZHANG Q W , ZHANG Y , et al. Effect of miR-495-3p on proliferation and differentiation of goat skeletal muscle cells[J]. China Animal Husbandry & Veterrinary Medicine, 2022, 49 (4): 1271- 1279.

[1]	吴少强, 刘雨帆, 韦义荣, 黄艳娜, 蒋钦杨. 白藜芦醇通过PROX1/SIRT1信号通路调控山羊肌纤维类型转化的机制研究[J]. 畜牧兽医学报, 2025, 56(1): 201-212.
[2]	贾宇航, 郭良富, 张茹楠, 赵阿勇, 刘玉芳, 储明星. miR-127调控绵羊骨骼肌细胞增殖分化及其转录因子PAX3筛选[J]. 畜牧兽医学报, 2024, 55(9): 3864-3875.
[3]	娄明, 罗昊玉, 牟芳, 李辉, 王宁. 鸡胰岛素信号通路的研究进展[J]. 畜牧兽医学报, 2024, 55(8): 3288-3296.
[4]	王子岩, 王亚慧, 吴天弋, 高晨, 杜振伟, 葛菲, 张晓贝, 赵文轩, 张路培, 高会江, 董焕声, 李俊雅. INTS11通过介导CDK2和CYCLIND1的转录促进牛成肌细胞增殖[J]. 畜牧兽医学报, 2024, 55(7): 2927-2939.
[5]	谢兵红, 刘一帆, 薛夫光, 单艳菊, 屠云洁, 姬改革, 巨晓军, 束婧婷, 吴红翔. 缺氧对鸡成肌细胞肌纤维类型转化作用的机制探究[J]. 畜牧兽医学报, 2024, 55(6): 2397-2408.
[6]	苏传琛, 焦静娅, 王永帅, 罗朋娜, 昌兴海, 黄艳群. 导入25%洛岛红鸡血缘对河南斗鸡相关指标的影响[J]. 畜牧兽医学报, 2024, 55(3): 1007-1018.
[7]	毛晓宇, 杜嘉伟, 汤嘉玉, 潘金海, 蒋蕾, 孙小磊, 昝林森, 王洪宝. 干扰和过表达CHRNG对牛成肌细胞增殖分化的影响[J]. 畜牧兽医学报, 2024, 55(10): 4360-4376.
[8]	苗舒, 安济山, 王祚, 肖定福, 兰欣怡, 刘磊, 沈维军, 万发春. 亮氨酸通过PI3K-AKT信号通路促进牛成肌细胞的增殖[J]. 畜牧兽医学报, 2024, 55(1): 142-152.
[9]	王开明, 喻宗岗, 徐雪莉, 艾妮妮, 李昕瞳, 何俊, 陶灯, 张硕, 马海明, 张跃博. 干扰Mtmr3基因对C2C12细胞增殖与分化的影响[J]. 畜牧兽医学报, 2023, 54(4): 1478-1489.
[10]	庞立川, 单艳菊, 刘一帆, 章明, 甘达峰, 屠云洁, 姬改革, 巨晓军, 束婧婷, 邹剑敏. METTL16在鸡不同类型肌肉中的表达规律及其对肌肉功能的调控作用[J]. 畜牧兽医学报, 2023, 54(2): 545-553.
[11]	孙金魁, 许厚强, 石鹏飞, 阮涌. 关岭牛MEF2A基因干扰载体构建及其转染对成肌细胞的影响[J]. 畜牧兽医学报, 2023, 54(2): 584-595.
[12]	孙严金, 薛亚男, 仲涛, 王林杰, 李利, 张红平, 占思远. HuR的功能及其对肌肉生长发育的调控作用[J]. 畜牧兽医学报, 2022, 53(5): 1345-1353.
[13]	李洁, 赵瑞鹏, 陈楚雯, 杨朝武, 吴锦波, 李志雄. 不同鸡种胚胎腿肌差异表达mRNA和lncRNA的筛选及其竞争性调控网络的构建[J]. 畜牧兽医学报, 2022, 53(12): 4207-4220.
[14]	马天文, 吕良钰, 于跃, 贾丽娜, 陈鸿, 汤继浪, 赵明超, 王新宇, 张建涛, 高利. 齐墩果酸抑制骨关节炎大鼠肌肉氧化损伤及软骨下骨异常骨重建[J]. 畜牧兽医学报, 2022, 53(11): 4081-4088.
[15]	王瑞秀, 陈中卫, 刘强, 庄苏. 合生素对樱桃谷肉鸭肌肉品质、机体抗氧化性能和免疫功能的影响[J]. 畜牧兽医学报, 2021, 52(9): 2522-2533.

miR-665靶向BCL2L11调控武安山羊成肌细胞增殖

miR-665 Targeting BCL2L11 Regulates the Myoblasts Proliferation of Wu'an Goat

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 31

相关文章 15

编辑推荐

Metrics

本文评价

引物名称 Primer	引物序列(5′→3′)Primer sequence	产物长度/bp Products length	退火温度/℃ Tm
BCL2L11	F: AACTCTATGGGAACAACGCAGCAG	98	60
BCL2L11	R: CAGAACCACACCAGCCACAGTC	98	60
XIAP	F: ATGGCAGATTACGAAGCACGGATC	147	60
XIAP	R: CCAATCATTTAGCCCTCCTCCACAG	147	60
FAS	F: CGGAAGAATGGTATGGAGGAAGCC	88	60
FAS	R: GGAGCAGTTGGACCTTCTGTTCAG	88	60
RPL-19	F: ATCGCCAATGCCAACTC	154	60
RPL-19	R: CCTTTCGCTTACCTATACC	154	60