CircCEP85L对牛肌肉干细胞增殖、成肌分化的调控

doi:10.3864/j.issn.0578-1752.2023.18.014

中国农业科学 ›› 2023, Vol. 56 ›› Issue (18): 3670-3681.doi: 10.3864/j.issn.0578-1752.2023.18.014

CircCEP85L对牛肌肉干细胞增殖、成肌分化的调控

韦瑶¹(), 张瑞门¹, 安强¹, 王乐怡¹, 张永旺¹, 邹超霞¹, 张尔康¹, 莫碧云², 石德顺¹, 杨素芳¹, 邓彦飞¹(), 韦英明³()

¹ 广西大学动物科学技术学院/广西畜禽繁育与疾病防控重点实验室，南宁 530004
² 钦州市动物疫病预防控制中心，广西钦州 535099
³ 广西大学农牧产业发展研究院，南宁 530004

收稿日期:2022-11-01 接受日期:2023-05-16 出版日期:2023-09-16 发布日期:2023-09-21
通信作者:
邓彦飞，E-mail：yanfei-dun@163.com
韦英明，E-mail：dkywym@163.com。
联系方式: 韦瑶，E-mail：1612985655@qq.com。
基金资助:
国家现代农业产业技术体系广西创新团队建设项目(nycytxgxcxtd-09-01); 国家重点研发计划(2021YFD110010); 广西自然科学基金重点项目(2021JJD130097); 巴马县人才科技计划项目(202101263)

CircCEP85L Regulates the Proliferation and Myogenic Differentiation of Bovine MuSCs

WEI Yao¹(), ZHANG RuiMen¹, AN Qiang¹, WANG LeYi¹, ZHANG YongWang¹, ZOU ChaoXia¹, ZHANG ErKang¹, MO BiYun², SHI DeShun¹, YANG SuFang¹, DENG YanFei¹(), WEI YingMing³()

¹ State Key Laborator for Conservation and Untilization of Subtropical Agro-bioresources /College of Animal Science and Technology, Guangxi University, Nanning 530004
² Qinzhou Center for Animal Disease Control and Prevention, Qinzhou 535099, Guangxi
³ Research Institute of Agriculture and Animal Husbandry Industry Development, Guangxi University, Nanning 530004

Received:2022-11-01 Accepted:2023-05-16 Published:2023-09-16 Online:2023-09-21

摘要/Abstract

摘要：

【目的】目前，越来越多的研究证明环状RNA（circRNA）在牛肌肉发育过程中扮演着重要的角色，但其分子调控机理尚未完善。通过挖掘与牛肌肉发育相关的circRNA的作用机制，为进一步阐明其调控牛肌肉发育的分子机制奠定基础。【方法】以前期分析的增殖期(GM)与成肌分化期（DM）黄牛肌肉干细胞（MuSCs）的RNA-seq测序结果为基础，筛选出显著差异表达的circRNA，circCEP85L。采集新鲜黄牛胎牛的心脏、肝脏、脾脏、肺脏、肾脏、肌肉、肠和胃的组织样本，分离培养黄牛肌肉干细胞并诱导成肌分化，收集黄牛体外培养的GM与DM细胞，分别提取RNA并反转录为cDNA。通过实时荧光定量PCR（quantitative real time PCR, qRT-PCR）检测circCEP85L在不同组织及不同细胞状态的表达规律。同时，设计特异性引物扩增circCEP85L全长，构建过表达载体p-circCEP85L，质粒转染MuSCs后收集过表达circCEP85L细胞样本。以过表达质粒pCD5-ciR细胞样本为对照，采用qRT-PCR、流式细胞术、Western Blot及免疫荧光等技术检测过表达circCEP85L对黄牛MuSCs增殖、凋亡及成肌分化的影响。【结果】PCR电泳结果证明circCEP85L环化位点真实存在。CircCEP85L在多种组织中表达，并在DM期的表达量显著高于GM期（P<0.001）；为了进一步探究对circCEP85L黄牛MuSCs的影响。将过表达载体p-circCEP85L与对照载体pCD5-ciR转染体外培养的黄牛MuSCs，继续培养24 h之后，EdU结果表明过表达circCEP85L能显著降低EdU阳性细胞比例（P<0.001）；流式周期检测结果表明，过表达circCEP85L增加了G0/G1期细胞比例，显著减少S期细胞比例（P<0.001）；流式凋亡结果表明，过表达circCEP85L显著抑制MuSCs的凋亡率（P<0.05）。QRT-PCR及Western blot检测黄牛MuSCs增殖及凋亡相关基因的表达情况,结果表明过表达circCEP85L后显著降低了黄牛MuSCs增殖及凋亡相关基因的mRNA表达水平（P<0.001）,凋亡蛋白BAX的表达量也显著降低（P<0.01）。此外，为了检测过表达circCEP85L对黄牛MuSCs成肌分化的影响，在转染24h后更换分化培养基诱导细胞分化，Western blot及免疫荧光结果显示过表达circCEP85L后显著促进分化标志基因MyH6的表达水平（P<0.001），细胞融合形成肌管的数量和大小均显著高于对照组。【结论】研究表明，circCEP85L通过抑制黄牛MuSCs增殖和凋亡、促进细胞成肌分化，从而影响黄牛骨骼肌的生长发育过程，为circRNA调控黄牛骨骼肌的生长发育机制研究奠定基础。

关键词: circCEP85L, 肌肉干细胞, 细胞增殖, 成肌分化

Abstract:

【Objective】At present, studies have proved that circRNA plays important roles in the development of bovine muscle, but its molecular regulation mechanism remain elusive. Screening circRNAs related to bovine muscle development can lay a foundation for further elucidating the molecular mechanism of bovine muscle development.【Method】In this study, RNA-seq sequencing results of proliferating (GM) and myogenic differentiation (DM) yellow bovine muscle stem cells (MuSCs) analyzed in the previous stage were used to screen for significantly differentially expressed circRNA, circCEP85L. Tissue samples of heart, liver, spleen, lung, kidney, muscle, intestine and stomach were collected aseptically from fresh yellow fetal calves, and yellow muscle stem cells were isolated and cultured and induced into myogenic differentiation. GM and DM cells cultured in vitro from yellow calves were collected, RNA was extracted and reverse transcribed into cDNA, respectively. Quantitative real time PCR (qRT-PCR) was used to detect the expression of circCEP85L in different tissues and different cell states. Meanwhile, specific primers were designed to amplify the full length of circCEP85L, and the overexpression vector p-circCEP85L was constructed. The plasmid was transfected into MuSCs and overexpressed circCEP85L cell samples were collected. Using overexpression plasmid pCD5-ciR cell samples as control, qRT-PCR, flow cytometry, Western Blot and immunofluorescence were used to detect the effects of overexpression of circCEP85L on proliferation, apoptosis and myogenic differentiation of yellow bovine MuSCs.【Result】The electrophoresis of PCR product proved the existence of circCEP85L. CircCEP85L was expressed in various tissues, and the expression level in DM stage was significantly higher than that in GM stage (P<0.001). To further investigate the effect on circCEP85L scallion MuSCs. The overexpression vector p-circCEP85L was transfected with the control vector pCD5-ciR in vitro cultured yellow bovine MuSCs and the EdU results showed that overexpression of circCEP85L significantly reduced the proportion of EdU positive cells (P<0.001) after continuing the culture for 24 h. Flow cycle analysis showed that overexpression of circCEP85L increased the proportion of cells in G0/G1 phase and significantly decreased the proportion of cells in S phase (P<0.001). Flow cytometry showed that overexpression of circCEP85L significantly inhibited the apoptosis rate of MuSCs (P<0.05). qRT-PCR and western blot were used to detect the expression of proliferation and apoptosis-related genes in MuSCs, respectively. The results showed that overexpression of circCEP85L significantly reduced the mRNA expression levels of proliferation and apoptosis-related genes in bovine MuSCs (P<0.001), and the expression of apoptotic protein BAX was also significantly reduced (P<0.01). In addition, in order to detect the effect of circCEP85L overexpression on myogenic differentiation of cattle MuSCs, the differentiation medium was replaced 24 hours after transfection to induce cell differentiation. Western blot and immunofluorescence results showed that overexpression of circCEP85L significantly promoted the expression level of differentiation marker gene MyH6 (P<0.001), and the number and size of myotubes formed by cell fusion were significantly higher than those of the control group.【Conclusion】The results of this study indicate that circCEP85L affects the growth and development process of skeletal muscle in cattle by inhibiting the proliferation and apoptosis of MuSCs and promoting myogenic differentiation of cells, which is expected to be a key circRNA for subsequent mechanistic studies to regulate the growth and development process of skeletal muscle in cattle.

Key words: circCEP85L, muscle stem cells, cell proliferation, myogenic differentiation

韦瑶, 张瑞门, 安强, 王乐怡, 张永旺, 邹超霞, 张尔康, 莫碧云, 石德顺, 杨素芳, 邓彦飞, 韦英明. CircCEP85L对牛肌肉干细胞增殖、成肌分化的调控[J]. 中国农业科学, 2023, 56(18): 3670-3681.

WEI Yao, ZHANG RuiMen, AN Qiang, WANG LeYi, ZHANG YongWang, ZOU ChaoXia, ZHANG ErKang, MO BiYun, SHI DeShun, YANG SuFang, DENG YanFei, WEI YingMing. CircCEP85L Regulates the Proliferation and Myogenic Differentiation of Bovine MuSCs[J]. Scientia Agricultura Sinica, 2023, 56(18): 3670-3681.

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基因名 Gene	序列号 Accession	引物序列（5′-3′） Primer sequence	产物大小 Product length（bp）
PCNA	NM_001034494	F: TCCAGAACAAGAGTATAGC R: TACAACAGCATCTCCAAT	94
CDK2	NM_001014934	F: TCTTTGCTGAGATGGTGACCC R: CATCTTCATCCAGGGGAGGC	201
CyclinD1	NM_001046273	F: AACCTCCTCAACGACCGAGT R: CGGTCCAGGTAGTTCATGGC	220
BAX	NM_173894	F: TGCAGAGGATGATCGCAGCTGT R: CCAATGTCCAGCCCATCATGGT	198
Caspase9	NM_001205504	F: GCTCCAGGCACGACATTCTT R: CAAAGGAAGGGCCTGACTCC	191
β-Actin	NM_173979	F: GCCCTGGCACCCAGCACAAT R: GGAGGGGCCGGACTCATCGT	188
circCEP85L		F: GAGACAACAAAATGAAAATGCC R: CTGGGAATGGGATGCTGACT	142

CircCEP85L对牛肌肉干细胞增殖、成肌分化的调控

CircCEP85L Regulates the Proliferation and Myogenic Differentiation of Bovine MuSCs

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