Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (5): 1502-1513.DOI: 10.1016/j.jia.2022.08.116

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收稿日期:2022-01-05 接受日期:2022-06-18 出版日期:2023-05-20 发布日期:2022-06-18

MicroRNA transcriptome of skeletal muscle during yak development reveals miR-652 regulates myoblasts differentiation and survival by targeting ISL1

ZHOU Xue-lan^{1, 2}, GUO Xian^{1, 2}, LIANG Chun-nian^{1, 2}, CHU Min^{1, 2}, WU Xiao-yun^{1, 2#}, YAN Ping^{1, 2#}

¹Key Laboratory of Yak Breeding Engineering of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, P.R.China
²Key Laboratory of Animal Genetics and Breeding on Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Lanzhou 730050, P.R.China

Received:2022-01-05 Accepted:2022-06-18 Online:2023-05-20 Published:2022-06-18
About author:ZHOU Xue-lan, E-mail: zhouxl17@lzu.edu.cn; #Correspondence YAN Ping, Tel: +86-931-2115288, E-mail: pingyanlz@163.com; WU Xiao-yun, Tel: +86-931-2115292, E-mail: wuxiaoyun@caas.cn
Supported by:
This study was supported by the Agricultural Science and Technology Innovation Program, CAAS (25-LZIHPS-01), the China Agriculture Research System of MOF and MARA (CARS-37) and the National Natural Science Foundation of China (32102500).

摘要/Abstract

摘要：

【背景】牦牛骨骼肌的生长发育决定其产肉量，进而影响经济收入。因此提高牦牛的产肉性能是发展牦牛产业的重要任务。骨骼肌发育过程受许多基因的调控，包括一些非编码RNA的调控，如miRNA。然而牦牛出生前后骨骼肌中miRNA的转录调控机制并不清楚。【目的】通过小RNA测序挖掘与牦牛骨骼肌生长发育有关的miRNA，并通过C2C12细胞对候选miRNA进行功能研究，为牦牛骨骼肌发育机制解析提供基础。【方法】对出生前后牦牛背最长肌（3个胎牛和3个成年牛）进行miRNA转录本测序，基于（log2 (差异倍数) |>1，P值≤0.05）筛选差异miRNAs，并通过Miranda和TargetScan算法预测差异miRNAs靶基因并求交集，对差异miRNA靶基因进行GO和KEGG富集分析。在C2C12细胞中通过干扰和过表达实验对候选miRNA进行功能分析，通过双荧光素酶实验验证候选miRNA的靶基因。【结果】本研究在胎牛和成年牦牛背最长肌中共鉴定到264个miRNAs。264个差异miRNAs共预测到5183个靶基因。GO和KEGG结果显示，差异miRNA的靶基因主要富集在能量平衡，蛋白激酶结合，ATP结合等GO条目，及一些与肌肉发育有关的信号通路，如MAPK，PI3K-Akt，Hippo等信号通路。其中候选miR-652在胎牛背最长肌中上调表达。通过在C2C12细胞中转染miR-652发现，miR-652可促进C2C12细胞的增殖和分化（P≤0.05），同时抑制C2C12细胞晚期凋亡（P≤0.001）。细胞周期实验结果显示，miR-652可导致C2C12细胞百分比在G1期下降（P≤0.001），S期和G2期上升（P≤0.01）。双荧光素酶实验结果提示ISL1是miR-652的一个靶基因。【结论】牦牛在出生前后骨骼肌中存在大量差异表达的miRNA，表明miRNA参与牦牛骨骼肌发育，miR-652可能通过靶向ISL1基因调控牦牛骨骼肌生长发育。

Abstract:

The growth and development of skeletal muscle also determine the meat production of yak, ultimately affecting the economic benefits. Hence, improving growth performance is a top priority in the yak industry. Skeletal muscle development is a complex process involving the regulation of several genes, including microRNAs (miRNAs). However, the transcription of miRNAs in yak skeletal muscle during prenatal to postnatal stages is unknown. We used small RNA sequencing (small RNA-Seq) to determine the global miRNAs of longissimus dorsi muscle from yak (the samples were collected from three fetuses and three adults). Totally 264 differently expressed miRNAs (|log₂(fold change)|>1 and P-value≤0.05) were detected between the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differently expressed miRNAs-targeted genes participated in pathways associated with muscle development, such as MAPK, PI3K-Akt, and Hippo signaling pathways, etc. MiR-652, which was up-regulated in the fetal group, was transfected into C2C12 myoblasts to examine its role. miR-652 promoted (P≤0.05) proliferation and differentiation, but inhibited (P≤0.001) apoptosis at early period. Furthermore, miR-652 reduced (P≤0.001) the proportion of C2C12 myoblasts in the G1 phase while increasing (P≤0.01) the proportion of cells in the S and G2 phases. Dual-luciferase reporter assays indicated that ISL1 served as a target of miR-652. In general, these findings expand our understanding of yak skeletal muscle miRNAs, and suggested that miR-652 probably regulated myogenesis by regulating ISL1.

Key words: skeletal muscle , small RNA Sequencing , miR-652 , C2C12 myoblast , ISL1

ZHOU Xue-lan, GUO Xian, LIANG Chun-nian, CHU Min, WU Xiao-yun, YAN Ping. [J]. Journal of Integrative Agriculture, 2023, 22(5): 1502-1513.

ZHOU Xue-lan, GUO Xian, LIANG Chun-nian, CHU Min, WU Xiao-yun, YAN Ping. MicroRNA transcriptome of skeletal muscle during yak development reveals miR-652 regulates myoblasts differentiation and survival by targeting ISL1[J]. Journal of Integrative Agriculture, 2023, 22(5): 1502-1513.

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