miR-138靶向PGC-1α调控牦牛肌内前体脂肪细胞增殖及分化

doi:10.11843/j.issn.0366-6964.2022.10.016

摘要/Abstract

摘要： 旨在分析miR-138在牦牛前体脂肪细胞分化过程中的调控作用。本研究设计合成miR-138 mimics和inhibitor以对细胞进行miR-138过表达及抑制表达，并通过油红O染色分析miR-138对牦牛肌内前体脂肪细胞脂质沉积的影响；利用CCK-8、划痕和流式细胞技术分析miR-138对细胞增殖的影响；通过对bta-miR-138进行生物信息学分析，筛选其在脂质沉积过程中的相关潜在靶基因；利用RT-qPCR测定miR-138潜在靶基因mRNA表达水平，并通过双荧光素酶报告试验确定靶向关系。结果显示，过表达miR-138后，细胞内脂滴沉积水平显著低于对照（NC）组（P<0.01），而抑制miR-138表达则显著增强脂肪细胞脂质沉积（P<0.01）；RT-qPCR结果表明，过表达miR-138可显著抑制脂肪分化标志基因PPARγ和c/EBPα的表达（P<0.01），抑制miR-138则上调PPARγ和c/EBPα表达水平（P<0.05）；此外，过表达miR-138后，潜在靶基因PTPN11、CREB1和ADCYAP1R1表达水平显著下调（P<0.05），而PGC-1α和SNAP25表达极显著下调（P<0.01）；抑制miR-138后MXLIP和SNAP25表达显著上调（P<0.05），PGC-1α和PTPN11表达极显著上调（P<0.01）；CCK-8和划痕试验结果表明，过表达miR-138后24~36 h，细胞增殖活性显著低于对照组（P<0.05），而抑制miR-138则表现为细胞增殖活性增强；流式分析结果显示过表达miR-138后，细胞出现G1-S期阻滞，细胞周期相关基因CCND1、CCNB1和增殖标志基因Ki67的mRNA表达显著降低；生物信息学分析预测获得263个miR-138公共靶基因，KEGG富集结果显示靶基因主要参与“轴突导引”、“胰岛素抵抗”、“胰岛素分泌”及“RNA降解”等通路，GO分析主要富集于“RNA聚合酶Ⅱ启动子对转录的正向调控”、“核染色质”、“染色质DNA结合”和“I型肺细胞分化”等功能；双荧光素酶报告试验结果显示，共转染miR-138 mimics和PGC-1α-Wt-PGL3-basic可显著降低细胞荧光强度（P<0.01）。以上结果表明，miR-138可通过靶向结合PGC-1α 3'UTR序列，降低PGC-1α的mRNA表达水平，抑制牦牛肌内前体脂肪细胞分化和脂质沉积，降低细胞增殖活性。本研究为阐明牦牛肉质性状的潜在分子机制提供参考。

关键词: 牦牛, miR-138, PGC-1α, 前体脂肪细胞, 增殖分化

Abstract: The study aimed to analyze the regulatory action of miR-138 during the yak preadipocytes differentiation. In this study, miR-138 mimics and inhibitor were designed and synthesized to overexpress or suppress miR-138 in preadipocytes. The oil red O staining assay were performed to analyze the effects of miR-138 on lipid deposition, while CCK-8, scratch test assays and flow cytometry were performed to analyze the effects of miR-138 on proliferation. Furthermore, the potential target genes of bta-miR-138 in the process of lipid deposition were screened by bioinformatics analysis; And the mRNA expression levels of miR-138 target genes were detected by RT-qPCR technology, in addition, dual-luciferase report assay were used to verify the interaction between miR-138 and its target. The results showed that overexpression of miR-138 significantly reduced lipid deposition level compared with the control (NC) group (P<0.01), and significantly increased in lipid deposition when miR-138 suppression (P<0.01). RT-qPCR results showed that, overexpression of miR-138 also significantly inhibited the expression of adipose differentiation markers (PPARγ and c/EBPα, P<0.01), whereas the expression of PPARγ and c/EBPα were enhanced when miR-138 suppression (P<0.05). In addition, overexpression of miR-138 significantly down-regulated the mRNA expression of potential targets PTPN11, CREB1, ADCYAP1R1, PGC-1α and SNAP25 (P<0.05 or P<0.01), while the mRNA expression of MXLIP, SNAP25, PGC-1α and PTPN11 significantly in creased (P<0.05 or P<0.01) when miR-138 suppression. The results of CCK-8 and scratch test showed that the cell proliferation activity decreased significantly (P<0.05) when miR-138 overexpression 24 to 36 h, whereas the proliferation activity was increased after miR-138 inhibition. The flow cytometry analysis results indicated that cell cycle were arrested at G1 to S phase, and mRNA level of cell cycle-related genes CCND1, CCNB1 and the proliferation marker gene Ki67 were significantly decreased after miR-138 overexpression. A total of 263 common targets of miR-138 were predicted by bioinformatics analysis. GO annotation results showed that those target genes significantly enriched in "Positive regulation of transcription from RNA polymerase II promoter", "Nuclear chromatin", "Chromatin DNA binding", and "Type I pneumocyte differentiation", KEGG enrichment results showed that these target genes mainly participated in "Axon guidance", "Insulin resistance", "Insulin secretion" and "RNA degradation" pathways. The dual-luciferase report assay showed that co-transfection of miR-138 mimics and PGC-1α-Wt-PGL3-basic significantly reduced the cell fluorescence activity (P<0.01). The present results suggest that miR-138 can decrease the mRNA expression level of PGC-1α by binding its 3' UTR sequence, and inhibit the differentiation and lipid deposition of intramuscular preadipocyte in yak, thus decrease the proliferation of cell. The current study may help clarify the mechanism underlying yak meat characters formation at molecular level.

Key words: yak, miR-138, PGC-1α, preadipocyte, proliferation and differentiation

中图分类号:

S823.85

冉宏标, 王会, 柴志欣, 王嘉博, 张明, 蔡欣, 钟金城. miR-138靶向PGC-1α调控牦牛肌内前体脂肪细胞增殖及分化[J]. 畜牧兽医学报, 2022, 53(10): 3434-3447.

RAN Hongbiao, WANG Hui, CHAI Zhixin, WANG Jiabo, ZHANG Ming, CAI Xin, ZHONG Jincheng. miR-138 Regulates Proliferation and Differentiation of Intramuscular Preadipocyte by Targeting PGC-1α in Yak[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3434-3447.

参考文献 34

[1]	张剑搏, 丁学智, AHMAD A A, 等.高原土著动物适应性进化的研究进展[J].畜牧兽医学报, 2019, 50(9):1723-1736.ZHANG J B, DING X Z, AHMAD A A, et al.Advances in research on adaptive evolution of native animals of Tibetan plateau[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(9):1723-1736.(in Chinese)
[2]	HUANG S L, LI X Z, ZHENG H, et al.Loss of super-enhancer-regulated circRNA Nfix induces cardiac regeneration after myocardial infarction in adult mice[J].Circulation, 2019, 139(25):2857-2876.
[3]	WANG L G, ZHOU Z Y, ZHANG T, et al.IRLnc:a novel functional noncoding RNA contributes to intramuscular fat deposition[J]. BMC Genomics, 2021, 22(1):95.
[4]	ZHANG C, WANG Q, ZHOU X W, et al.MicroRNA-138 modulates glioma cell growth, apoptosis and invasion through the suppression of the AKT/mTOR signalling pathway by targeting CREB1[J].Oncol Rep, 2020, 44(6):2559-2568.
[5]	LI J B, WANG H Y, YAO Y, et al.Overexpression of microRNA-138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway[J].J Cell Mol Med, 2017, 21(8):1482-1491.
[6]	OLIVO-MARSTON S E, HURSTING S D, PERKINS S N, et al.Effects of calorie restriction and diet-induced obesity on murine colon carcinogenesis, growth and inflammatory factors, and microRNA expression[J].PLoS One, 2014, 9(4):e94765.
[7]	WU L P, DAI X J, ZHAN J F, et al.Profiling peripheral microRNAs in obesity and type 2 diabetes mellitus[J].APMIS, 2015, 123(7):580-585.
[8]	NARDELLI C, GRANATA I, IAFFALDANO L, et al.miR-138/miR-222 overexpression characterizes the miRNome of amniotic mesenchymal stem cells in obesity[J].Stem Cells Dev, 2017, 26(1):4-14.
[9]	YANG Z, BIAN C J, ZHOU H, et al.MicroRNA hsa-miR-138 inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells through adenovirus EID-1[J].Stem Cells Dev, 2011, 20(2):259-267.
[10]	WANG Y H, BOWER N I, REVERTER A, et al.Gene expression patterns during intramuscular fat development in cattle[J].J Anim Sci, 2009, 87(1):119-130.
[11]	纪会, 王会, 柴志欣, 等.牦牛miR-378前体克隆及组织表达分析[J].生物技术通报, 2019, 35(1):58-66.JI H, WANG H, CHAI Z X, et al.Precursor cloning and tissue expression analysis of yak miR-378[J].Biotechnology Bulletin, 2019, 35(1):58-66.(in Chinese)
[12]	赵丽玲.lncFAM200B对牦牛前体脂肪细胞分化的作用研究[D].成都:西南民族大学, 2021.ZHAO L L.Studys of lncFAM200B on the differentiation in yak pre-adipocytes[D].Chengdu:Southwest Minzu University, 2021.(in Chinese)
[13]	GUO S Q, MA B J, JIANG X K, et al.Astragalus polysaccharides inhibits tumorigenesis and lipid metabolism through miR-138-5p/SIRT1/SREBP1 pathway in prostate cancer[J].Front Pharmacol, 2020, 11:598.
[14]	DANG Y Q, XU J J, ZHU M Z, et al.Gan-Jiang-Ling-Zhu decoction alleviates hepatic steatosis in rats by the miR-138-5p/CPT1B axis[J].Biomed Pharmacother, 2020, 127:110127.
[15]	WANG Y T, LIN L X, HUANG Y, et al.MicroRNA-138 suppresses adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells by targeting lipoprotein lipase[J].Yonsei Med J, 2019, 60(12):1187-1194.
[16]	BAI X M, SHAO J F, ZHOU S J, et al.Inhibition of lung cancer growth and metastasis by DHA and its metabolite, RvD1, through miR-138-5p/FOXC1 pathway[J].J Exp Clin Cancer Res, 2019, 38(1):479.
[17]	LIU Z Z, TIAN Y F, WU H, et al.LncRNA H19 promotes glioma angiogenesis through miR-138/HIF-1α/VEGF axis[J]. Neoplasma, 2020, 67(1):111-118.
[18]	YOU C X, JIN L M, XU Q, et al.Expression of miR-21 and miR-138 in colon cancer and its effect on cell proliferation and prognosis[J].Oncol Lett, 2019, 17(2):2271-2277.
[19]	WANG J, LI J X, YANG J, et al.MicroRNA-138-5p regulates neural stem cell proliferation and differentiation in vitro by targeting TRIP6 expression[J].Mol Med Rep, 2017, 16(5):7261-7266.
[20]	ZHENG H J, RAMNARAIGN D, ANDERSON B A, et al.MicroRNA-138 inhibits osteogenic differentiation and mineralization of human dedifferentiated chondrocytes by regulating RhoC and the actin cytoskeleton[J].JBMR Plus, 2019, 3(2):e10071.
[21]	RU W X, QI A, SHEN X M, et al.The circular RNA circCPE regulates myoblast development by sponging miR-138[J].J Anim Sci Biotechnol, 2021, 12(1):102.
[22]	LIU J J, ZHANG H, XING F, et al.MicroRNA-138 promotes proliferation and suppresses mitochondrial depolarization in human pulmonary artery smooth muscle cells through targeting TASK-1[J].Mol Med Rep, 2018, 17(2):3021-3027.
[23]	XU J, LI L, YUN H F, et al.MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1[J].Biochem Biophys Res Commun, 2015, 463(4):1159-1164.
[24]	HAO J J, HAO C, ZHANG L J, et al.OM2, a novel oligomannuronate-chromium(III) complex, promotes mitochondrial biogenesis and lipid metabolism in 3T3-L1 adipocytes via the AMPK-PGC1α pathway[J].PLoS One, 2015, 10(7):e0131930.
[25]	CHENG C F, KU H C, LIN H.PGC-1α as a pivotal factor in lipid and metabolic regulation[J].Int J Mol Sci, 2018, 19(11):3447.
[26]	YU B, HUO L H, LIU Y S, et al.PGC-1α controls skeletal stem cell fate and bone-fat balance in osteoporosis and skeletal aging by inducing TAZ[J].Cell Stem Cell, 2018, 23(2):193-209.E5.
[27]	丁玲.淫羊藿苷促小鼠胚胎干细胞体外分化为心肌细胞的相关机制研究[D].杭州:浙江大学, 2007.DING L.Mechanisms involved in icariin induced cardiomyocyte differentiation of murine embryonic stem cells in vitro[D]. Hangzhou:Zhejiang University, 2007.(in Chinese)
[28]	ZHANG Y, SHEN L C, ZHU H L, et al.PGC-1α regulates autophagy to promote fibroblast activation and tissue fibrosis[J].Ann Rheum Dis, 2020, 79(9):1227-1233.
[29]	MA J, WANG Z, ZHAO J Q, et al.Resveratrol attenuates lipopolysaccharides (LPS)-induced inhibition of osteoblast differentiation in MC3T3-E1 cells[J].Med Sci Monit, 2018, 24:2045-2052.
[30]	YOON S Y, AHN D, KIM J K, et al.Nepetin acts as a multi-targeting inhibitor of protein tyrosine phosphatases relevant to insulin resistance[J].Chem Biodivers, 2021, 19(1):e202100600.
[31]	PURI P, WALKER W H.The regulation of male fertility by the PTPN11 tyrosine phosphatase[J].Semin Cell Dev Biol, 2016, 59:27-34.
[32]	ZHOU R P, DENG M T, CHEN L Y, et al.Shp2 regulates chlorogenic acid-induced proliferation and adipogenic differentiation of bone marrow-derived mesenchymal stem cells in adipogenesis[J].Mol Med Rep, 2015, 11(6):4489-4495.
[33]	HUANG Q Z, LIAN C L, DONG Y Y, et al.SNAP25 inhibits glioma progression by regulating synapse plasticity via GLS-mediated glutaminolysis[J].Front Oncol, 2021, 11:698835.
[34]	XU C L, WANG C, MENG Q Y, et al.miR-153 promotes neural differentiation in the mouse hippocampal HT-22 cell line and increases the expression of neuron-specific enolase[J].Mol Med Rep, 2019, 20(2):1725-1735.