miR-186-5p调控猪原代前体脂肪细胞增殖和分化的研究

doi:10.11843/j.issn.0366-6964.2021.05.011

摘要/Abstract

摘要： 旨在探究miR-186-5p对猪原代前体脂肪细胞增殖和成脂分化的调控作用及机制。本研究采集7日龄健康马身猪公猪的颈部皮下脂肪，分离培养马身猪原代前体脂肪细胞；猪原代前体脂肪细胞分为4组，分别转染miR-186-5p模拟物（mimics）及其对照组（mimics NC），miR-186-5p抑制剂（inhibitor）及其对照组（inhibitor NC），用CCK8和划痕试验分析猪原代前体脂肪细胞的增殖效果，油红O染色检测其成脂分化能力，qPCR检测其增殖和成脂分化相关基因的表达水平；预测miR-186-5p的靶基因，用双荧光素酶报告试验检测miR-186-5p与靶基因的相互作用。结果，当猪原代前体脂肪细胞中转染mimics时，miR-186-5p的表达量极显著升高（P<0.01），细胞增殖能力和增殖相关基因：增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、周期蛋白依赖性激酶4（cyclin-dependent kinases 4，CDK4）的表达量以及预测靶基因去乙酰化酶2（sirtuins 2，SIRT2）的表达量都极显著降低（P<0.01），而细胞成脂分化能力和成脂分化相关基因：过氧化物酶体增殖剂激活受体γ（peroxisome proliferators-activated receptor γ，PPARγ）、固醇调节元件结合转录因子1（sterol regulatory element binding transcription factor 1，SREBF1）、CCAAT增强子结合蛋白β（CCAAT enhancer binding protein β，C/EBPβ）、脂肪酸结合蛋白4（fatty acid-binding protein 4，FABP4）、脂蛋白脂肪酶（lipoprotein lipase，LPL）的表达量都极显著升高（P<0.01）；当猪原代前体脂肪细胞转染inhibitor时，miR-186-5p的表达量极显著降低（P<0.01），细胞增殖能力以及增殖相关基因PCNA和CDK4的表达量、预测靶基因SIRT2的表达量都显著或极显著升高（P<0.05、P<0.01），而细胞成脂分化能力以及成脂分化相关基因PPARγ、SREBF1、C/EBPβ、FABP4、LPL的表达量都极显著降低（P<0.01）。转染miR-186-5p mimics可以显著抑制SIRT2 3'UTR野生型双荧光素酶报告载体（SIRT2-Wt-psiCHECK-2）中荧光素酶的活性，但不影响SIRT2 3'UTR突变型双荧光素酶报告载体（SIRT2-Mut-psiCHECK-2）中荧光素酶的活性。miR-186-5p可以靶向结合SIRT2的3'UTR序列，降低SIRT2的表达，抑制马身猪原代前体脂肪细胞增殖，促进其成脂分化。

关键词: miR-186-5p, 马身猪, 原代前体脂肪细胞, 增殖, 成脂分化, 靶基因

Abstract: The study aimed to explore the regulation and mechanism of miR-186-5p on the proliferation and adipogenic differentiation of porcine primary preadipocytes. The neck subcutaneous fat of 7-day-old healthy Mashen boar was collected and the primary preadipocytes were isolated and cultured. The primary preadipocytes were divided into 4 groups, which were transfected with miR-186-5p mimics (mimics) and its control group (mimics NC), miR-186-5p inhibitor (inhibitor) and its control group (inhibitor NC). CCK8 and scratch test were used to analyze the proliferation effect of porcine primary preadipocytes. Oil red O staining was used to detect the adipogenic differentiation ability. qPCR was used to detect the expression of proliferation and adipogenic differentiation related genes. The target genes of miR-186-5p were predicted, and the interaction between miR-186-5p and target genes was detected by dual luciferase reporter assay. When mimics were transfected into porcine primary preadipocytes, the expression of miR-186-5p significantly increased(P<0.01); meanwhile the cell proliferation ability and expression of proliferation related genes, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinases 4 (CDK4), and the target gene sirtuins 2 (SIRT2) significantly decreased(P<0.01); however, the adipogenic differentiation ability and expression of adipogenic differentiation related genes, peroxisome proliferators-activated receptor γ (PPARγ), sterol regulatory element binding transcription factor 1 (SREBF1), CCAAT enhancer binding protein β (C/EBPβ), fatty acid-binding protein 4 (FABP4) and lipoprotein lipase (LPL) significantly increased(P<0.01). When the primary preadipocytes were transfected with inhibitor, the expression of miR-186-5p significantly decreased(P<0.01); meanwhile the cell proliferation ability and expression of PCNA, CDK4 and SIRT2 significantly increased(P<0.05 or P<0.01); while the adipogenic differentiation ability and expression of PPARγ, SREBF1, C/EBPβ, FABP4 and LPL significantly decreased(P<0.01). Transfection of miR-186-5p mimics significantly inhibited the activity of luciferase in SIRT2-Wt-psiCHECK-2, but did not affect the activity of luciferase in SIRT2-Mut-psiCHECK-2. The results show that miR-186-5p can target the 3'UTR of SIRT2 and decrease the expression of SIRT2, which can inhibit the proliferation of Mashen pig primary preadipocytes and promote their adipogenic differentiation.

Key words: miR-186-5p, Mashen pigs, primary preadipocytes, proliferation, adipogenic differentiation, target gene

中图分类号:

S828.2

蔡春波, 刘敏, 杨阳, 张万锋, 高鹏飞, 曹果清, 李步高, 郭晓红. miR-186-5p调控猪原代前体脂肪细胞增殖和分化的研究[J]. 畜牧兽医学报, 2021, 52(5): 1247-1257.

CAI Chunbo, LIU Min, YANG Yang, ZHANG Wanfeng, GAO Pengfei, CAO Guoqing, LI Bugao, GUO Xiaohong. Regulation of miR-186-5p on Proliferation and Differentiation of Porcine Primary Preadipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1247-1257.

参考文献 37

[1]	ZHANG Y F,ZHANG J J,GONG H F,et al.Genetic correlation of fatty acid composition with growth,carcass,fat deposition and meat quality traits based on GWAS data in six pig populations[J].Meat Sci,2019,150:47-55.
[2]	BREMER A A,JIALAL I.Adipose tissue dysfunction in nascent metabolic syndrome[J].J Obes,2013,2013:393192.
[3]	YANG J H,WU W Z,WU M H,et al.Long noncoding RNA ADPGK-AS1 promotes cell pro-liferation,migration,and EMT process through regulating miR-3196/OTX1 axis in breast cancer[J].In Vitro Cell Dev Biol Anim,2019,55(7):522-532.
[4]	单留群,方征,刘洪,等.miR-92a通过下调RAB3B表达促进肝癌细胞增殖、迁移的机制分析[J].临床和试验医学杂志, 2019, 18(12):1266-1270.SHAN L Q,FANG Z,LIU H,et al.Mechanism of miR-92a in promoting proliferation and migration of hepatoma cells by down-regulating expression of RAB3B[J].Journal of Clinical and Experimental Medicine,2019,18(12):1266-1270.(in Chinese)
[5]	洪嵩,蔡东峰,张靖,等.miR-552靶向调控Wnt抑制因子-1促进MG-63骨肉瘤细胞增殖、侵袭、迁移的实验研究[J].临床肿瘤学杂志,2019,24(6):494-499.HONG S,CAI D F,ZHANG J,et al.Effect of miR-552 on proliferation,migration and invasion of osteosarcoma cell lines MG-63 via targeting WIF1[J].Chinese Clinical Oncology,2019,24(6):494-499.(in Chinese)
[6]	时军利,王磊,王春青,等.miR-9-5p通过靶向FOXO1基因调控食管癌细胞增殖、侵袭和迁移[J].胃肠病学和肝病学杂志,2019,28(6):644-649.SHI J L,WANG L,WANG C Q,et al.MiR-9-5p regulates proliferation,invasion and migration of esophageal cancer cells by targeting FOXO1 gene[J].Chinese Journal of Gastroenterology and Hepatology,2019,28(6):644-649.(in Chinese)
[7]	王桂冬,侯媛溪,王宁,等.miR-34a-3p通过靶基因YAP1调控黑色素瘤A375细胞的增殖和凋亡[J].临床和实验医学杂志, 2019,18(6):594-598.WANG G D,HOU Y X,WANG N,et al.The effect of miR-34a-3p on proliferation and apoptosis of melanoma A375 cells by targeting YAP1[J].Journal of Clinical and Experimental Medicine,2019,18(6):594-598.(in Chinese)
[8]	FENG L L,LIU T H,YANG Y Y,et al.Metformin promotes proliferation and suppresses apoptosis in Ox-LDL stimulated macrophages by regulating the miR-34a/Bcl2 axis[J].RSC Adv,2019,9(26):14670-14676.
[9]	HU X,TANG J,HU X,et al.MiR-27b impairs adipocyte differentiation of human adipose tissue-derived mesenchymal stem cells by targeting LPL[J].Cell Physiol Biochem,2018,47(2):545-555.
[10]	LI F,LI D H,ZHANG M,et al.MiRNA-223 targets the GPAM gene and regulates the differentiation of intramuscular adipocytes[J].Gene,2019,685:106-113.
[11]	MA X Y,WEI D W,CHENG G,et al.Bta-miR-130a/b regulates preadipocyte differentiation by targeting PPARG and CYP2U1 in beef cattle[J].Mol Cell Probes,2018,42:10-17.
[12]	SUN G R,LI F,MA X F,et al.Gga-miRNA-18b-3p inhibits intramuscular adipocytes differentiation in chicken by targeting the ACOT13 gene[J].Cells,2019,8(6):556.
[13]	ZHANG Y Y,WANG Y N,WANG H B,et al.MicroRNA-224 impairs adipogenic differentiation of bovine preadipocytes by targeting LPL[J].Mol Cell Probes,2019,44:29-36.
[14]	贺小云,刘秋月,储明星.miRNA调控哺乳动物卵泡发育和卵母细胞成熟的研究进展[J].畜牧兽医学报, 2019,50(11):2175-2185.HE X Y,LIU Q Y,CHU M X.Advances in miRNA regulating mammalian follicular development and oocyte maturation[J].Acta Veterinaria et Zootechnica Sinica,2019,50(11):2175-2185.(in Chinese)
[15]	蔡萌,南雪梅,熊本海,等.脂肪酸调控奶牛乳腺microRNAs研究进展[J].畜牧兽医学报,2020,51(12):2934-2941.CAI M,NAN X M,XIONG B H,et al.Research progress of fatty acid regulation of microRNAs in dairy cow mammary glands[J].Acta Veterinaria et Zootechnica Sinica,2020,51(12):2934-2941.(in Chinese)
[16]	CHEN T,CUI J X,MA L X,et al.The effect of microRNA-331-3p on preadipocytes proliferation and differentiation and fatty acid accumulation in Laiwu pigs[J].Biomed Res Int,2019,2019:9287804.
[17]	OUYANG D,YE Y Q,GUO D G,et al.MicroRNA-125b-5p inhibits proliferation and promotes adi-pogenic differentiation in 3T3-L1 preadipocytes[J].Acta Biochim Biophys Sin,2015,47(5):355-361.
[18]	DU J J,ZHANG P W,GAN M L,et al.MicroRNA-204-5p regulates 3T3-L1 preadipocyte proliferation,apoptosis and differentiation[J].Gene,2018,668:1-7.
[19]	ZHANG Z Q,ZHANG W,MAO J S,et al.MiR-186-5p functions as a tumor suppressor in human osteosarcoma by targeting FOXK1[J]. Cell Physiol Biochem,2019,52(3):553-564.
[20]	LI J L,XIA L M,ZHOU Z H,et al.MiR-186-5p upregulation inhibits proliferation,metastasis and epithelial-to-mesenchymal transition of colorectal cancer cell by targeting ZEB1[J].Arch Biochem Biophys,2018,640:53-60.
[21]	LIAO G Q,LIU X P,WU D H,et al.MORC2 promotes cell growth and metastasis in human cholangiocarcinoma and is negatively regulated by miR-186-5p[J].Aging,2019,11(11):3639-3649.
[22]	ZHU K,SU Y L,XU B,et al.MicroRNA-186-5p represses neuroblastoma cell growth via down-regulation of Eg5[J].Am J Transl Res,2019,11(4):2245-2256.
[23]	FENG H X,ZHANG Z R,QING X,et al.MiR-186-5p promotes cell growth,migration and invasion of lung adenocarcinoma by targeting PTEN[J].Exp Mol Pathol,2019,108:105-113.
[24]	姚克,陈涛,王冬,等.miR-186靶向调控骨桥蛋白基因表达对骨髓间充质干细胞成骨分化的影响[J].蚌埠医学院学报, 2017, 42(1):30-33.YAO K,CHEN T,WANG D,et al.Effect of the miR-186 target-regulating the osteopontin expression on the osteogenic differentiation of bone marrow mesenchymal stem cells[J].Journal of Bengbu Medical College,2017,42(1):30-33.(in Chinese)
[25]	苏欣.miR-186对C2C12中myogenin基因表达的抑制研究[J].中国饲料,2015(23):15-17.SU X.Study on miR-186 inhabit myogenin expression in C2C12[J].China Feed,2015(23):15-17.(in Chinese)
[26]	ANTONIOU A,MASTROYIANNOPOULOS N P,UNEY J B,et al.miR-186 inhibits muscle cell differentiation through myogenin regulation[J].J Biol Chem,2014,289(7):3923-3935.
[27]	辜浩,郭鑫宇,堵晶晶,等.MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究[J].中国生物工程杂志,2020,40(3):21-30.GU H,GUO X Y,DU J J,et al.The effect of miR-186-5p on the proliferation and differentiation of 3T3-L1 preadipocyte[J].China Biotechnology,2020,40(3):21-30.(in Chinese)
[28]	CAO Q H,WANG Z,WANG Y,et al.TBL1XR1 promotes migration and invasion in osteosarcoma cells and is negatively regulated by miR-186-5p[J].Am J Cancer Res,2018,8(12):2481-2493.
[29]	许冬梅,赵宇军,杜斌,等.miR-186-5p对绵羊黑色素细胞迁移增殖的影响[J].畜牧兽医学报,2017,48(11):2068-2075.XU D M,ZHAO Y J,DU B,et al.The influences of miR-186-5p on migration and proliferation of sheep melanocytes[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2068-2075.(in Chinese)
[30]	刘炳婷.Sirt2在猪前体脂肪细胞分化中的作用及其机理研究[D].杨凌:西北农林科技大学,2010.LIU B T.Effects and molecular mechanism of SIRT2 during porcine preadipocytes differentiation[D].Yangling:Northwest A&F University,2010.(in Chinese)
[31]	WILSON J M,LE V Q,ZIMMERMAN C,et al.Nuclear export modulates the cytoplasmic Sir2 homologue Hst2[J].EMBO Rep,2006,7(12):1247-1251.
[32]	DENG A L,NING Q Y,ZHOU L,et al.SIRT2 is an unfavorable prognostic biomarker in patients with acute myeloid leukemia[J]. Sci Rep,2016,6:27694.
[33]	JING H,HU J,HE B,et al.A SIRT2-selective inhibitor promotes c-Myc oncoprotein degradation and exhibits broad anticancer activity[J].Cancer Cell,2016,29(3):297-310.
[34]	房晓欢,杜明,李飒,等.Sirtuins对雌性动物生殖的影响研究进展[J].畜牧兽医学报,2019,50(12):2379-2386.FANG X H,DU M,LI S,et al.Research progress on the effects of Sirtuins on female animal reproduction[J].Acta Veterinaria et Zootechnica Sinica,2019,50(12):2379-2386.(in Chinese)
[35]	JING E X,GESTA S,KAHN C R.SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation[J].Cell Metab,2007,6(2):105-114.
[36]	WANG F,TONG Q.SIRT2 suppresses adipocyte differentiation by deacetylating FOXO1 and enhancing FOXO1’s repressive interaction with PPARγ[J].Mol Biol Cell,2009,20(3):801-808.
[37]	刘炳婷,白亮,刘飞,等.过表达Sirt2抑制猪前体脂肪细胞的分化[J].中国生物化学与分子生物学报,2010,26(6):575-580.LIU B T,BAI L,LIU F,et al.Overexpression of Sirt2 suppresses porcine preadipocyte differentiation[J].Chinese Journal of Biochemistry and Molecular Biology,2010,26(6):575-580.(in Chinese)