Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (10): 2047-2056.doi: 10.3864/j.issn.0578-1752.2022.10.014

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

The miR-221 Inhibits the Viability and Proliferation of Ovine Mammary Epithelial Cells by Targeting IRS1

KE Na(),HAO ZhiYun,WANG JianQing,ZHEN HuiMin,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin,ZHAO ZhiDong,HUANG ZhaoChun,LIANG WeiWei,WANG JiQing()   

  1. College of Animal Science and Technology/Gansu Key Laboratory of Herbivorous Animal Biotechnology/Gansu Engineering Lab of Genetic Improvement in Ruminants, Gansu Agricultural University, Lanzhou 730070
  • Received:2021-04-09 Accepted:2022-03-07 Online:2022-05-16 Published:2022-06-02
  • Contact: JiQing WANG E-mail:ken@st.gsau.edu.cn;wangjq@gsau.edu.cn

RichHTML

29

PDF

82

Abstract:

【Background】MicroRNAs (miRNA) are a type of small RNAs (18-23 nt) that are widely involved in the regulation of mammogenesis and milk traits in livestock animals. In our previous research, the expression level of miR-221 in non-lactating mammary gland was found to be 3.6-time higher than in mammary gland at lactation period in Small-Tailed Han sheep by using RNA-Seq. However, the regulatory mechanism of miR-221 on ovine mammary gland development is still unclear. 【Objective】The aim of this study was to investigate the inhibition of miR-221 on the viability and proliferation of ovine mammary epithelial cells by targeting insulin receptor substrate 1 (IRS1) gene, so as to provide a theoretical reference for revealing the molecular regulation mechanism of miR-221 on ovine lactation performance.【Method】In the study, mammary gland, heart, liver, kidney, spleen, lung, Longissimus dorsi muscle and ovary tissues were collected in Small-Tailed Han sheep, and the expression profiles of miR-221 were constructed in ovine eight tissues by using reverse transcription-quantitative PCR (RT-qPCR). The effects of miR-221 on the viability and proliferation of ovine mammary epithelial cells (OMECs) were investigated by using cell transfection, CCK-8 and Edu assays. The miRDB and miRanda were used to predict the target genes of miR-221. Based on functional enrichment analysis, an investigated target gene was screened. The target relationship between miR-221 and the predicted target gene was investigated by constructing wild-type and mutant-type report vectors for the target gene by using dual luciferase reporter assay. Finally, the effects of over-expressed and silenced miR-221 on expression levels of the target gene and other functional genes in downstream signaling pathways were detected.【Result】The miR-221 was expressed in ovine eight tissues including mammary glands, with the highest expression levels in lung and spleen, and the lowest expression levels in Longissimus dorsi muscle and kidney. The CCK-8 assay result revealed that miR-221 mimic inhibited the viability of OMECs, whereas miR-221 inhibitor promoted the viability of OMECs. The Edu result found that miR-221 mimic reduced the number of Edu-labeled positive OMECs. On the contrary, miR-221 inhibitor increased the number of Edu-labeled positive OMECs. The result from dual luciferase reporter assays showed that the miR-221 mimics reduced the luciferase activity of the 3′UTR region of IRS1, while miR-221 inhibitor increased the luciferase activity. This suggested that IRS1 was a target gene of miR-221. The results from RT-qPCR further found that over-expressed miR-221 reduced expression levels of IRS1 and PIK3R1 in OMECs (P<0.05), while silenced miR-221 enhanced the levels of the two genes in expression (P<0.05). No effect on IGF1R was found for over-expressed and silenced miR-221 in OMECs (P>0.05).【Conclusion】The miR-221 inhibited the viability and proliferation of OMECs by reducing IRS1 expression.

Key words: sheep, miR-221, insulin receptor substrate 1, mammary epithelial cells.

Table 1

Primer sequence"

引物名称 Primer 引物序列（5'$\to$3'） Primer sequence (5'$\to$3') 目的 Purpose
miR-221 F: AGCTACATTGTCTGCTGGTTT RT-qPCR
R: TGGTGTCGTGGAGTCG
U6 F: GGAACGATACAGAGAAGATTAGC RT-qPCR
R: TGGAACGCTTCACGAATTTGCG
18sRNA F: GTGGTGTTGAGGAAAGCAGACA RT-qPCR
R: TGATCACACGTTCCACCTCATC
IRS1 F: AAGGACTTCAAACAGTGCCCT RT-qPCR
R: AGGTCATTTAGGTCTTCATTC
IGF1R F: CTTGTCCCAAAGTGTGT RT-qPCR
R: CTCCAGTTCCGAAGCGA
PIK3R1 F: CACCATGACGAGAAGAC RT-qPCR
R: CCTGTTTACTGCTTTCCC
β-actin F: AGCCTTCCTTCCTGGGCATGGA RT-qPCR
R: GGACAGCACCGTGTTGGCGTAGA
IRS1 F: CGCTCGAGCGTTGGGTGGAGAGAGTTT 3′UTR野生型载体
3′UTR Wild-type vector
R: TTTGCGGCCGCGGAAGGGGGAGAAGAAGA
IRS1 F: TTTACATTGCTTGATGTTGAGCTGAGATCCTTACA 3′UTR突变型载体
3′UTR Mutant vector
R: CAAGCAATGTAAACAAGAGATAGTATCAGCAAATAGAACTATAC

Fig. 1

The expression abundances of miR-221 in ovine eight organs (A) and mammary gland tissues during different development periods (B) Significant difference is indicated with a, b, c and d (P<0.05). ** P<0.01 and * P<0.05. The same below"

Fig. 2

The effect of miR-221 on the viability and proliferation of ovine mammary epithelial cells A: The viability of cells measured using CCK8 assay when miR-221 mimic, mimic-NC, inhibitor and inhibitor-NC were transfected into ovine mammary epithelial cells for 48 h; B: The proliferation of cells analyzed using Edu assay when miR-221 mimic, mimic-NC, inhibitor and inhibitor-NC were transfected into ovine mammary epithelial cells for 48 h"

Fig. 3

The results of agarose gel electrophoresis (A) and sequencing (B and C) for dual luciferase vectors constructed Lane 1 (Fig. A) and Fig. B were the identified results of wild-type dual luciferase vector; Lane 2 (Fig. A) and Fig. C were the identified results of mutated dual luciferase vector; M: Marker"

Fig. 4

The effect of miR-221 on the luciferase activity of IRS1 in wild-type (A) and mutated (B) dual luciferase vectors"

Fig. 5

The effect of miR-221 on the expression levels of important functional genes"

[1] 孙海云. 外源重组Wnt3a蛋白对体外培养的奶牛乳腺上皮细胞数量及分泌活性影响的研究[D]. 保定: 河北农业大学, 2014.
SUN H Y. Effects of exogenous recombinant Wnt3a protein on cell number and secretion activity in cuitured bovine mammary epithelial cells[D]. Baoding: Hebei Agricultural University, 2014. (in Chinese)
[2] 孟凯. Wnt信号通路激活剂BIO在山羊乳腺上皮细胞形成腺泡样结构中的调控作用[D]. 杨凌: 西北农林科技大学, 2015.
MENG K. The effect of Wnt activator-bio on the formation of acinar-like structure of goat mammary epithelial cells[D]. Yangling: Northwest A&F University, 2015. (in Chinese)
[3] 王建清. 小尾寒羊miR-221和miR-329b-3p功能研究及其与IRS1基因的靶向关系验证[D]. 兰州: 甘肃农业大学, 2020.
WANG J Q. Study on the Function of miR-221 and miR-329b-3p and verification of their targeted relationship with IRS1 in Small-Tailed Han sheep[D]. Lanzhou: Gansu Agricultural University, 2020. (in Chinese)
[4] 李学忠. 过表达lncRNA H19对奶牛乳腺上皮细胞生物学特性和功能的影响及作用机制[D]. 杨凌: 西北农林科技大学, 2018.
LI X Z. The mechanism and effects of of lncRNA H19 overexpression on biological characteristics and functions of bovine mammary epithelial cells[D]. Yangling: Northwest A&F University, 2018. (in Chinese)
[5] LU P, LI L, WANG F, GU Y. Effects of long non‐coding RNA HOST2 on cell migration and invasion by regulating MicroRNA let‐7b in breast cancer. Journal of Cellular Biochemistry, 2017, 119(6): 4570-4580.
doi: 10.1002/jcb.26606
[6] AMBROS V. The functions of animal microRNAs. Nature, 2004, 431(7006): 350-355.
doi: 10.1038/nature02871
[7] HUANG Y, SHEN X, ZOU Q, WANG S, TANG S, ZHANG G. Biological functions of microRNAs: a review. Journal of Physiology and Biochemistry, 2011, 67(1): 129-139.
doi: 10.1007/s13105-010-0050-6
[8] WANG X, ZHANG L, JIN J, XIA A, WANG C, CUI Y, QU B, LI Q, SHENG C. Comparative transcriptome analysis to investigate the potential role of miRNAs in milk protein/fat quality. Scientific Reports, 2018, 8(1): 6250.
doi: 10.1038/s41598-018-24727-y
[9] UCAR A, VAFAIZADEH V, JARRY H, FIEDLER J, KLEMMT P A B, THUM T, GRONER B, CHOWDHURY K. miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development. Nature Genetics, 2010, 42(12): 1101-1108.
doi: 10.1038/ng.709
[10] IBARRA I, ERLICH Y, MUTHUSWAMY S K, SACHIDANANDAM R, HANNON G J. A role for microRNAs in maintenance of mouse mammary epithelial progenitor cells. Genes & Development, 2007, 21(24): 3238-3243.
doi: 10.1101/gad.1616307
[11] LI D, XIE X, WANG J, BIAN Y, LI Q, GAO X, WANG C. miR-486 Regulates Lactation and Targets the PTEN Gene in Cow Mammary Glands. PLoS One, 2015, 10(3): e0118284.
doi: 10.1371/journal.pone.0118284
[12] CUI W, LI Q, FENG L, DING W. miR-126-3p regulates progesterone receptors and involves development and lactation of mouse mammary gland. Molecular and Cellular Biochemistry, 2011, 355(1-2): 17-25.
doi: 10.1007/s11010-011-0834-1
[13] CHU M, ZHAO Y, YU S, HAO Y, ZHANG P, FENG Y, ZHANG H, MA D, LIU J, CHENG M, LI L, SHEN W, GAO H, LI L, MIN L. MicroRNA-221 may be involved in lipid metabolism in mammary epithelial cells. International Journal of Biochemistry and Cell Biology, 2018, 97: 118-127.
doi: 10.1016/j.biocel.2018.02.014
[14] 包黎娟, 刘育含, 马毅, 安小鹏, 张月, 张梦, 王建刚, 堵斌, 李广, 曹斌云. miR-92a对奶山羊乳腺上皮细胞增殖及凋亡的调控分析. 畜牧兽医学报, 2020, 51(01): 137-149.
BAO L J, LIU Y H, MA Y, AN X P, ZHANG Y, ZHANG M, WANG J G, DU B, LI G, CAO B Y. The Regulatory of miR-92a on proliferation and apoptosis of dairy goat mammary epithelial cells. Chinese Journal of Animal and Veterinary Sciences, 2020, 51(01): 137-149. (in Chinese)
[15] 王褚悦, 王春梅, 邵丽, 张莉, 林叶, 崔英俊, 高学军, 李庆章. miR-142-3p对奶山羊乳腺上皮细胞泌乳功能的影响. 中国畜牧兽医, 2015, 42(07): 1823-1829.
WANG C Y, WANG C M, SHAO L, ZHANG L, LIN Y, CUI Y J, GAO X J, LI Q Z. Effect of miR-142-3p on lactation function in dairy goat mammary epithelial cells. China Animal Husbandry & Veterinary Medicine, 2015, 42(07): 1823-1829. (in Chinese)
[16] WANG J, HAO Z, HU J, LIU X, LI S, WANG J, SHEN J, SONG Y, KE N, LUO Y. Small RNA deep sequencing reveals the expressions of microRNAs in ovine mammary gland development at peak-lactation and during the non-lactating period. Genomics, 2021, 113(1-2): 637-646.
doi: 10.1016/j.ygeno.2020.09.060
[17] HAO Z, WANG J, LUO Y, LIU X, LI S, ZHAO M, JIN X, SHEN J, KE N, SONG Y, QIAO L. Deep small RNA-Seq reveals microRNAs expression profiles in lactating mammary gland of 2 sheep breeds with different milk performance. Domestic Animal Endocrinology, 2021, 74: 106561.
doi: 10.1016/j.domaniend.2020.106561
[18] LI Z, LIU H, JIN X, LO L, LIU J. Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation. BMC Genomics, 2012, 13(2): 731.
doi: 10.1186/1471-2164-13-731
[19] 帅维, 陈琼, 王懿春. miR-221在人肺纤维化组织和TGFβ1干预A549细胞中的表达变化. 中国医师杂志, 2018, 20(02): 187-190.
SHUAI W, CHEN Q, WANG Y C. Examination of expression of miR-221 in human pulmonary fibrosis tissues and in A549 cells treated with TGFβ1. Journal of Chinese Physician, 2018, 20(02): 187-190. (in Chinese)
[20] OGAWA T, ENOMOTO M, FUJII H, SEKIYA Y, YOSHIZATO K, IKEDA K, KAWADA N. MicroRNA- 221/222 upregulation indicates, the activation of stellate cells and the progression of liver fibrosis. Gut, 2012, 61(11): 1600-1609.
doi: 10.1136/gutjnl-2011-300717
[21] 李庆章. 乳腺发育与泌乳生物学. 北京: 科学出版社, 2009.
LI Q Z. Mammary gland development and lactation biology. Beijing: Science Press, 2009. (in Chinese)
[22] 初美强. MiR-15b, miR-126, miR-221影响乳腺上皮细胞脂类代谢及分子机制探究[D]. 青岛: 青岛农业大学, 2017.
CHU M Q. MiR-15b, miR-126, miR-221 involved in lipid metabolism in mammary epithelial cells and the underlying mechanism[D]. Qingdao: Qingdao Agricultural University, 2017. (in Chinese)
[23] BOUTINAUD M, GUINARD-FLAMENTA J, JAMMES H. The number and activity of mammary epithelial cells, determining factors for milk production. Reproduction Nutrition Development, 2004, 44(5): 499-508.
doi: 10.1051/rnd:2004054
[24] 陆黎敏, 李庆章, 王春梅, 李晔, 高学军. miR-221对小鼠乳腺上皮细胞增殖和泌乳功能的影响. 中国生物化学与分子生物学报, 2009, 25(5): 454-458.
LU L M, LI Q Z, WANG C M, LI Y, GAO X J. Impact of miR-221 on mouse mammary epithelial cells and lactation. Chinese Journal of Biochemistry and Molecular Biology, 2009, 25(5): 454-458. (in Chinese)
[25] KEENAN T W, WINTER S, RACKWITZ H R, HEID H W. Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells. Biochimica et Biophysica Acta, 2000, 1523: 84-90.
[26] JIAO B, ZHANG X, WANG S, WANG L, LUO Z, ZHAO H, KHATIB H, WANG X. MicroRNA-221 regulates proliferation of bovine mammary gland epithelial cells by targeting the STAT5a and IRS1 genes. Journal of Dairy Science, 2018, 102(1): 426-435.
doi: 10.3168/jds.2018-15108
[27] ZHAO J, LIN J, YANG H, KONG W, HE L, MA X, COPPOLA D, CHENG J. MicroRNA-221/ 222 Negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer. The Journal of Biological Chemistry, 2008, 283(45): 31079-31086.
doi: 10.1074/jbc.M806041200
[28] 焦蓓蕾. MiR-221通过靶向STAT5a和IRS1基因抑制奶牛乳腺上皮细胞增殖的研究[D]. 杨凌: 西北农林科技大学, 2018.
JIAO B L. MiR-221 regulates cell proliferation of bovine mammary epithelial cells by targeting STAT5A and IRS1 genes[D]. Yangling: Northwest A&F University, 2018. (in Chinese)
[29] LAVAN B E, LANE W S, LIENHARD G E. The 60-kDa phosphotyrosine protein in insulin-treated adipocytes is a new member of the insulin receptor substrate family. The Journal of Biological Chemistry, 1997, 272(17): 11439-11443.
doi: 10.1074/jbc.272.17.11439
[30] RÄDLER P D, WEHDE B L, WAGNER K U. Crosstalk between STAT5 activation and PI3K/AKT functions in normal and transformed mammary epithelial cells. Molecular and Cellular Endocrinology, 2017, 451: 31-39.
doi: 10.1016/j.mce.2017.04.025
[31] 舒适, 宋菊敏. 胰岛素受体底物-1/-2与胰岛素信号转导. 医学综述, 2008, 14(5): 723-725.
SHU S, SONG J M. Insulin receptor substrate-1/-2 and insulin resistance. Medical Recapitulate, 2008, 14(5): 723-725. (in Chinese)
[32] MENZIES K K, LEFÈVRE C, MACMILLAN K L, NICHOLAS K R. Insulin regulates milk protein synthesis at multiple levels in the bovine mammary gland. Functional & Integrative Genomics, 2009, 9(2): 197-217.
[33] MENZIES K K, LEE H J, LEFÈVRE C, ORMANDY C J, MACMILLAN K L, NICHOLAS K R. Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis in murine mammary explants. Functional & Integrative Genomics, 2010, 10(1): 87-95.
[34] CHEN Q, ZHAO F, REN Y, HAN J, LIU J, LI Y, LIU H. Parenterally delivered methionyl-methionine dipeptide during pregnancy enhances mammogenesis and lactation performance over free methionine by activating PI3K-AKT signaling in methionine- deficient mice. Journal of Nutrition, 2020, 150(5): 1186-1195.
doi: 10.1093/jn/nxaa005
[35] LI Q, TIAN Y, LIANG Y, LI C. CircHIPK3/miR-876-5p/PIK3R1 axis regulates regulation proliferation, migration, invasion, and glutaminolysis in gastric cancer cells. Cancer Cell International, 2020, 20: 391.
doi: 10.1186/s12935-020-01455-w
[1] LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing. 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.
[2] CHE DaLu,ZHAO LiChen,CHENG SuCai,LIU AiYu,LI XiaoYu,ZHAO ShouPei,WANG JianCheng,WANG Yuan,GAO YuHong,SUN XinSheng. Effect of Litter Bed on Growth Performance and Odor Emission in Fattening Lamb [J]. Scientia Agricultura Sinica, 2022, 55(24): 4943-4956.
[3] SONG ShuZhen, GAO LiangShuang, LI Hong, GONG XuYin, LIU LiShan, WEI YuBing. Effects of Feeding Levels on Muscle Tissue Structure and Muscle Fiber Composition Related Genes in Sheep [J]. Scientia Agricultura Sinica, 2022, 55(21): 4304-4314.
[4] ChunTao ZHANG,Tao MA,Yan TU,QiYu DIAO. Effects of Circadian Rhythm on Rumen Fermentation and Nutrient Digestion of Mutton Sheep [J]. Scientia Agricultura Sinica, 2022, 55(18): 3664-3674.
[5] LIU WangJing,TANG DeFu,AO ChangJin. Effect of Allium mongolicum Regel and Its Extracts on the Growth Performance, Carcass Characteristics, Meat Quality and Serum Biochemical Indices of Captive Small-Tailed Han Sheep [J]. Scientia Agricultura Sinica, 2022, 55(17): 3461-3472.
[6] LIANG Peng,ZHANG TianWen,MENG Ke,SHAO ShunCheng,ZOU ShiFan,RONG Xuan,QIANG Hao,FENG DengZhen. Association Analysis of the ADIPOQ Variation with Sheep Growth Traits [J]. Scientia Agricultura Sinica, 2022, 55(11): 2239-2256.
[7] WANG Qian,LI Zheng,ZHAO ShanShan,QIE MengJie,ZHANG JiuKai,WANG MingLin,GUO Jun,ZHAO Yan. Application of Stable Isotope Technology in the Origin Traceability of Sheep [J]. Scientia Agricultura Sinica, 2021, 54(2): 392-399.
[8] LI SongMei,QIU YuGe,CHEN ShengNan,WANG XiaoMeng,WANG ChunSheng. CRISPR/Cas9 Mediated Exogenous Gene Knock-in at ROSA26 Locus in Sheep Umbilical Cord Mesenchymal Stem Cells [J]. Scientia Agricultura Sinica, 2021, 54(2): 400-411.
[9] WANG Chen,ZHANG HongWei,WANG HuCheng,SUN XiaoPing,LI FaDi,YANG BoHui. Energy and Protein Requirements of Alpine Merino Growing Sheep [J]. Scientia Agricultura Sinica, 2021, 54(16): 3537-3548.
[10] WANG JiQing,HAO ZhiYun,SHEN JiYuan,KE Na,HUANG ZhaoChun,LIANG WeiWei,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin. Screening, Identification and Functional Analysis of Important LncRNAs for Lactation Traits in Small-Tailed Han Sheep [J]. Scientia Agricultura Sinica, 2021, 54(14): 3113-3123.
[11] ZHANG Wei,WANG ShiYin,GAO Li,YANG LiWei,DENG ShuangYi,LIU XiaoNa,SHI GuoQing,GAN ShangQuan. Investigation of miR-486 Target Genes in Skeletal Muscle of Bashbay Sheep in Different Development Periods [J]. Scientia Agricultura Sinica, 2021, 54(14): 3134-3148.
[12] LI RunTing,CHEN LongXin,ZHANG LiMeng,HE HaiYing,WANG Yong,YANG RuoChen,DUAN ChunHui,LIU YueQin,WANG YuQin,ZHANG YingJie. Transient Expression and the Effect on Proliferation and Apoptosis of Granule Cell Stimulating Factor in Ovarian Fibroblasts [J]. Scientia Agricultura Sinica, 2021, 54(11): 2434-2444.
[13] LI WenJuan,TAO Hui,ZHANG NaiFeng,MA Tao,DIAO QiYu. Effects of High-Fat Diet on Energy Metabolism and Slaughter Performance of Early-Weaning Lambs [J]. Scientia Agricultura Sinica, 2021, 54(10): 2206-2216.
[14] SHI TianPei,WANG XinYue,HOU HaoBin,ZHAO ZhiDa,SHANG MingYu,ZHANG Li. Analysis and Identification of circRNAs of Skeletal Muscle at Different Stages of Sheep Embryos Based on Whole Transcriptome Sequencing [J]. Scientia Agricultura Sinica, 2020, 53(3): 642-657.
[15] ZHANG DeYin,ZHANG XiaoXue,LI FaDi,LI Chong,LI GuoZe,ZHANG YuKun,LI XiaoLong,SONG QiZhi,ZHAO Yuan,LIU XiaoQing,MA LiangQiang,WANG WeiMin. Association of Rumen Histomorphology of Sheep with Different Feed Efficiencies [J]. Scientia Agricultura Sinica, 2020, 53(24): 5115-5124.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd