miR-221靶向IRS1抑制绵羊乳腺上皮细胞活力和增殖  

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

作　　者：柯娜 郝志云 王建清 甄慧敏 罗玉柱 胡江 刘秀 李少斌 赵志东 黄兆春 梁维炜 王继卿 KE Na;HAO ZhiYun;WANG JianQing;ZHEN HuiMin;LUO YuZhu;HU Jiang;LIU Xiu;LI ShaoBin;ZHAO ZhiDong;HUANG ZhaoChun;LIANG WeiWei;WANG JiQing(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)

机构地区：[1]甘肃农业大学动物科学技术学院/甘肃省草食动物生物技术重点实验室/甘肃省牛羊基因改良工程实验室,兰州730070

出　　处：《中国农业科学》2022年第10期2047-2056,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32060746和31860635);甘肃农业大学青年导师扶持基金(GAU-QDFC-2020-01);甘肃农业大学伏羲青年英才培育计划(Gaufx-02Y02);甘肃省基础研究创新群体项目(18JR3RA190)。

摘　　要：【背景】MicroRNAs(miRNA)是一类小RNA分子(18—23nt),广泛参与了家畜乳腺发育和泌乳性能的调控。项目组前期在小尾寒羊上应用RNA-Seq研究发现,miR-221在空怀期乳腺组织中的表达量是泌乳期的3.6倍,但是尚不清楚miR-221对绵羊乳腺发育的调控机制。【目的】探讨miR-221是否通过靶向基因IRS1抑制绵羊乳腺上皮细胞的活力和增殖数量,为揭示miR-221对绵羊泌乳性能的分子调控机理提供理论参考。【方法】采集小尾寒羊乳腺、心脏、肝脏、肾脏、脾脏、肺脏、背最长肌和卵巢等8个组织样本,采用实时荧光定量PCR(reverse transcription-quantitative PCR,RT-qPCR)技术,构建miR-221在绵羊8个组织中的表达谱。采用细胞转染、CCK-8和Edu等方法,研究miR-221对绵羊乳腺上皮细胞活力和增殖的影响。利用miRDB和miRanda数据库,预测miR-221的靶基因,结合功能富集分析,确定目标靶基因,构建靶基因的野生型和突变型载体,进而用双荧光素酶报告实验,验证miR-221与预测靶基因间的靶向关系。分析过表达和沉默miR-221对靶基因及其信号通路下游功能基因的影响。【结果】RT-qPCR结果表明,miR-221在绵羊乳腺等8个组织中均表达,其中在肺脏和脾脏中的表达量最高,在背最长肌和肾脏中的表达量最低。CCK-8结果表明,miR-221模拟物抑制了绵羊乳腺上皮细胞的活力(P<0.01),而miR-221抑制剂提高了乳腺上皮细胞的活力(P<0.05)。Edu试验发现,miR-221模拟物减少了Edu标记的阳性乳腺上皮细胞数量(P<0.01),而miR-221抑制剂增加了Edu标记的阳性乳腺上皮细胞数量(P<0.01)。双荧光素酶报告实验结果表明,miR-221模拟物抑制了胰岛素受体底物1(insulin receptor substrate 1,IRS1)基因3′UTR区域的双荧光素酶活性(P<0.01),而miR-221抑制剂提高了该基因的活性(P<0.05),表明IRS1是miR-221的一个靶基因。RT-qPCR结果进一步发现,过表达miR-221降低了绵羊乳腺上皮细胞中IRS1【Background】Micro RNAs(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 mi R-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 in

关 键 词：绵羊 MIR-221 胰岛素受体底物1 乳腺上皮细胞 

分 类 号：S826[农业科学—畜牧学]