miR-221靶向IRS1抑制绵羊乳腺上皮细胞活力和增殖

doi:10.3864/j.issn.0578-1752.2022.10.014

中国农业科学 ›› 2022, Vol. 55 ›› Issue (10): 2047-2056.doi: 10.3864/j.issn.0578-1752.2022.10.014

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

miR-221靶向IRS1抑制绵羊乳腺上皮细胞活力和增殖

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()

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

收稿日期:2021-04-09 接受日期:2022-03-07 出版日期:2022-05-16 发布日期:2022-06-02
通讯作者: 王继卿
作者简介:柯娜,E-mail： ken@st.gsau.edu.cn。
基金资助:
国家自然科学基金(32060746);国家自然科学基金(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)

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()

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

摘要/Abstract

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和PIK3R1的表达量（P<0.05）,沉默miR-221则提高了这2个基因的表达量（P<0.05）。过表达或沉默miR-221对乳腺上皮细胞中IGF1R的表达量没有显著影响（P>0.05）。【结论】miR-221通过抑制靶基因IRS1的表达量,最终抑制了绵羊乳腺上皮细胞的活力和增殖数量。

关键词: 绵羊, miR-221, 胰岛素受体底物1, 乳腺上皮细胞

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.

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. miR-221靶向IRS1抑制绵羊乳腺上皮细胞活力和增殖[J]. 中国农业科学, 2022, 55(10): 2047-2056.

KE Na,HAO ZhiYun,WANG JianQing,ZHEN HuiMin,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin,ZHAO ZhiDong,HUANG ZhaoChun,LIANG WeiWei,WANG JiQing. The miR-221 Inhibits the Viability and Proliferation of Ovine Mammary Epithelial Cells by Targeting IRS1[J]. Scientia Agricultura Sinica, 2022, 55(10): 2047-2056.

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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
miR-221	R: TGGTGTCGTGGAGTCG	RT-qPCR
U6	F: GGAACGATACAGAGAAGATTAGC	RT-qPCR
U6	R: TGGAACGCTTCACGAATTTGCG	RT-qPCR
18sRNA	F: GTGGTGTTGAGGAAAGCAGACA	RT-qPCR
18sRNA	R: TGATCACACGTTCCACCTCATC	RT-qPCR
IRS1	F: AAGGACTTCAAACAGTGCCCT	RT-qPCR
IRS1	R: AGGTCATTTAGGTCTTCATTC	RT-qPCR
IGF1R	F: CTTGTCCCAAAGTGTGT	RT-qPCR
IGF1R	R: CTCCAGTTCCGAAGCGA	RT-qPCR
PIK3R1	F: CACCATGACGAGAAGAC	RT-qPCR
PIK3R1	R: CCTGTTTACTGCTTTCCC	RT-qPCR
β-actin	F: AGCCTTCCTTCCTGGGCATGGA	RT-qPCR
β-actin	R: GGACAGCACCGTGTTGGCGTAGA	RT-qPCR
IRS1	F: CGCTCGAGCGTTGGGTGGAGAGAGTTT	3′UTR野生型载体 3′UTR Wild-type vector
IRS1	R: TTTGCGGCCGCGGAAGGGGGAGAAGAAGA	3′UTR野生型载体 3′UTR Wild-type vector
IRS1	F: TTTACATTGCTTGATGTTGAGCTGAGATCCTTACA	3′UTR突变型载体 3′UTR Mutant vector
IRS1	R: CAAGCAATGTAAACAAGAGATAGTATCAGCAAATAGAACTATAC	3′UTR突变型载体 3′UTR Mutant vector

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

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