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

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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"

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