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 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 effect of miR-221 on the viability and proliferation of sheep mammary epithelial cells by targeting IRS1 gene, and to provide a theoretical reference for revealing the molecular regulation mechanism of miR-221 on sheep 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 using reverse transcription-quantitative PCR (RT-qPCR). The effect of miR-221 on the viability and proliferation of ovine mammary epithelial cells (OMECs) were investigated 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 using dual luciferase reporter assay. Finally, the effect of over-expressed and silenced miR-221 on expression levels of the target gene and other functional genes in downstream signaling pathways was 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 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 insulin receptor substrate 1 (IRS1). On contrary, miR-221 inhibitor increased the luciferase activity. This suggests that IRS1 is a target gene of miR-221. The results from RT-qPCR further found that the over-expressed miR-221 reduced expression levels of IRS1 and PIK3R1 in OMECs (P<0.05), while silenced miR-221 enhanced the levels of these 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 results show that 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