Study On The Function Of MiR-221 And MiR-329b-3p And Verification Of Their Targeted Relationship With IRS1 In Small-Tailed Han Sheep

The lactation trait of sheep is an important economic trait,which significantly affects the survival rate and growth rate of the lambs.The mammary gland development and the number and secretory activity of mammary epithelial cells directly determine the milk production and milk composition of ewes.MicroRNAs(miRNAs)are a class of non-coding RNA that are widely present in eukaryotes.Previous studies have shown that miRNAs play an important role in the regulation of lactation traits in animals.In our previous study,mammary gland tissues were collected from the peak-lactating and non-lactating Small-tailed Han ewes,and then small RNA-Seq were carried out.23 differentially expressed miRNAs were found between the two periods,of which miR-221 and miR-329b-3p were up-regulated in the non-lactation period.In this study,miR-221 and miR-329b-3p were selected.Firstly,their expression levels were verified by reverse transcription quantitative PCR(RT-qPCR),and the effect of miR-221 and miR-329b-3p on proliferation of mammary epithelial cells was detected by CCK-8.Secondly,the target genes of the two miRNAs were predicted and the GO and KEGG pathways of target genes were analyzed.Finally,the dual luciferase report experiment and RT-qPCR were used to verify the targeted relationship between miR-221 and miR-329b-3p and the predicted target gene IRS1.The effect of miR-221 and miR-329b-3p on target IRS1 and other important genes on the downstream functional genes of PI3K/AKT and MAPK signaling pathways was investigated.The main results are as follows:1.The expression levels of miR-221 and miR-329b-3p in the non-lactation mammary gland tissue were 2.23 and 2.15-fold higher than those in the peak-lactation period,respectively.2.MiR-221 and miR-329b-3p was predicted to have 33 and 19 target genes using miRDB and miRnada databases,respectively.IRS1 was a common target gene for the two miRNAs.3.It was found that overexpression of miR-221 can inhibit the proliferation of ovine mammary epithelial cells(P<0.01),while silenced miR-221 can promote the proliferation of sheep mammary epithelial cells(P<0.01)using CCK-8 kit.MiR-329b-3p has the same effect on ovine mammary epithelial cells as miR-221.4.Respectively overexpression of miR-221 and miR-329b-3p inhibited the relative activity of the dual-luciferase of IRS1 3?UTR in the wild-type vector(P<0.05),while silenced miR-221 and miR-329b-3p increased the relative activity of the dual-luciferase of IRS1 3?UTR in the wild type vector(P<0.05).Compared with the negative control group,overexpressed or silenced miR-221 and miR-329b-3p had no significantly effect on the relative activity of the dual luciferase of IRS1 3?UTR in the mutant-type vector(P>0.05).This indicates that IRS1 was a target gene of miR-221 and miR-329b-3p.5.Overexpression of miR-221 reduced the expression level of IRS1 in mammary epithelial cells(P<0.05),while silenced miR-221 increased the expression level of IRS1(P<0.05).The effect of miR-329b-3p on IRS1 gene expression was the same as that of miR-221.6.Overexpression of miR-221 reduced the expression level of MAPK8 and PIK3R1(P<0.05),while silenced miR-221 increased the expression of MAPK8 and PIK3R1(P<0.05).Overexpression of miR-329b-3p decreased the expression level of MAPK8 and IGF1R(P<0.05),however,silenced miR-329b-3p increased the expression level of MAPK8 and IGF1R(P<0.05).7.The CDS of ovine IRS1 is 3708 bp in length and would encode 1235 amino acids.The gene was expressed in eight tissues including mammary gland in the Small-tailed Han sheep,and showed obvious tissue and spatiotenoiral expression pattern.In summary,miR-221 and miR-329b-3p not only inhibited the proliferation of ovine mammary epithelial cells,but also inhibited the expression level of target IRS1 and other genes IGF1 R,PIK3R1 and MAPK8 in the signaling pathways.