| The mammary gland is a dynamic organ that continuously grows,develops,and degrades in mammals,and is crucial for the growth,development,and survival of offspring.The ability of the mammary gland to secrete milk is mainly measured by the amount of milk produced and the composition of the milk,depending on the number and vitality of the mammary epithelial cells that secrete milk.Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs that play an important role in organisms.They play a post transcriptional regulatory role mainly by complementary binding with m RNA.Mi RNAs are widely involved in various biological processes in the body,and also have important regulatory roles in breast development and lactation.Based on the previous sequencing of the research group,this study selected miR-21,which is highly expressed in the mammary gland tissue of small-tailed Han sheep,as the research object.Using methods such as CCK8,Ed U and triglyceride content detection kit,the effects of miR-21 on the viability and triglyceride content of ovine mammary epithelial cells were measured.Subsequently,the target genes of miR-21 were predicted,and the targeting relationship between them was further verified by constructing a dual luciferase vector.The effect of miR-21 on the expression level of the target genes was studied.The main research results are as follows:1.Sequence alignment results showed that the mature sequence of miR-21 had the same sequence among different species;The phylogenetic analysis of miR-21 precursor sequences showed that ovine miR-21 sequences were most closely related to goat miR-21 sequences,followed by mouse and rat.2.The results of CCK8,Ed U,and triglyceride content detection showed that compared to the negative control group,the treatment of ovine mammary epithelial cells with miR-21 mimic significantly enhanced cell viability,increased cell number,and increased triglyceride content in ovine mammary epithelial cells(P<0.05);On the contrary,treatment of ovine mammary epithelial cells with miR-21 inhibitor resulted in a significant decrease in cell viability and cell number(P<0.05).3.The prediction results of online prediction software show that miR-21 can target469 m RNA,including PDCD4 and STAT3.4.The results of double luciferase reporter gene detection showed that overexpression of miR-21 could significantly reduce the activity of renilla enzyme/firefly enzyme in PDCD4 and STAT3 wild-type dual luciferase vectors(P<0.05).However,overexpression of miR-21 did not significantly affect the activity of the mutant dual luciferase vector(P>0.05).5.RT-q PCR analysis showed that treatment of ovine mammary epithelial cells with miR-21 mimic significantly reduced the expression level of PDCD4 and STAT3genes(P<0.01).On the contrary,the treatment of ovine mammary epithelial cells with miR-21 inhibitor significantly promoted the expression level of STAT3 gene(P<0.01),but had no significant effect on the expression level of PDCD4 gene(P>0.05).In summary,miR-21 can promote the proliferation of ovine mammary epithelial cells,improve the vitality and triglyceride content of ovine mammary epithelial cells,and inhibit the expression of target genes PDCD4 and STAT3. |
