Regulation Of MiR-186-5p On The Proliferation,Migration And Differentiation Of Porcine Precursor Adipocytes

Micro RNAs(mi RNAs)is a type of single-stranded endogenous non-coding RNA with a length of about22 nt.A mi RNA regulates the expression of the target gene through combining with its end of 3′UTR,and then participates in the regulation of cell proliferation,migration and differentiation.Previous research results showed that mi R-186-5p participated in many biological processes including cell proliferation and apoptosis.The role and specific mechanism of mi R-186-5p in the proliferation and differentiation of porcine precursor adipocytes is currently unknown.Studying the role and specific mechanism of mi R-186-5p in the proliferation and differentiation of porcine precursor adipocytes can provide a theoretical basis for further revealing the mechanism of mi R-186-5p regulating porcine fat deposition.To explore the role and specific mechanism of mi R-186-5p in the proliferation and differentiation of porcine precursor adipocytes,this experiment first used the subcutaneous adipose tissue of the back and neck of 8-day-old piglets,and the collagenase digestion method was used to isolate the primary pig precursors.Adipocytes were tested for the effects of mi R-186-5p on the proliferation and migration of porcine precursor adipocytes using MTT method,CCK8 method and scratch test.Oil red O staining and triglyceride content were used to observe the effects of mi R-186-5p on pigs.Effect of lipid droplet formation after induction of precursor adipocytes,q PCR method was used to detect m RNA expression levels of related genes during the proliferation and differentiation of porcine precursor adipocytes,and dual luciferase activity was used to identify mi R-186-5p and target genes.Targeting relationship.The results are as follows:1.After transfection of mi R-186-5p mimics and inhibitor,the cell proliferation was detected at 72 h by MTT method.It was found that the cell viability of the mimics group was significantly reduced(P<0.05).The inhibitor group cell growth viability increased significantly(P<0.05);the effect of mi R-186-5p on the proliferation of precursor adipocytes at different time points was detected by the CCK8 method,and the results were consistent with the results of the MTT test.At 24 h,48 h,and 72 h,mimics Cell growth was significantly inhibited in the group(P<0.01),while cell growth in the inhibitor group was significantly promoted(P<0.05);The scratch test result is consistent with this result;q PCR results showed that the expression of PCNA and CDK4 related to cell proliferation in the mimics group was significantly reduced(P<0.01)The expression of PCNA and CDK4 in the inhibitor group increased significantly(P<0.01).The results showed that mi R-186-5p can inhibit the proliferation and migration of porcine precursor adipocytes.2.Transfection of mi R-186-5p mimics and inhibitors induced differentiation of precursor adipocytes into mature adipocytes.It was found that the intracellular triglyceride content and lipid droplet number of mimics group were significantly increased,and the m RNA expression of adipogenesis-related genes PPARγ,SREBF1,C/EBPβ,FABP4,and LPL was significantly increased(P<0.01),while the results in the inhibitor group were reversed,indicating that mi R-186-5p can inhibit the differentiation of porcine precursor adipocytes.3.The q PCR results showed that the expression of SIRT2 in the over-expression mi R-186-5p group was significantly reduced(P<0.01),and the expression of SIRT2 was significantly increased after the inhibition of mi R-186-5p(P<0.01).Double luciferase reporter vector activity study found that overexpression of mi R-186-5p significantly inhibited the activity of SIRT2 wild-type luciferase reporter vector.From the above experimental results,it is speculated that the effect of mi R-186-5p on porcine precursor adipocytes is achieved to some extent by regulating the expression of SIRT2.In summary,this research systematically analyzed the regulation of mi R-186-5p on the proliferation,migration and differentiation of porcine precursor adipocytes,and finally proved that mi R-186-5p can inhibit the proliferation,migration and promotion of porcine precursor adipocytes Its differentiation.The results of this study provide a theoretical basis for studying mi RNAs in regulating pig fat development and improving pork quality.