Effects Of MiR-1271,miR-381,miR-17-3p And MiR-22-3p On Differentiation Of Yanbian Yellow Cattle Precursor Adipocytes

MicroRNA(miRNA)is a type of non-coding single-stranded RNA molecule with a length of about 20-24 nucleotides encoded by an endogenous gene.It can completely or partially combine with the target gene through RNA-induced silencing complex to degrade mRNA or hinder translation,thereby regulating protein expression.It is a type of small molecule RNA that has been favored by researchers in recent years and participates in the regulation and control of many physiological processes.Researchers have also demonstrated that miRNA plays a vital role in lipid metabolism through more molecular or biological experiments.Based on the previous research group’s analysis of miRNA-seq and RNA-seq technology,we screened out miRNAs and genes that were differentially expressed in the high and low fat groups of Yanbian yellow cattle.Choose miR-1271,miR-381,miR-17-3p and miR-22-3p to study.Use TargetScan and other miRNA target gene bioinformatics prediction sites to select target genes from differentially expressed genes.The effects and mechanisms of the four miRNAs were studied using dual-luciferase reporter gene detection,qRT-PCR,Western blot,and oil red O staining.The results of this study are as follows:1.miR-1271 targets ATF3 to regulate adipocyte differentiation in Yanbian cattleComparing the miR-1271 sequences of different species,we found that the mature sequence of miR-1271 is highly conserved in mammals.The miRNA target gene prediction website found that among the differentially expressed genes obtained from RNA-seq,the ATF3 gene may be the target gene of miR-1271.Through the verification analysis of the dual-luciferase reporter gene,the overexpressed miR-1271 significantly inhibited the activity of the ATF3-3’UTR dual-luciferase reporter gene vector.Overexpression of miR-1271 will increase the content of lipid droplets in Yanbian yellow cattle fat cells.Similarly,it will also increase the mRNA and protein expression of lipid metabolism marker genes PPARy and C/EBPa.The mRNA and protein expression levels of miR-1271 candidate target gene ATF3 will also be inhibited by miR-1271.The inhibition of miR-1271 expression had the opposite result.2.MiR-381 and miR-17-3p simultaneously target KCTD15 to regulate adipocyte differentiation in Yanbian yellow cattleComparing the miR-381 and miR-17-3p sequences of different species,it is found that the mature sequences of miR-381 and miR-17-3p are highly conserved in mammals.The miRNA target gene prediction website found that among the differentially expressed genes obtained from RNA-seq,the KCTD15 gene may be the target gene of miR-381 and miR-17-3p.Through dual-luciferase reporter gene verification analysis,overexpressed miR-381 and miR-17-3p significantly inhibited the activity of KCTD15-3’UTR dual-luciferase reporter gene vector.The content of lipid droplets in adipocytes after transfection with miR-381 was significantly higher than that in the negative control group.The mRNA and protein expression levels of the overexpressed miR-381 and miR-17-3p experimental group lipid metabolism marker genes PPARy and C/EBPa were also significantly higher than those of the control group.Inhibiting the expression of miR-381 and miR-17-3p had the opposite result.Overexpressed miR-381 and miR-17-3p also inhibit the expression of mRNA and protein of the candidate target gene KCTD15.3.miR-22-3p targets WFIKKN2 to regulate adipocyte differentiation in Yanbian cattleThrough the comparison of miR-22-3p sequences between different species,it was found that the mature sequence of miR-22-3p is highly conserved in mammals.According to the miRNA target gene prediction website,it was found that among the differentially expressed genes obtained by RNA-seq,the WFIKKN2 gene may be a target gene of miR-22-3p.According to the verification analysis of the dual-luciferase reporter gene,miR-22-3p significantly inhibited the activity of the WFIKKN2-3’UTR dual-luciferase reporter gene vector.Over-expressed miR-22-3p can significantly reduce the lipid droplet content of adipocytes in Yanbian yellow cattle,and also reduce the mRNA and protein expression of the lipid metabolism markers PPARy and C/EBPa.In contrast,inhibition of miR-22-3p expression had opposite results.The expression level of the candidate target gene WFIKKN2 was suppressed due to the overexpression of miR-22-3p.In summary,miR-1271,miR-381 and miR-17-3p may promote the differentiation of Yanbian yellow cattle precursor adipocytes by targeting ATF3 and KCTD15,respectively;miR-22-3p may inhibit bovine precursor adipocyte differentiation by targeting WFIKKN2.