Negative Functions Of MiR-128a During The Differentation Of Rat Pre-adipogenesis And Identification Of Its Target Genes

MicroRNAs are19-24nt long non-coding single strand RNA, regulating the translation of protein by binding with the specific region of mRNA. Former researches had shown that miRNAs play an important role in cell differentiation, apoptosis, cancer, metabolism. Plenty of miRNAs such as miR-375, miR-143, miR-103and miR-378were identified with an promotions effect on preadipocyte differentiation, while miR-27, miR-138, miR-130and let-7were reported to have an opposite effect. Seeking for the new regulators during the adipogenesis, we took out solexa sequencing with the back fat of7d and240d of the Rongchang pig and found that miR-128a had a notable difference expression level between these two tissues. Through prediction for the target genes, we found that a lot of potential target genes of miR-128a are the key genes during adipocyte differentiation; so we regard miR-128a as our study object. This study provides new supplement and further understanding for the construction of miRNA regulatory network of preadipocyte differentiation, also present theoretical foundation for genetic breeding research; the main results of this study are listed as below:(1) PAd-miR-128a vector was constructed and transfected into293A cell by Lipofecta mineTM2000, after packaging, Ad-miR-128a viruses were harvested and infected the rat preadipocytes, the miR-128a expression level was detected48h later, and the result shown that the expression level was5.31folds higher than the control group.(2) The rat preadipocytes were infected by adenovirus and induced to differentiation, the results shown that the expression level of PPARy and aP2in miR-128a overexpression group were significantly lower than the control group, and the lipid was also less than the control group, which implied that miR-128a has a negative effect on adipocyte differention.(3) The targets gene of miR-128a were predicted by Pictar and TargetScan, dual luciferase report carriers were constructed and examined, the results confirmed that IRS1is a target gene of miR-128a.