Effects Of MiR-196a On Porcine Adipocyte Proliferation And Differentiation

MicroRNAs are non-coding RNA that regulate gene expression at poat-transcription level. They participate various biological processes, including cell differentiation, apoptosis, energy metabolism and cancer development. Previous studies have been identified some miRNAs that participate adipocyte formation, for example miR-143 (Esau et al.2004) and miR-17-92 cluster(Wang et al.2008) promote adipocyte differentiation, let-7(Sun et al.2009) and miR-27(Lin et al.2009) inhibit adipocyte differentiation, and miR-8(Jennifer et al.2008) promotes marrow stromal cells differentiation into adipocytes. Besides, miR-196a is differentially expressed during adipocyte differentiation, indicating a role in the regulation of adipocyte formation. Previous studies showed that miR-196a play an important role in the regulation of osteogenic differentiation (Kim et al 2009),limb development(Hornstein et al. 2005),tail regeneration (Sehm et al.2009) and tumorgenesis(Schimanski et al.2009). However, it is still unclear of the role of miR-196a in adipocyte differentiation. With regard to the conservation of miRNA across species, we hypothesize that miR-196a regulates adipocyte differentiation. To test our hypothesis, porcine adipocytes were used to study the role of miR-196a during porcine adipocyte proliferation and differentiation through over-expression of miR-196a. These results provide evidence for animal breeding and the further investigation of microRNA regulation of adipogenesis. The miR-196a expression plasmid will be a new tool for the further investigation of the role of miR-196a in gene regulation and mechanism of metabolic diseases.1. Tissue distribution of miR-196aThe expression level of miR-196a was detected by real-time PCR from 3 days or 6 months pig tissues. The result showed that miR-196a was widely expressed in all detected pig tissues. It's worth noting that the expression level of miR-196a was lowest in adipose tissue of 3 days pig, but kept the highest level in adipose tissue of 6 months pig. These results indicated that miR-196a may regulate pig adipose tissue development.2. The effects of miR-196a on porcine pre-adipocyte proliferation and differentiationThe precursor of miR-196a was amplified by polymerase chain reaction(PCR), and the miR-196a expression vector pcDNA3.0-miR-196a was constructed. Then the pcDNA3.0-miR-196a was transfected into porcine pre-adipocytes, and the adipocytes proliferation, key transcription factors of adipogenesis and lipid droplet were separately detected by MTT, real-time PCR and Oil Red O staining. The result of MTT analysis showed that miR-196a cannnot have significant effect on porcine adipocytes proliferation. The results of real-time PCR and Oil Red O staining showed that over-expression of miR-196a promoted key transcription factors of adipocytes differentiation, including PPARy, C/EBPa and LPL, and lipid droplet accumulation. These results indicated that miR-196a had no effect on adipocyte proliferation, but it may play a role in the regulation of adipocyte differentiation. 3. The bioinformatics analysis of miR-196a targetsThe potential targets of miR-196a were predicted by RNA22 prediction software, including sirtl, wnt3a, wntlOa and wntlOb. There are two target sites in sirtl, wnt3a and wntlOa, and four target sites in wntlOb. Furthermore, the key transcription factors of adipocytes differentiation, including PPARy, C/EBPa, LPL, aP2 and SREBP genes did not have any miR-196a target site. This result showed miR-196a regulates PPARy, C/EBPa and LPL expression may through targeting those potential targets, and then promotes adipocyte differentiation.