Investigating The Role Of Microrna-199a-5p In Porcine Preadipocyte Proliferation And Differentiation

Based on the fact that miR-199a-5p is upregulated in obese white adipose tissue whilebeing significantly downregulated in early3T3-L1differentiation, we proposed thatmiR-199a-5p might play a role during preadipocyte differentiation. Although miR-199a-5pwas once shown to suppress aP2level in adipogenic-differentiated hMSCs, little furtherinvestigation has been reported since then. Both the function of miR-199a-5p and its potentialtarget genes in adipogenesis still require further elucidation. This study exployed the pig, afine model animal of adipogenesis research. First, we established an in vitro adipogenicmodel of porcine preadipocytes, then the function of miR-199a-5p in porcine preadipocytedifferentiation and its possible target genes were systematically explored. Also, we hereindisplayed the first evidence about miR-199a-5p’s role in porcine preadipocyte proliferation.Taken together, our study provided evidence for the functions of miR-199a-5p and its targetgene in adipogenesis and theoretic supportings for animal breeding practice. The main resultsof this study is presented as follows:1. MiR-199a-5p is abundant in porcine white subcutaneous fat and downregulatedduring early porcine preadipocyte differentiation.We fist tested the tissue distribution of miR-199a-5p in pig and found the highexpression level of miR-199a-5p in subcutaneous adipose tissue, and this abundance ofmiR-199a-5p may have functional consequence. Then we established an in vitro adipogenicmodel of porcine preadipocytes to find by real-time qPCR that miR-199a-5p level wasrapidly downregulated in the early stage of porcine preadipocyte differentiation while risingback in the late differentiation.2. MiR-199a-5p impaired porcine preadipocyte differentiation while promoted itsproliferative activity.We performed miR-199a-5p overexpression in porcine preadipocytes followed byadipogenic induction. Using real-time qPCR and Western Blot, we observed that theadipogenic markers were repressed by elevated miR-199a-5p level. Oil Red O staining and extraction assay displayed the impaired lipid accumulation of miR-199a-5p transfectedadipocytes. We also performed miR-199a-5p overexpression in porcine preadipocytes, andtested using flow cytometry the cell cycle alterations during the proliferative stage. Also, wedectected the cell number changes upon miR-199a-5p overexpression by nulei staining. Theresults showed that miR-199a-5p was a facilitator of porcine preadipocyte proliferation.3. Cav-1was targeted by miR-199a-5p in porcine adipocytes.We predicted the target genes of miR-199a-5p using online solfwares. By consulting theliterature, we found that among the predicted targets, Cav-1has been previously shown to beindispensable for adipocyte differentiation, which made Cav-1a desirable candidate formiR-199a-5p target gene validation. Real-time qPCR, western blot, and luciferase reporterassay were perfomed to experimentally confirmed that miR-199a-5p was capable of targetingCav-1in porcine adipocytes. Taken together, we suggest that miR-199a-5p exerts its functionin adipogenesis at least partially by targeting Cav-1.