MiR-148a-3p Promotes Rabbit Preadipocyte Differentiation By Targeting PTEN

At present,studies have shown that miR-148a-3p can regulate the differentiation of the preadipocytes and the formation of lipid;PTEN can inhibit the differentiation of the preadipocytes by inhibiting the PI3K-AKT signaling pathway;PTEN is a potential target gene of miR-148a-3p.The aim of this study is to establish a rabbit model of differentiation in vitro and verify that miR-148a-3p can target PTEN to regulate adipocyte differentiation of rabbit adipocytes.Results:(1)It was found that the expression of miR-148a-3p in the perinenal adipose tissue of 5-month-old rabbits was significantly higher than that in the primary rabbit.(2)After inducing preadipocytes differentiation,a large number of small lipid droplets appeared on the second day,and the aggregation of lipid droplets appeared on the sixth day.On the tenth day,it showed the characteristics of mature adipocytes with lipid droplets increasing significantly.The marker gene assay found that it accords with the rules of preadipocytes differentiation.The expression of miR-148a-3p increased gradually with the time of preadipocyte differentiation.(3)After transfection of miR-148a-3p analogue mimic,the expression of miR-148a-3p was significantly up-regulated(P<0.01).The results showed that mimic could promote the preadipocytes differentiation,and the content of fatty acid and triglyceride increased significantly(P<0.01),and the mRNA level of PPARγ,C/EBPα and FABP4 increased significantly(P<0.05).Transfection of miR-148a-3p inhibitor significantly reduced the expression of miR-148a-3p in cells(P<0.05).The results showed that inhibitor could inhibit lipid differentiation of the preadipocytes,and the content of fatty acid and triglyceride decreased significantly(P<0.05),and the mRNA levels of PPARγ,C/EBPα and FABP4 genes of differentiation related genes were significantly decreased(P<0.05).(4)Based on target gene prediction and a luciferase reporter assay we further demonstrated that miR-148a-3p directly targeted one of the 3’ untranslated regions of PTEN(P<0.01).(5)Overexpression of miR-148a-3p led to a very obvious decrease in PTEN mRNA and protein levels(P<0.05).In contrast,when endogenous miR-148a-3p was knocked down,PTEN mRNA and protein levels increased compared with the NC group(P<0.05).(6)SiRNA was utilized to knockdown endogenous PTEN in rabbit preadipocytes.Results from Oil Red-O staining and TG measurement showed that knockdown PTEN might promote adipogenesis in rabbit preadipocytes(P<0.01).Furthermore,the expression patterns of PPARγ,C/EBPα and FABP4 and the PPARγ protein increased significantly(P<0.05),which were also consistent with above results from miR-148a-3p overexpression experiments.All above results have indicated that the induced differentiation model of rabbit in vitro was successfully established and during the differentiation of rabbit preadipocyte,miR-148a-3p promotes rabbit preadipocyte differentiation by inhibiting expression of PTEN at both transcriptional and translational levels.