Study Of MiR-125a-3p On Adipogenic Differentiation And Inflammation Of Mouse Subcutaneous Preadipocytes By Targeting FSTL1

The main characteristics of adipose tissue in obesity state are the excessive proliferation and differentiation of adipocytes,which can induce macrophages infiltration and increase the expression of inflammatory cytokines,leading to the occurrence of chronic inflammation.Studies have shown that numerous genes and micro RNAs are involved in the regulation of this process,but the specific mechanism of obesity-induced chronic inflammation of adipose tissue is still not fully understood.In this study,the constructed pc DNA3.1-FSTL1 recombinant vector and synthetic si RNAs were transfected into the mouse subcutaneous preadipocytes,and Oil Red O staining was used to examine the accumulation of lipid droplets,while q PCR and Western blotting were applied to detect the relative mRNA and protein expression levels of adipocyte differentiation-related genes and inflammatory factors.In order to investigate the specific mechanism of miRNA and FSTL1 regulating the inflammation induced by preadipocyte differentiation,we predicted miRNA targeting FSTL1 by software prediction and validated through luciferase reporter assay,and then miRNA Mimic and Inhibitor were transfected into mouse subcutaneous preadipocytes.Moreover,the co-culture system of mouse subcutaneous preadipocytes and marrow-derived macrophages was established to further explore the relationship between adipogenesis and inflammation.This study provided a theoretical basis for obesity-linked metabolic diseases and improvement of animal productivity.The main results were as follows:(1)The mouse subcutaneous preadipocytes were isolated successfully,and Oil Red O staining showed significant accumulation of lipid droplets and strong adipogenic differentiation potential.(2)The results showed that overexpression of FSTL1 significantly inhibited the formation of lipids,the adipogenic marker genes PPARγ/FABP4 mRNA levels were significantly decreased(P<0.05,P<0.01),and FABP4 protein levels were also markedly reduced(P<0.01).However,the mRNA expression levels of inflammatory genes IL-1β/CCL2 and protein levels of IL-1β were significantly upregulated(P<0.05,P<0.01).Conversely,knockdown of FSTL1 promoted mouse subcutaneous preadipocyte differentiation and suppressed the mRNA and protein levels of inflammatory factors.(3)Software prediction showed that miR-125a-3p could bind with FSTL1 3’ UTR,and dual luciferase reporter assay indicated that miR-125a-3p directly targeted FSTL1 and remarkably decreased luciferase activity(P<0.01);Moreover,upregulation of miR-125a-3p significantly inhibited mRNA and protein levels of FSTL1,while inhibition of miR-125a-3p dramatically upregulated FSTL1 expression at mRNA and protein levels(P<0.01).(4)Through transfection of miR-125a-3p Mimic and Inhibitor into mouse subcutaneous preadipocytes,we found that overexpression of miR-125a-3p accelerated the mRNA levels of PPARγ/FABP4 and FABP4 protein levels(P<0.05,P<0.01),while the mRNA and protein levels of inflammatory factors IL-1β and CCL2 were markedly down-regulated(P<0.01);In contrast,downregulation of miR-125a-3p repressed the differentiation process and facilitated the expression of inflammatory genes.(5)The direct co-culture system of mouse subcutaneous preadipocytes and bone marrow-derived macrophages in mice was established successfully;Under co-culture conditions,the mRNA expression levels of pro-inflammatory cytokines IL-1β and i NOS in bone marrow-derived macrophages induced by adipogenesis were markedly increased(P<0.01);Instead,bone marrow-derived macrophages could phagocytize lipid droplets produced by preadipocyte differentiation,the mRNA expression levels of PPARγ and LPL were significantly reduced(P<0.01),and decelerated the differentiation process.Taken together,this study revealed that miR-125a-3p could promote mouse subcutaneous preadipocyte differentiation and inhibit inflammation by targeting FSTL1 gene.