The Effect Of MiR-214-3p On The Differentiation Of 3T3-L1 Preadipocytes And The Mechanism Of Action

Adipogenesis is a complex development process from preadipocytes to mature adipocytes,which is essential for mammalian fat formation and adipose tissue metabolism.This complex process is regulated by many regulatory factors.MicroRNAs(miRNAs)are a family of non-coding small RNA molecules of approximately 22 nucleotides in length that are involved in the regulation of many life processes through transcriptional regulation of transcription factors and other genes,including adipocyte differentiation.After treatment of 3T3-L1 preadipocytes with LiCl,miR-214-3p was found to be significantly down-regulated.Meanwhile,miR-214-3p was highly conserved among multiple species such as mice,humans and pigs.Therefore,this study used the 3T3-L1 cell line as a research material to explore the role and mechanism of miR-214-3p in adipogenic differentiation of 3T3-L1 preadipocytes.In this study,miR-214-3p agomir or antagomir was transfected into 3T3-L1 cells to study the effect of miR-214-3p on proliferation and differentiation of 3T3-L1 cells;The potential target genes of miR-214-3p in 3T3-L1 differentiation were predicted and validated by bioinformatics software analysis and dual luciferase reporter assays.The main findings obtained are as follows:1.miR-214-3p promotes the proliferation of 3T3-L1 preadipocytesOverexpression of miR-214-3p in 3T3-L1 cells significantly up-regulated cell proliferation-related genes CyclinB,CyclinD(P<0.05)and CDK4,especially CyclinE(P<0.05);EdU staining also showed that overexpression of miR-214-3p resulted in a significant increase in the number of EdU positive cells(P<0.01).In contrast,the proliferation of 3T3-L1 preadipocytes was significantly inhibited after inhibition of miR-214-3p(P<0.05).2.miR-214-3p promotes differentiation of 3T3-L1 preadipocytesOverexpression of miR-214-3p significantly up-regulated the mRNA and protein levels of the adipogenic marker genes FAS,PPAR?,Adiponectin and aP2(P<0.05);Through Bodipy(P<0.01)and oil red O staining(P<0.05)experiments,it was found that overexpression of miR-214-3p can significantly enhance lipid droplet formation.In contrast,inhibition of miR-214-3p significantly inhibited the expression of adipogenic genes as well as lipid deposition(P<0.05).3.miR-214-3p directly targets Ctnnb1By constructing the wild-type and mutant dual luciferase reporter vectors of Ctnnb1 and co-transfecting 3T3-L1 cells with miR-214-3p agomir,it was found that miR-214-3p significantly inhibited the luciferase activity of the wild-type reporter vector of Ctnnb1(P<0.01),and has no effect on the mutant.4.miR-214-3p promotes adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting ?-catenin-mediated Wnt/?-catenin pathwayOverexpression of miR-214-3p significantly inhibited the expression of Ctnnb1 mRNA(P<0.05)and protein(P<0.01)during 3T3-L1 differentiation.In conclusion,miR-214-3p specifically target Ctnnb1 promotes the adipogenic differentiation of 3T3-L1 preadipocytes by interfering with ?-catenin-mediated Wnt/?-catenin signaling pathway.In addition,miR-214-3p can promote the proliferation of 3T3-L1 preadipocytes.