Studies On The Effects Of MiR-224, MiR-146B, MiR-215and MiR-135a On Preadipocyte Differentiation And Lipid Metabolism

In recent years, more and more studies indicate that miRNA plays an important role in adipocyte differentiation and lipid metabolism by targeting the key adipogenic transcription factors and the key genes of lipid metabolism. This study aimed to investigate the effect of miRNA on adipocyte differentiation and lipid metabolism based on the differentional expressions miRNAs of the backfat tissues of150-day-old Large White and Meishan pigs by Solexa sequencing. Then, these miRNAs with differentional expressions were subjected to sequence analysis, target prediction and function analysis and miR-224, miR-146b, miR-215and miR-135a became the object of our study. The four miRNAs were studied by using the dual luciferase reporter gene analysis, Q-PCR assay and Western blotting etc. The main results as follows:1. mR-224regulates adipocyte differentiation and fatty acid metabolismSsc-miR-224is located on Chromosome X and the same miRNA of the other mammals is also present in the same location, the intron of GABRE, using UCSC website. Furthermore, the pre-miRNAs and the mature miRNAs of miR-224are highly conserved in many mammals.Several bioinformatics target prediction softwares were used to predict targets of miR-224. The results showed that EGR2and ACSL4may be the targets of miR-224. miR-224-5p significantly inhibited the luciferase activity of EGR2and ACSL43’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.miR-224-5p abundance decreases first and then increases during adipogenesis of3T3-L1cells. The content of triglyceride was lower in miR-224-5p group, and meanwhile the expression levels of adipogenic maker genes (PPARy, aP2) were also decreased.The mRNA expression levels of EGR2was suppressed by miR-224-5p during early adipogenesis and the downstream gene C/EBPβ was also suppressed. miR-224-5p could suppress EGR2expression by directly targeting it at least in part through indirect suppression of the C/EBPβ expression, decreasing the expressions of PPARy, aP2and accumulation of triglyceride, resulting in the inhibition of adipocyte differentiation. The mRNA and protein expression levels of ACSL4were both suppressed by miR-224-5p at terminal differentiation, inhibiting fatty acid activation of fatty acid β-oxidation and fatty acid β-oxidation, regulating fatty acid metabolism. We first found that miR-224plays different roles on different stages of adipogenesis by directly targeting EGR2and ACSL4.2. miR-146b regulates adipocyte differentiationThe mature miRNAs of miR-146b are highly conserved in many mammals through sequence alignment.Several bioinformatics target prediction softwares were used to predict targets of miR-146b. The results showed that Runxltl and Smad4may be the targets of miR-146b. miR-146b-5p significantly inhibited the luciferase activity of Runxltl and Smad43’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.The content of triglyceride was higher in miR-146b-5p group, and meanwhile the mRNA and protein expression levels of adipogenic maker genes(C/EBPa, PPARy, aP2) were also upregulated.miR-146b could suppress Runxltl expression by directly targeting it and may control the interaction of Runxltl and C/EBPβ, then prevent the transcriptional activation of the C/EBPa promoter and promote adipocyte differentiation. Furthermore, miR-146b could suppress Smad4expression by directly targeting it at least in part through suppression of the CTGF expression which is the downstream gene of TGFβ signaling pathway, resulting in promotion of adipocyte differentiation.3. miR-215regulates adipocyte differentiationThe mature miRNAs of miR-215are highly conserved in many mammals through sequence alignment. Several bioinformatics target prediction softwares were used to predict targets of miR-215. The results showed that FNDC3B and CTNNBIP1may be the targets of miR-215. miR-215-5p significantly inhibited the luciferase activity of FNDC3B and CTNNBIP13’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.The content of triglyceride was lower in miR-215-5p group, and meanwhile the mRNA and protein expression levels of adipogenic maker genes(C/EBPa, PPARy, aP2) were also decreased.miR-215-5p could suppress FNDC3B expression by directly targeting it and impair adipocyte differentiation. Furthermore, miR-215-5p could suppress CTNNBIP1expression by directly targeting it through promotion of the c-myc and CCND1expression which is the downstream gene of Wnt/β-catenin signaling pathway, resulting in inhibition of adipocyte differentiation. 4. miR-135a regulates fatty acid elongationThe mature miRNAs of miR-135a are highly conserved in many mammals through sequence alignment. Several bioinformatics target prediction softwares were used to predict targets of miR-135a. The results showed that ELOVL2and ELOVL6may be the targets of miR-135a. miR-135a-5p significantly inhibited the luciferase activity of ELOVL2and ELOVL63’UTR plasmid when compared to control groups through the dual luciferase reporter gene analysis.miR-215-5p could suppress ELOVL6expression in mouse3T3-L1adipocytes and miR-215-5p also could suppress ELOVL2and ELOVL6in mouse Hepa1-6hepatocytes by using Q-PCR. miR-135a may regulate fatty acid elongation.In summary, the conclusions of this study are:①miR-224could suppress EGR2expression by directly targeting it and impair adipocyte differentiation during early adipogenesis; miR-224also could suppress ACSL4expression by directly targeting it and regulate fatty acid metabolism.②miR-146b could suppress Runxltl and Smad4expression by directly targeting them and promote adipocyte differentiation during early adipogenesis.③miR-215could suppress FNDC3B and CTNNBIP1expression by directly targeting them and impair adipocyte differentiation during early adipogenesis.④miR-135a may regulate fatty acid elongation.