Studies On The Regulatory Effects Of MiR-135a And MiR-183on3T3-L1Preadipocyte Differentiation And Adipogenesis

This study aimed to investigate the effect of miRNA on adipocyte differentiation, and clarify the molecular mechanisms of miRNA regulating adipocyte differentiation and adipogenesis. Firstly, we chose the backfat tissues of150-day-old Large White and Meishan pigs as samples, and then constructed two small RNA libraries. High-throuput Solexa sequencing method and bio informatics softwares were used to identify miRNAs, analyze miRNAs with differentional expressions and predict novel miRNAs in both libraries. Then, these miRNAs with differentional expressions were subjected to validation, target prediction and function analysis, and miRNAs potentially correlated with preadipocyte differentiation, adipogenesis and lipid metabolism would be selected. Afterwards the analysis results found that miR-135a and miR-183may affect adipogenesis by regulating the canonical Wnt/β-catenin singnaling pathway. We hereby synthesized oligonucleotide miRNA mimics and inhibitor which simulate "gain-of-function" and "loss-of-function" to investigate the effects of miRNAs on triglyceride content and adipogenic marker genes expression levels of adipocyte. Meanwhile, the dual luciferase reporter gene analysis, QPCR assay and Western blotting examination were all adopted to validate the predicted targets. Finally, the flanking sequences of mmu-miR-183were experienced to predict by several bio informatics softwares. The core promoter of mir-183was subsequently isolated and cloned. ChlP-PCR experiment verified that GATA3is a transcription factor of mmu-mir-183, binding of GATA3with mir-183core promoter negatively regulated the transcriptional activity of pri-miR-183and maturity of miR-183. The main results as follows:1. Screening and identification of miRNAs in porcine backfatThe analyzed results by high-throughput Solexa sequencing showed that:①21,911,830and22,384,940small RNA raw reads were generated in backfat tissues of Large White and Meishan libraries, respectively, of which19,510,268and19,952,323were high-quality reads, accounting for89.04%and89.13%of raw reads.19,271,999(accounted for98.78%of high-quality reads) and19,613,350(accounted for98.30%of high-quality reads) clean reads were obtained.②121mature miRNAs were detected in both libraries comprising72known porcine miRNAs and49miRNA*were first identified in pigs which had homologs in other species.9conserved miRNAs with significantly differential expressions were found, of which the expressions of ssc-miR-135, ssc-miR-224, ssc-miR-146b and ssc-miR-215were higher in Large White library, opposite to the patterns shown by ssc-miR-la, ssc-miR-122, ssc-miR-133a, ssc-miR-183and ssc-miR-204.③140novelly predicted miRNAs (corresponding to156independent genomic loci) were identified in both libraries, of which87miRNAs were found in Large White library and102in Meishan library. Almost all novel miRNAs could be considered pig-specific except ssc-miR-1343, miR-2320, miR-2326, miR-2411and miR-2483which had homologs in Bos taurus.2. Expression pattern analysis and function prediction of miRNAs in procine backfatThe stem-loop QPCR method was utilized to analyzed6conserved miRNAs with significantly differential expressions and4novelly predicted miRNAs. The QPCR results validated the sequencing results and they were consistent but miR-la was an exception.Several bioinformatics target prediction softwares were used to predict targets of mouse miR-135a and miR-183. The results showed that3’UTR of APC gene contained two potential binding sites with miR-135a, GSK3β and LRP6may also be the separate target of miR-135a and miR-183. KEGG software analysis results showed that APC, GSK3β and LRP6are all important members of and participate in the canonical Wnt/β-catenin singaling pathway. We hereby speculated that miR-135a and miR-183may involve in the canonical Wnt/β-catenin singaling pathway by separately targeting APC, GSK3β and LRP6, and then regulate adipogenesis.3. Effect of miR-135a on3T3-L1preadipocyte differentiation and adipogenesisThe expression level of miR-135a decreased almost3times during3T3-L1preadipocyte adipogenic differentitation. The content of triglyceride was lower in miR-135a group, and meanwhile the expression levels of adipogenic maker genes (PPARy, C/EBPa, ap2and FAS) were also decreased.Validation of predicted target APC by several methods:miR-135a significantly inhibited the luciferase activity of APC3’UTR plasmid when compared to control groups (p<0.01). The mRNA and protein expression levels of endogenous APC were both suppressed by miR-135a, and viceversa, miR-135a inhibitor increased the expression of APC protein. Futhermore, mRNA expression levels of CCND1, c-myc and protein expression levels of nuclear P-catenin downstream canonical Wnt/β-catenin singaling pathway were increased in miR-135a group. Therefore, miR-135a activated the canonical Wnt/β-catenin signaling pathway by directly targeting APC, and then impaired3T3-L1preadipocyte differentiation and adipogenesis. 4. Influence of miR-183on adipogenesis of3T3-L1preadipocyte differentiationThe mRNA expression levels of adipogenic marker genes (C/EBPa, PPARy, adiponectin and FAS) were higher in miR-183group when compared to control groups after3T3-L1cells experienced to adipogenic differentiation for2days and4days. The amount of lip id droplets and triglyceride content were both increased in miR-183group at5days. Moreover, miR-183inhibitor also decreased the expressions of PPARy and C/EBPa and accumulation of triglyceride during3T3-L1adipogenic induction.Validation of predicted target LRP6:miR-183significantly inhibited the luciferase activity of LRP63’UTR plasmid when compared to control groups (p<0.01). The mRNA and protein expression levels of endogenous LRP6were both suppressed by miR-183. Futhermore, mRNA expression levels of c-myc and protein expression levels of nuclear P-catenin were impaired in miR-183group. Therefore, miR-183inhibited canonical Wnt/β-catenin signaling pathway by targeting LRP6, and then facilitated adipogenesis.5. Study of transcriptional activity of mmu-mir-183promoterThe sequences of mouse mir-183were predicted and analyzed by several online websites and softwares. The results displayed that the upstream sequences of miR-183have potential transcriptional strat sites, numerous transcription factor binding sites, CpG islands (about1200bp) and many EST data reports. We thus speculated that mir-183may have its own transcription unite.The promoter1(2309bp) and upstream fragment promoter2(1927bp) of miR-183were amplified, respectively. The results of dual luciferase reporter gene analysis showed that pGL3-mir-183promoter1has no luciferase activity, and pGL3-mir-183promoter2could significantly enhance the luciferase activity in3T3-L1, C2C12and BHK cells (p<0.01). Further experiments demonstrated that the176bp fragment in the proximal5’end of mir-183promoter2significantly enhanced the luciferase activity (p<0.01) and this176bp area may be the core promoter of mir-183. The transcription factors sp1and GATA3, which are related to adipogenesis, were found may bind with the core promoter predicted by online TFSEARCH and TESS softwares. The results of dual luciferase reporter gene analysis demonstrated that GATA3significantly decreased the luciferase activity of core promoter (p<0.01). Furthermore, the luciferase activity of core promoter with mutated GATA3binding sites was significantly increased when compared to wild core promoter (p<0.05). QPCR analysis results indicated that the expression level of mature miR-183was significantly inhibited by transcription factor GATA3. ChIP-PCR experiment verified binding of transcription factor GATA3with mir-183core promoter. These outcomes confirmed that binding of transcription factor GATA3with mmu-mir-183core promoter may inhibit the transcriptional activity of pri-miR-183and generation of mature miR-183and thus decreased its expression.Conclusion:①Identificaiton of more porcine miRNAs, and enrich the repertoire of miRNAs in pigs; Our experimental results displayed a high level of concordance with that of Solexa sequencing, showing that the results of high-throughput Solexa sequencing strategy are accurate, true and reliable.②miR-135a promoted the translocation of β-catenin protein from cytoplasm to nuclear by directly targeting APC, activated the canonical Wnt/β-catenin singaling pathway, inhibited the accumulation of triglyceride and expressions of adipogenic marker genes, and then impaired the3T3-L1adipogenic differentiation and adipogenesis.③miR-183inhibited canonical Wnt/β-catenin singaling pathway by directly targeting cellular surface receptor gene LRP6, acclerated adipocyte differentiation and accumulation of triglyceride, and then supported the adipogenesis of3T3-L1cells.④Binding of transcription factor GATA3with mmu-mir-183core promoter inhibited the transcriptional activity of pri-miR-183and generation of mature miR-183, and then led to the final decreased miR-183expression.