A deep investigation into the adipogenesis mechanism: Profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/β-catenin signaling pathway

Abstract

BackgroundMicroRNAs (miRNAs) are a large class of tiny non-coding RNAs (~22-24 nt) that regulate diverse biological processes at the posttranscriptional level by controlling mRNA stability or translation. As a molecular switch, the canonical Wnt/β-catenin signaling pathway should be suppressed during the adipogenesis; However, activation of this pathway leads to the inhibition of lipid depots formation. The aim of our studies was to identify miRNAs that might be involved in adipogenesis by modulating WNT signaling pathway. Here we established two types of cell model, activation and repression of WNT signaling, and investigated the expression profile of microRNAs using microarray assay.ResultsThe high throughput microarray data revealed 18 miRNAs that might promote adipogenesis by repressing WNT signaling: miR-210, miR-148a, miR-194, miR-322 etc. Meanwhile, we also identified 29 miRNAs that might have negative effect on adipogenesis by activating WNT signaling: miR-344, miR-27 and miR-181 etc. The targets of these miRNAs were also analysed by bioinformatics. To validate the predicted targets and the potential functions of these identified miRNAs, the mimics of miR-210 were transfected into 3T3-L1 cells and enlarged cells with distinct lipid droplets were observed; Meanwhile, transfection with the inhibitor of miR-210 could markedly decrease differentiation-specific factors at the transcription level, which suggested the specific role of miR-210 in promoting adipogenesis. Tcf7l2, the predicted target of miR-210, is a transcription factor triggering the downstream responsive genes of WNT signaling, was blocked at transcription level. Furthermore, the activity of luciferase reporter bearing Tcf7l2 mRNA 3' UTR was decreased after co-transfection with miR-210 in HEK-293FT cells. Last but not least, the protein expression level of β-catenin was increased in the lithium (LiCl) treated 3T3-L1 cells after transfection with miR-210. These findings suggested that miR-210 could promote adipogenesis by repressing WNT signaling through targeting Tcf7l2.ConclusionsThe results suggest the presence of miRNAs in two cell models, providing insights into WNT pathway-specific miRNAs that can be further characterized for their potential roles in adipogenesis. To our knowledge, present study represents the first attempt to unveil the profile of miRNAs involed in adipogenesis by modulating WNT signaling pathway, which contributed to deeper investigation of the mechanism of adipogenesis.

Fine-mapping quantitative trait loci affecting murine external ear tissue regeneration in the LG/J by SM/J advanced intercross line

“…Interestingly, the microRNA 344 family is found in our Q7.1 locus, is differentially expressed, and may increase canonical WNT signaling and affect Wnt3a expression (Qin et al, 2010). Another candidate gene, Lgr6, enhances Wnt/b-catenin, and thus WNT3a, signaling through increased LGR6 phosphorylation by high affinity binding to R-spondins 1-3 (Gong et al, 2012).…”

MicroRNA-30c promotes human adipocyte differentiation and co-repressesPAI-1andALK2

“…9 Collectively, these results suggest a strong involvement of miRNAs in the process of adipogenesis. In line with this, several in vitro studies have already revealed distinct miRNAs to steer murine adipocyte differentiation, 6,[10][11][12][13][14][15][16][17] while only a few studies did so for human. [18][19][20][21] We are interested in miRNAs with a regulatory role in adipocyte differentiation and function, particularly in human.…”

“…Protease Inhibitor Cocktail (PIC, #1836170) was obtained from Roche. Trypsin (#15400054), Phosphate buffered saline (PBS, #10010015), 1M HEPES (#15630122), L-Glutamine (#25030024), TRIzol reagent (#15596018), SuperScript II Reverse Transcriptase Kit (#18064014), dNTP mix (#10297018), random hexamer primers (#48190011), oligo (dT) [12][13][14][15][16][17][18] primers (#18418012), Platinum SYBR Green qPCR SuperMix-UDG w/ROX (#11744500) and RNase OUT Recombinant Ribonuclease Inhibitor (#10777019) were purchased from Invitrogen. Universal cDNA Synthesis Kit (#203300), SYBR Green master mix (#203400) and primers for detection of miR-30c (#204783) and 5S rRNA (#203906) were purchased from Exiqon.…”

“…Total RNA was obtained using TRIzol. 0.5-1 μg of RNA were DNase digested with RQ1 RNase-Free DNase and cDNA synthesis was performed with random hexamer primers and oligo (dT) [12][13][14][15][16][17][18] primers using SuperScript II Reverse Transcriptase following manufacturer's instructions. The qRT-PCR reaction volume was 18 μL, consisting of 4.5 ng reverse transcribed RNA in water, 200 nM forward and reverse primer (sequences in online Table S1) and Platinum SYBR Green qPCR SuperMix-UDG with ROX.…”

miR-135a-5p inhibits 3T3-L1 adipogenesis through activation of canonical Wnt/β-catenin signaling

“…For example, miR-143, the miR-17-92 cluster, and miR-375 accelerate 3T3-L1 adipogenic differentiation (Esau et al 2004, Wang et al 2008, Ling et al 2011, Yi et al 2011, while let-7 and the miR-27 family impair preadipocyte differentiation (Karbiener et al 2009, Sun et al 2009, Kim et al 2010. In addition, miR-210 has been reported to promote adipogenesis by inhibiting the canonical Wnt/b-catenin signaling pathway (Qin et al 2010).…”