Dataset Information

Next Generation Sequencing Facilitates Quantitative Analysis of the control 3T3-L1 (3T3-L1-NC) and LIGHT (tnfsf14) overexpression 3T3-L1 cells (3T3-L1-LIGHT) before and after differentiation into beige adipocytes

ABSTRACT: Purpose: The goals of this study are to compare NGS-derived 3T3-L1-NC transcriptome profiling (RNA-seq) to LIGHT overexpression 3T3-L1 cells and find out the DEGs Methods: mRNA profiles of 3T3-L1-NC and 3T3-L1-LIGHT before and after differentiation into beige adipocytes were generated by deep sequencing, in triplicate, using Illumina HiSeq. The sequence reads that passed quality filters were analyzed at the transcript isoform level. Results: Using an optimized data analysis workflow, we mapped about 6.53 Gb data per sample. The average genome mapping rate is 87.27% and the average gene mapping rate is 80.18%. 18,255 genes were identified in which 17,367 of them are known genes and 1,001 of them are novel genes. 13,086 novel transcipts were identified in which 9,304 of them are previously unknown splicing event for known genes, 1,001 of them are novel coding transcripts without any known features, and the remaining 2,781 are long noncoding RNA. Conclusions: Our study represents the first detailed analysis of the impact of LIGHT on gene expression in process of beige adipocytes biogenesis with biologic replicates, generated by RNA-seq technology.

ORGANISM(S): Mus musculus

PROVIDER: GSE114690 | GEO | 2018/05/22

REPOSITORIES: GEO

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Project description:Snail1 is a transcriptional repressor required for a correct embryonic development. In cancer, Snail1 promotes the epithelial to mesenchymal transition in tumorigenic epithelial cells. In this work, we have analyzed the control of Snail1 in the differentiation of the 3T3-L1 cell line derived from murine embryo cells. The activation by snail of 3T3-L1 induced typical markers of cancer-activated fibroblasts as S100A4 or CD44. We generated 3T3-L1 cells stably over expressing Snail1 (3T3L1/Snail1) and control (3T3-L1/control) cells. We used SILAC quantitative approach to identify and characterize protein alterations induced by Snail1. Cells were fractionated in 5 subcellular fractions. The nuclear fraction of the cells was separated by 10% SDS-PAGE. Gels with forward and reverse experiments were stained with Coomassie Blue and cut into 18 slices prior to reduction, alkylation and digestion with trypsin. Tryptic peptides were scanned and fragmented with a linear ion trap-Orbitrap Velos (ThermoScientific). We identified a total of 3108 proteins, with 2572 quantified proteins, and 565 proteins modulated >1.5-fold by Snail1 overexpression. Among them, we found interesting up-regulated proteins associated to early differentiation of adipogenesis (C/EBPβ) and down-regulated proteins implicated in the final stages of differentiation to adipocytes (Fatty acid-binding protein or Fatty acid synthase). We also observed as down-regulated proteins important mediators of PPARγ pathway. We also observed downregulation of proteins implicated in mTOR, SRC and JAK/STAT pathway. We validated these proteomics data by western blot and qPCR in 3T3-L1 cells and other types of fibroblasts with capable to differentiate to terminal mesenchymal phenotypes, as well as in mesenchymal stem cells (MSC). This work provided insight into novel proteins with potential roles in the regulation of differentiation of the 3T3-L1 and MSCs as Nr2F6, ASC-1, Prrx1 or Cbx6. These candidates are down regulated due to the overexpression of Snail1 in 3T3-L1 cells. We next investigated the potential binding of Snail1 to promoter of these candidates. In silico analysis with MatInspector program revealed various putative E-box consensus motifs for Snail1. We performed ChIP and Luciferase assay to validate Snail1 binds to different E-box motifs of our candidates. Additionally, we analyzed the ability to prevent the differentiation to adipocytes of the 3T3-L1 cells using siRNAs. This work provided insight into novel proteins with potential roles in the regulation of differentiation to adipocytes of the 3T3-L1 and mMSC cells as Nr2F6, ASC-1, Prrx1 or Cbx6 controlled by Snail1.

Dataset Information

Next Generation Sequencing Facilitates Quantitative Analysis of the control 3T3-L1 (3T3-L1-NC) and LIGHT (tnfsf14) overexpression 3T3-L1 cells (3T3-L1-LIGHT) before and after differentiation into beige adipocytes

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