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Expression profile of select glucose and lipid metabolic genes mediated by STIM1-Snail1 circuit in SMMC7721 cells

ABSTRACT: To investigate the altered glucose and lipid metabolic genes after Snail1 overexpression, Snail1 plus STIM1 double overexpression in SMMC7721 cells. Snail1 overexpression (Snail1 OE)- and Snail1 plus STIM1 double overexpression(Snail1+STIM1 dOE)- SMMC7721 cells were established via lentiviral Infection. The cells mentioned above were cultured in DMEM supplemented with 10% (v/v) FBS at 37°C in 5% CO2. When the cells grow to 60~70% confluent, Total RNA was extracted by using Trizol (TAKARA, Japan). Reverse transcription was performed from 1μg total RNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA), Quantitative real-time PCR were performed (CFX386, BioRad) with 40 cycles at 95 oC for 10 seconds, 59 oC for 20seconds and 72 oC for 30 seconds.

ORGANISM(S): Homo sapiens

PROVIDER: GSE135901 | GEO | 2019/08/17

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.

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Expression profile of select glucose and lipid metabolic genes mediated by STIM1-Snail1 circuit in SMMC7721 cells

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