![]() ![]() 1 TGF-β-mediated EMT is associated with proliferative arrest. Overall, these results provide evidences for proliferative arrest coupled to TGF-β-mediated EMT differentiation in Huh7 cells.įig. The protein levels of E-Cad also showed a decrease validating the EMT of Huh7 cells ( Supplementary Figure S2). In accordance to above, analysis of RNA sequencing data from TGF-β exposed Huh7 cells also revealed an elevated expression level of mesenchymal candidate genes like Vimentin and N-Cad coupled to a decreased expression of epithelial cell marker- Epithelial-Cadherin (E-Cad) transcript level ( Supplementary Figure S1). Moreover, a clear enhancement in fluorescence signal and spatial re-arrangement of Vimentin was observed post TGF-β treatment ( Fig. Similar results were also observed at the protein level for N-Cad and Vimentin ( Fig. To further validate a differentiated phenotype, we analysed the transcript levels of the well-acknowledged EMT markers like Neural-Cadherin ( N-Cad) and Vimentin which also showed a significant increase with TGF-β exposure ( Fig. Importantly, the cortical actin network also showed considerable alterations upon cytokine treatment. As expected, the Huh7 cells attained a comparatively elongated phenotype post TGF-β exposure reflective of their probable mesenchymal differentiation, as observed through confocal imaging with F-actin phalloidin staining ( Fig. We initially monitored the overall morphology of the cells after treatment. This putative proliferative arrest was coupled to the canonical feature of TGF-β-induced effect- EMT. In addition, a significant decrease in expression of both CCNB1 and Cyclin A2 (CCNA2) was observed through real-time PCR suggesting a deregulation of cell cycle in Huh7 cells after exposure to the cytokine- TGF-β ( Fig. On the similar note, deep sequencing of total RNA from TGF-β exposed Huh7 cells revealed a 2-fold decrease in transcript level of cell cycle associated genes like, Cyclin B1 transcript (CCNB1) ( Fig. This was coupled with a significant decrease in protein expression of cellular proliferation associated molecular markers like, Proliferating Cell Nuclear Antigen (PCNA) after TGF-β administration ( Fig. ![]() Importantly, in our study, TGF-β exposure in Huh7 cells led to a considerable decrease in cell density in comparison to the untreated cells as observed through Crystal Violet staining ( Fig. Our results provide better understanding of cellular invasive features which can lead to development of novel therapeutic strategies. Overall, our findings reflect that HCC cells undergoing EMT, attain cytostasis and modulate metabolic demands to efficiently facilitate the EMT differentiation switch, and these events are regulated at the epigenomic level through TGF-β-mediated signalling. Importantly, TGF-β-downstream signalling mediator- SMAD and chromatin repressive complex member-enhancer of zeste homolog 2 (EZH2) were found to co-immunoprecipitate and were required for the above effects. An overall increase in total histone repressive mark (H3K27me3) associated with a specific enrichment of H3K27me3 at the upstream promoter region of CCNA2 and GOT1 was observed after TGF-β exposure, leading to their down-regulation. TGF-β resulted in down-regulation of cell cycle-associated transcripts, like Cyclin A2 (CCNA2), and metabolic genes, like Glutamic-oxaloacetic transaminase 1 (GOT1) through epigenetic silencing. ![]() Interestingly, EMT triggered by TGF-β was found to be associated with cytostasis and altered cellular metabolism. Therefore, as part of this study, we treated HCC cells with TGF-β and characterized the cellular processes associated with EMT. In spite of its clinical relevance, the cellular events associated with TGF-β-induced EMT and its molecular regulation is poorly elucidated. One of its classical functions involve facilitation of epithelial to mesenchymal transition (EMT), in tumour cells, promoting an invasive phenotype. In this context, one of the major cytokines present in the HCC tumour milieu is the transforming growth factor-β (TGF-β). A better understanding of the cytokine functions and their contributions to disease development is key to design of future therapeutic strategies and reduction of global HCC burden. Hepatocellular carcinoma (HCC) frequently unfolds under an inflammatory condition, which is a hub for a plethora of cytokines. ![]()
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