Study Of The Role And Relative Mechanism Of HSF1 In EMT-Associated Migration Which Effected By Low Glucose In HCC Cells

Reprogramming energy metabolism has been defined as the ninth hallmark of cancer and glucose deprivation might be a novel,feasible and effective approach for cancer treatment.However,the comprehensive illustration of behavior alteration of hepatocellular carcinoma(HCC)cells induced by glucose restriction is lacking and associated molecular mechanism is still unclear.In this study,the HCC cells were cultured with low glucose(LG)and control high glucose(Control)medium respectively,cell migration ability was assessed by the Transwell migration assay and wound healing assay;Then,the role of HSF1 in LG-induced inhibition of epithelial-mesenchymal transition(EMT)-associated migration was investigated by the RNA interference(sh RNA)and HSF1 expression rescue(sh RES)strategy;Bioinformatics analysis,Chromatin Immunoprecipitation(CHIP)and Luciferase reporter assay were used to further explore the associated molecular mechanism,which may provide a novel and effective therapeutic strategy for the treatment of HCC.PART ONE THE INFLUENCE OF LOW GLUCOSE ON EMT AND THE MIGRATION ABILITY OF HCC CELLSTransforming growth factor ?1(TGF-?1)can function as a potent inducer of epithelial plasticity leading to EMT.After treatment with 5ng/m L TGF-?1 for48 h,the morphology of HCC cells with Control medium was converted to a diffused fibroblast-like morphology,presented as the characteristic of EMT;in contrast,the morphology of HCC cell with LG culture had no significant change,as compared without TGF-?1 treated cells.Moreover,it was observed that significant up-regulation of E-cadherin and down-regulation of N-cadherin both in m RNA levels and protein levels in LG medium,compared with Control medium.In addition,the number of cells crossing the chambers was significantly decreased and the wound-healing closure of cells was conspicuously slower in LG medium,compared with those in Control medium,and cells were epithelial-like at the edge of wound lines in LG medium meanwhile cells were fibroblast-like in Control medium.This part showed that low glucose culture hampered typical EMT-like morphological change,“cadherin switching” and cell migration ability of HCC cells.PART TWO THE ROLE OF HSF1 IN THE INHIBITION OF EMT-ASSOCIATED MIGRATION BY LOW GLUCOSEHSF1 is a multifaceted transcription factor of carcinogenesis,which participates in the glucose metabolism of tumor cells and promotes malignant phenotype as well as metastasis.The result of RT-PCR and western blot showed that LG stimulation induced persistent downregulation of HSF1,speculating that HSF1 may be involved in the effects of glucose starvation on malignant phenotype of HCC cells.To clarify whether HSF1 plays a role inEMT-associated migration of HCC cells with LG culture,HCC cells were treated with sh NT or individual sh RNA against HSF1,besides,RT-PCR and western blot were used for verification.The results showed that in both Control and LG culture conditions,silencing the expression of HSF1 resulted significantly in low HSF1 expression,which verified that the knockdown of HSF1 gene was reliable.In addition,the specificity of sh HSF1-mediated effects was further documented by reintroducing HSF1-c DNA(sh RES)engineered to be insensitive to sh RNA.The results from Transwell migration assay and wound-healing showed that,comparing with Control condition,the numbers of HCC cells in control and sh NT groups crossing the chambers decreased significantly when cultured with LG condition.Notably,the significant increase of sh HSF1-treated HCC cells crossing the chambers was found as compared with sh NT-treated HCC cells in LG condition.Similar effects were observed in wound-healing assay,the closure of sh HSF1 cells was significantly faster as compared with sh NT-treated HCC cells in LG condition.These results indicated that HSF1 is critical for maintaining LG-mediated inhibition of HCC cells' EMT and migration ability.PART THREE THE RELATIVED MECHANISM OF HSF1 IN THE INHIBITION OF EMT-ASSOCIATED MIGRATION BY LOW GLUCOSETranscription factor Snail is the key molecule that triggers the process of EMT.Combined TRED database and Promoter 2.0 Prediction Server showed that the promoter sequence was located at-700 bp to 299 bp upstream of Snail gene.Moreover,a 5?-n GAAn-3? core sequence(heat shock element,HSE)and other related sequences of HSF1 binding sites were found in the promoter regionof Snail gene based on the prediction by Transcription Factors Database.We suggested that Snail may be downstream target molecule of HSF1 and HSF1 can directly regulate the transcriptional level of Snail.Chromatin immunoprecipitation(CHIP)and Luciferase reporter assay were used to further verified,the results showed that compared with Control group,relative luciferase activity was obviously enhanced in LG group,furthermore,DNA product combined with HSF1 in LG group was significantly higher than Control group.Results indicated that HSF1 can combine with the promoter of Snail and directly regulate its expression in transcriptional level under LG condition;in contrast,the binding force of HSF1 and Snail promoter was significantly weakened in Control condition.CONCLUSIONS1.Low glucose hampered typical EMT-like morphological change,“cadherin switching,” and cell migration of HCC cells.2.Low glucose stimulation induced persistent downregulation of HSF1 and HSF1 was required for migration of HCC cells with low glucose culture.3.HSF1 modulates the expression of EMT markers through directly regulation of snail transcription in glucose restriction medium.