Molecular Mechanism Of Histone Demethylase PHF8 Promoting Autophagy And HCC Progression

BackgroundHepatocellular carcinoma(HCC)is the most common type of primary liver cancer with high morbidity and mortality per year.Notwithstanding the rapid development of precise surgery and multiple discipline treatment,the long-term outcome of HCC still remains poor and 5-year overall survival is less than 50%.High malignancy with rapid progression,recurrence and metastasis are the major barriers for HCC treatment.To elucidate the underlying mechanism of development,invasion,metastasis and self-protect of tumor cells is critical for improving therapeutic effect in HCC.ObjectiveThis study aimed to explore the expression pattern of PHF8 as well as its clinicopathological significance,and followed by PHF8 biological function and relevant molecular mechanism on regulating cell proliferation,apoptosis,invasion and metastasis,and autophagy in HCC.Materials and Methods1.Differently expressed genes were screened from RNA-seq detection for 10 pairs of HCC with or without PVTT.Transcriptome datasets from public database including TCGA,GEO and Oncomine were obtained to compare the expression of PHF8 between HCC and normal liver tissues.2.RT-q PCR,western-blot and immunohistochemistry(IHC)were used to examine the expression of PHF8 in 198 pairs of HCC and corresponding adjacent tissues,which were collected from the Department of Hepatopancreatobiliary Surgery,First Affiliated Hospital,School of Medicine,Zhejiang University.Furthermore,the association of PHF8 expression with clinicopathological parameters and outcome was analyzed based on the results of IHC.3.Establish cell model with stable PHF8-knockdown in SMMC-7721 and Huh7 cell lines via transfection of sh RNA-lentivirus,and cell model with PHF8 overexpression in Hep G2 and SK-Hep-1 cell lines by plasmid transfection.Assess the regulation of PHF8 on liver cancer cell proliferation through CCK8 and colony formation in vitro and tumor cell xenograft subcutaneously in mice.Validate the suppression of PHF8 on apoptosis of liver cancer cells via flow cytometry and western-blot to measure the amounts of apoptotic markers.Combine Transwell assay,scratch/ wound healing assay and mice model with lung metastases to detect the migration,invasion and metastasis of liver cancer cells.Examine autophagy based on transfection of adenovirus with expression of m Cherry-GFP-LC3 fusion protein and measurement of autophagy related markers by western-blot.4.Examine the expression of EMT related genes through RT-q PCR in liver cancer cells with PHF8-knockdown.Verify the protein expression in liver cancer cells with PHF8-knockdown or-overexpression.Study the mechanism of PHF8 on regulating E-cadherin by SNAIL1 expression in rescue expression of combining PHF8-overexpression and SNAIL1-knockdown.5.Analyze the relationship between PHF8 level and expression of autophagy related genes(ATGs)based on the data from online databases GEPIA and ULACAN.6.Examine expression of FIP200,the ATG with the closest link to PHF8,by RT-q PCR and western-blot in liver cancer cells with PHF8-knockdown or-overexpression.7.Validate the contribution of PHF8-mediated FIP200 upregulation to autophagy,migration and invasion of liver cancer cells in the rescue experiment of combining PHF8-knockdown and FIP200-overexpression.Based on this rescue experiment,the effect of PHF8-FIP200-autophagy pathway on E-cadherin degradation was assessed through the treatment of CHX,or together with protease inhibitor MG132 or autophagy inhibitor CQ.8.Study the relationship among the expression of PHF8,FIP200 and E-cadherin in HCC tissues via IHC.9.Examine the amount of H3K4me3?H3K9me1/2?H3K27me2 and H4K20me1 in liver cancer cells with PHF8-knockdown or-overexpression.Detect the occupancy of PHF8 and histone H3K4me3,H3K9me1/2,H3K27me2 and H4K20me1 on promoter region of FIP200,SNAIL1,VIM,E-cadherin and N-cadherin.Analyze the ability of PHF8 and H3K4me3 to bind with the promoter region of FIP200,SNAIL1,VIM,E-cadherin and N-cadherin as well as the overlap promoter regions bound with PHF8 and H3K4me3.Results1.PHF8 upregulation was most significant among the differently expressed genes in HCC tissues with PVTT in comparison to those without PVTT.Then,higher expression of PHF8 expression was verified in HCC tissues than adjacent normal liver tissues according to the analysis of transcriptome datasets from public databases,and the results of RT-q PCR,IHC and western-blot based on HCC samples.Furthermore,PHF8 overexpression was positively correlated with tumor size,incomplete capsule,low grade of differention,advanced stage and poor outcome.PHF8 upregulation was an independent risk factor for predicting worse outcome.A remarkable increase in PHF8 m RNA and protein level was found in liver cancer cell lines by comparing with normal hepatocytes in vitro.2.PHF8-knockdown was able to suppress cell proliferation,colony formation,tumor growth and size,migration,invasion,rate of wound healing and autophagy,and increase the number of apoptotic cells and the amount the apoptotic proteins,while PHF8-overexpression led to opposite results.3.PHF8-knockdown had the ability to alter the expression of EMT related genes.Among them,SNAIL1,VIM and N-cadherin were downregulated while E-cadherin level increased.Opposite phenomena were observed in liver cancer cells with PHF8-overexpression.Specifically,E-cadherin exhibited more susceptible to PHF8 abnormal expression in protein level than m RNA level.4.The results of rescue experiment of combining PHF8-overexpression and SNAIL1-knockdown showed that the inhibition of E-cadherin by PHF8-overexpression could be ablated by SNAIL1-knockdown,which had little impact on the attenuation of E-cadherin protein mediated by PHF8-overexpression.5.Bioinformatic analysis demonstrated that PHF8 level was mostly related to FIP200 expression by comparison to other ATGs(r=0.47,P<0.0001).Moreover,PHF8-knockdown resulted in FIP200 decrease,while PHF8-overexpression elevated FIP200 level.6.The results of rescue experiment of combining PHF8-knockdown and FIP200-overexpression displayed that FIP200 could reverse PHF8-knockdonwnmediated suppression of autophagy formation,migration and invasion of liver cancer cells.7.According to the results of E-cadherin degradation experiment,the degradation of E-cadherin was merely affected by protease inhibitor MG132,but dramatically delayed by autophagy inhibitor CQ and PHF8-knockdown.Additionally,PHF8-knockdown-mediated inhibitory effect on E-cadherin degradation could be counteracted by FIP200-overexpression.8.IHC results showed positive relationship between expression of PHF8 and FIP200(r=0.6016,P<0.0001),both which exhibited negative association with E-cadherin expression(r=-0.3017 and-0.3750,respectively;both P<0.0001).9.PHF8-knockdown was able to reduce the protein amount of H3K4me3 while raise the protein level of H3K9me1/2,H3K27me2 and H4K20me1.The protein expressions of these histones were altered in opposite direction once PHF8-overexpression.10.The analysis of ENCODE database demonstrated that PHF8 and H3K4me3 were able to bind to the promoter region of FIP200,SNAIL1 and VIM.These promotor regions bound with PHF8 or H3K4me3 were highly overlapping.Ch IP results showed that both PHF8 and H3K4me3 could bind to promoter region of FIP200,SNAIL1 and VIM,and the binding process can be impeded by PHF8-knockdown.In addition,the promoter region of FIP200 could be occupied by H3K9me1/2,while the promoter region of SNAIL1 was able to bind with H3K9me1/2 and H3K27me2.Their binding processes were significantly enhanced by PHF8-knockdown.Conclusions1.PHF8 expression is significantly upregulated,indicating the malignant progression and poor outcome in HCC.2.PHF8 can promote cell proliferation,invasion and metastasis,and autophagy as well as suppress apoptosis in liver cancer cells.3.The enhancement of autophagy by PHF8 is reliable on FIP200 upregulation.Moreover,PHF8-FIP200-autophagy pathway contributes to E-cadherin degradation and metastasis of liver cancer cells.4.PHF8 contributes to transcriptional activity of FIP200,SNAIL1 and VIM,and this process is probably dependent on PHF8-mediated H3K4me3 upregulation and inhibition of H3K9m1/2 and H3K27me2.