BPTF Promotes Hepatocellular Carcinoma Progression By Targeting HTERT

Objective:As a core subunit of Nucleosome-remodeling factor(NURF)complex,the Bromodomain PHD Finger Transcription Factor(BPTF)plays important roles in chromatin remodeling.Recently,its function in tumor progression has attracted increased attention.However,its precise function and molecular mechanisms in hepatocellular carcinoma(HCC)development is still poorly defined.Here,we demonstrated the tumor-promoting role of BPTF in HCC progression.BPTF was found to be highly expressed in HCC cells and tissues compared with normal liver cells and tissues.Its silencing led to the inhibited proliferative capacity,colony formation ability and stem cell-like traits in HCC cells.Furthermore,BPTF knockdown effectively sensitized the anti-tumor effect of chemotherapeutic drugs and led to more apoptosis induction in HCC cells.Consistently,the silencing of BPTF in xenografted human HCC cells decreased tumor growth and metastasis accompanied by the down-regulated levels of cancer stem cells(CSC)-related protein markers.Of note,our study provided mechanistic insights into the tumor-promoting role of BPTF in HCC by transcriptionally regulating hTERT expression,thereby elevating hTERT-mediated HCC cell survival and stemness maintenance.HCC patients with high BPTF expression also displayed high hTERT expression,and high BPTF or hTERT expression level was positively correlated with advanced malignancy and poor survival in HCC patients.In summary,our results demonstrate the protumorigenic function for BPTF in HCC progression and also suggest the novel BPTF-hTERT axis in HCC with potential therapeutic and prognostic significance.Methods:1.Using Western blot analysis,we studied the expression of BPTF in liver cancer cells and liver cancer tissue;using Oncomine public database,we studied the expression of BPTF mRNA in liver and liver cancer tissues;Through the TCGA database,Changes of DNA copy number in liver and liver cancer tissues were studied.2.Knocking down BPTF,The effect of BPTF on cell proliferation were detected by MTT assay and colony formation assay.The effect of BPTF on cell migration and invasion were evaluated by scratch assay and Transwell assay.3.Silencing BPTF was used to detect the effect of BPTF on the hepatocellular carcinoma cells stemness by tumor sphere formation assay,detection of stemness marker CD24,CD44 by flow cytometry and Western blot.4.Detecting the IC50 values of antitumor drugs DDP and 5-Fu and the sensitivity of chemotherapeutic drugs to liver cancer cells by AOEB assay,flow cytometry and Western blot by Knocking down BPTF.5.The effect of BPTF on the luciferase reporter gene activity of hTERT promoter was detected by knockdown of BPTF.The interaction between BPTF and h TERT was detected by chromatin immunoprecipitation.The knockdown of BPTF and overexpression of hTERT were performed to colony formation assay,tumor sphere formation assay,Western blot assay and detected whether BPTF can affect the proliferation and stemness of liver cancer by targeting hTERT.6.Animal experiments,through the establishment of stable cell lines,the cells were injected subcutaneously to observe the formation of subcutaneous xenografts in mice;the stable cell lines were injected into the tail vein of nude mice to observe the lung metastatic ability of liver cancer cells.7.To verify the relationship between the BPTF expression and TNM stage,and the expression of BPTF and hTERT on the survival rate of patients with hepatocellular carcinoma through the analysis of array tissue containing 81 hepatocellular carcinoma samples.Results:1.Compared with liver tissues,BPTF protein expression of hepatocellular carcinoma tissue is high;through the database,we found that BPTF mRNA expression and DNA copy numbers in liver cancer tissues were higher than the liver tissue.2.The study found that BPTF knockdown reduced the proliferation,migration,invasion,stemness characteristic and increased sensitivity to chemotherapy drugs.3.BPTF bound to specific hTERT promoter region and interacts with hTERT,and promoted the proliferation and maintenance of hepatocellular carcinoma cells by regulating the expression of cancer stem cell(CSC)-related proteins.4.The silencing of PTTF reduced the proliferation ability of xenograft tumor and lung metastasis ability,accompanied by the decreased expression of cancer stem cell(CSC)-related protein marker in xenograft and lung metastases.5.Tissue microarray analysis showed that the expression of BPTF was correlated with TNM stage.The Kaplan-Meier survival curve showed that the high expression level of BPTF and hTERT predicted the poor prognosis of patients with liver cancer.6.Through the analysis of clinical samples,BPTF and hTERT have a great correlation with the protein expression level.Conclusion:Our data provide evidence of functional and mechanistic evidence that BPTF contributes to the progression of HCC via transcriptional activation of hTERT expression,including promoting tumor cell proliferation,maintaining stem cell properties and increasing tumor metastasis.Our data also demonstrate the contribution of BPTF in regulating the sensitivity of hepatocellular cells to chemotherapeutic drugs,such as knockdown of BPTF increases the sensitivity of hepatocellular cells to DDP and 5-FU as well as cellular apoptosis.All results indicate that activation of BPTF may serve as a biomarker to promote tumor progression,predict poor prognosis,and serve as a promising therapeutic target for HCC patients.