| Background and objectiveHepatocellular carcinoma(HCC)is the 2nd leading cause of global cancer morbidity and mortality,and brings about 750,000 new patients and 695,000 deaths per year.Therefore,the elucidation of the molecular mechanisms of HCC pathogenesis and progression would provide a strong theoretical basis for targeted therapies and drug design.The local microenvironment is essential for the development and progression of the hepatocellular carcinoma(HCC).Due to insufficient blood supply,a prominent feature of solid tumors like HCC is hypoxia that induces cancer cells to change their signaling pathways and metabolic processes to adapt to the hypoxic challenge.Hypoxia inducible factor-1α(HIF-1α),a well-defined hypoxia responsive factor,activates diverse pathways that regulate cellular metabolism,angiogenesis,proliferation,and drug resistance.In a normal oxygen environment,HIF-1α is hydroxylated and rapidly degraded by the ubiquitin-proteasome pathway.In hypoxic conditions HIF-1α is no longer degraded but translocates into the nucleus and binds HIF-1β to activate the transcription of downstream genes.It has been shown that the up-regulation of HIF-1α activity promotes tumor-associated angiogenesis,and hence the survival and proliferation of tumor cells in solid tumors.Consequently,due to the basis for the transcriptional regulation of HIF1A expression in HCC is still unclear.It is crucial to elucidate the regulatory pathway leading to angiogenesis in the HCC context,starting at HIF-1α.This study focused on the Bcl-2 related transcription factor 1(BCLAF1).Up to date,BCLAP1 has been reported in many cell biological processes,such as the role of RNA regulation and tumor cell proliferation promotion.Previous studies have indicated that BCLAF1 associated with tumor angiogenesis.Our RNA-sequencing result revealed that depletion of Bclafl associates with the inhibition of the angiogenesis pathway and down-regulation of HIF1A mRNA levels.Thus,it is interesting to explore the effect of Bclafl on HIF-1α transcription and protein levels and the biological significance of Bclafl in HCC angiogenesis and tumor progression under hypoxic conditions.Methods1.The Aortic ring assay and Tube formation assay to detect angiogenesis2.CCK8 to test cell proliferation;Trypan blue staining to detect cell activity;Crystal violet to detect cell clone number3.The RT-PCR to detect gene expression in the cell4.Western Blotting,immunofluorescence to detect protein expression in the cell5.The dual-Luciferase assay to detect relative luciferase activity6.Immunohistochemical staining to detect tumors in protein expression7.The plasmid transformation and cell transfection8.Chromatin immune co-precipitation(ChIP)to detect the combination of protein and chromatin9.Nude mouse tumor formation to detect tumor growth in vivo10.Statistical AnalysisResultsBclafl levels are highly correlated with HIF-1α levels in HCC tissues,and that knockdown of Bclafl in HCC cell lines significantly reduces hypoxia-induced HIF1A expression.Furthermore,we found that Bclafl promotes HIF1A transcription via its bZIP domain,leading subsequently to increased transcription of the HIF-1αdownstream targets VEGFA,TGFB and EPO that in turn promote HCC-associated angiogenesis and thus survival and thriving of HCC cells.Moreover,we demonstrate that HIF-1α levels and microvessel density decreases after shRNA-mediated Bclafl knockdown in xenograft tumors.Finally,we found that Bclafl levels increase in hypoxia in a HIF-1α dependent manner.Therefore,our study identifies Bclafl as a novel positive regulator of HIF-1α in the hypoxic microenvironment.Conclusion1.Bclafl knockdown inhibits hypoxia and angiogenesis pathways2.Hypoxia enhances Bclafl-mediated growth promotion and angiogenesis3.Expression of Bclafl correlates with HIF1A expression in the HCC4.Bclafl regulates HIF1A transcription under hypoxia5.HIF1A transcription is controlled by Bclafl through its bZIP DNA-binding domain6.Bclafl is required for HIF-1α-mediated expression of angiogenic genes... |
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