BCLAF1 Regulates The Expression Of HIF-1 Alpha And Promotes Hepatocellular Carcinoma Cells Proliferation Under Hypoxia Condition

Background and objectiveAccording to the data from the International Cancer Center in 2014,the estimated mortality of liver cancer had increased as many as 19.6 percent in men and 13 percent in women in China.And the new cases of men had been up to 293318.All of these indicates the importance and the urgency of primary liver cancer research in China.Hepatocellular carcinoma(HCC)is one of the most harmful and highest incidence of malignant tumors in our country.The typical micro-environment of liver tumor is characterized by hypoxia,which is dueing to the vascular growth can't keep up with the tumor growth,multiplication rate,leading to the insufficient of blood supply for tumors.When the oxygen concentration was as low as 1%,the stress effects of hypoxia shows up,stimulates the tumor to produce stress protein Hypoxia Inducible Factor-1alpha HIF-1alpha(HIF-1?).It is the key transcriptional regulation factors in adaptation response,related signaling pathways activation.HIF-la plays crucial roles in maintaining the metabolism of tumor cells,so the regulation pathways of HIF-1 alpha is important for the research of liver cancer.This study focused on the Bcl-2 related transcription factor 1(BCLAF1).Up to date,BCLAF1 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.Since HIF-1 alpha is tightly related to tumor angiogenesis,whether there is a certain regulation relationship between BCLAF1 and HIF-1 alpha?So,this study discussed the relationship between BCLAF1 and HIF-1 alpha in liver cancer cells from in vivo and in vitro experiments,and the regulatory mechanism.Therefore,this study intended to explore the mechanism of BCLAF1 involved in liver cancer proliferation under hypoxia,then provide a new possibility for the BCLAF1 related diagnosis and therapyt in Hepatocellular carcinoma patients.Methods1.CCK8 to test cell proliferation;Crystal violet to detect cell clone number;Trypan blue staining to detect cell activity2.The Annexin V-FITC and PI staining to detect cell apoptosis3.The RT-PCR to detect gene expression in the cell4.Western Blotting,immunofluorescence to detect protein expression in the cell5.The immune co-precipitation(Co-IP)to detect the protein interaction relations6.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.The dual luciferase to detect the report gene10.The experiment data were described as mean±S.D.The differences between groups were analyzed by One-Way ANOVA,Dunnett,Student's t-test or simple regression analysis with SPSS 16.0 according to data feature.Significant level is p?0.05.Results1.The expression of BCLAF1 has a positive correlation with the expression of HIF-1?High expression of BCLAF1 and HIF-1? in liver cancer tissue samples from HCC patients,and there is an apparently positive correlation between BCLAF1 and HIF-1?.The expression level of two proteins in HepG2 and Huh7 cells has time-dependented under the hypoxia2.BCLAF1 regulates the expression of HIF-1? under hypoxia condition via down-regulating the expression and activity of HSF1Under the hypoxia the down-regulation of BCLAF1 by siRNA transfection in HepG2 and Huh7 cells reduced HSF1 expression and activity,as demonstrated by Western Blot and the dual luciferase detection.There is a HSF1 binding sites on HIF-la promoter.It could be reduced the combination of HSF1 and HIF-1? promoter after knocking down BCLAF1,and then the expression of HIF-1? was reduced including protein and mRNA.3.HIF-1? regulates the expression of BCLAF1 under the hypoxiaUnder the hypoxia the down-regulation of HIF-1? by siRNA transfection in HepG2 and Huh7 cells reduced the protein expression of BCLAF1 but the mRNA expression level was higher than the cotrol group.Investigating its reason:there is a HSF1 binding sites on BCLAF promoter,it could be increased the combination of HSF1 and BCLAF1 promoter after knocking down HIF-1?,that's the reason of high mRNA expression level;And because of the reduction of HSF1 and HSP70 which have the function of stablizing protein,the protein expression of BCLAF1 was lower than the cotrol group.And then cells knocking down HIF-1? were co-incubated with CHX?MG132 respectively,BCLAF1 protein expression has a recovery after co-incubated with MG132,and is lower after co-incubated with CHX4.The proliferation inhibition effect of BCLAF1 on hepatocellular carcinoma(HCC)To inhibit BCLAF1 could inhibit the tumor cell proferation in hypoxia,as dem-onstrated by CCK8 and crystal violet assay.At the same time knocking down BCLAF1 in tumor tissues of orthotopic hepatocarcinoma xenograft nude mice can down-regulate the expression of HIF-1?.Conclusion1.This study found that BCLAF1 is upregulated in hypoxic tumor and correlates significantly with HIF-1?2.There is the interaction between BCLAF1 and HSF1,and these two proteins were localized in the nucleus region.BCLAF1 can regulates the expression and activity of HSF1.3.BCLAF1 could regulate HIF-la expression under Hypoxia via mediating HSF1 in vitro and in vivo4.HIF-la could influnce the expression of BCLAF1 under Hypoxia via mediating HSF15.BCLAF1 impacts the tumor cell proferation in hypoxia.