Effects Of Aberrant Methylation And Hypoxia On The Expression Of BNIP3in Esophageal Squamous Cell Cancer

Objective:Bcl-2/adenovirus E1B19kDa interacting protein3(BNIP3) is a mitochondrial proapoptotic protein. The expression and the function of BNIP3in different tumors and different development stages were not the same. Our previous study found that the expression of BNIP3in esophageal squamous carcinoma tissues was significantly lower than normal esophageal tissues. But the exact mechanism of low expression of BNIP3in esophageal cancer is not yet clear. In this present study, the aim was to detect the expression of BNIP3and to investigate the association of DNA methylation with histone H3lysine9(H3-K9) methylation and the expression of BNIP3in human esophageal cancer cells, and to identify the epigenetic regulatory mechanism on the expression of BNIP3in esophageal cancer cells. At the same time, cells were cultured in hypoxic microenvironment. We investigated the effects of hypoxia and silencing hypoxia inducible factor-la (HIF-la) gene by RNAi on the expression of BNIP3in esophageal cancer cells.Methods:1. KE4, Kyse-140, Caes-17and TE-7cell lines were cultured in vitro. Reverse transcription Polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression lever of BNIP3mRNA and protein. Furthermore, the promoter methylation status of BNIP3gene was detected by methylation specific PCR (MSP). The corresponding relationship between the expression of BNIP3and the methylation status of BNIP3gene was analyzed. At the same time, we selected the cell lines that were unmethylated or highly methylated in the promoter region of BNIP3gene respectively.2. Methyltransferase inhibitor5-azacytidine-2’-deoxvcytidines (5-Aza-CdR) was used to treat the cell lines that were highly methylated in the promoter region of BNIP3. RT-PCR, Western blotting and MSP were used to detect the expression of BNIP3and the methylation status of BNIP3in the cell lines before and after treated with5-Aza-CdR, and to identify the methylation regulatory mechanism on the expression of BNIP3in esophageal cancer cells. 3. Chromatin immunoprecipitation (ChIP) assay was used to assess the association between the status of histone H3lysine9(H3-K9) methylation and DNA methylation in the promoter regions of BNIP3gene and the expression of BNIP3before and after treated with5-Aza-CdR, and to identify the histone H3-K9methylation regulatory mechanism on the expression of BNIP3in esophageal cancer cells.4. To identify the effect of hypoxia on the expression of BNIP3, Modular Incubator Chamber (MIC-101) was used to mimic tumor hypoxic microenvironment. The expression of BNIP3mRNA and protein was detected by using fluorescence quantitative polymerase chain reaction (FQ-PCR) and western blot under normoxia, hypoxia(1%O2) or transfected with HIF-la-siRNA in esophageal cancer cells.Results:1. RT-PCR and Western blot showed that the expression of BNIP3was high in KE4and Caes-17cell lines, however, low expression was found in Kyse-140and TE-7cell lines. Hypermethylation status of BNIP3gene promoter region was observed in Kyse-140and TE-7cell lines by MSP.2. After treated with5-Aza-CdR, it was found to effectively reverse BNIP3gene methylation status of BNIP3gene promoter region in Kyse-140and TE-7cell lines and strongly up-regulate the expression lever of BNIP3.3. In Kyse-140cell line, BNIP3gene was characterized by histone H3-K9methylation, DNA methylation and lower expression.5-Aza-CdR was able to reduce histone H3-K9methylation(P<0.05) and reverse DNA methylation status of BNIP3gene and up-regulate the expression of BNIP3.4. In Caes-17cell line that was unmethylation of BNIP3gene, the expression of BNIP3mRNA and protein were increased under hypoxia than those under normoxia (P<0.05). After HIF-la-siRNA transfection, the HIF-la was down-regulated efficiently in Caes-17cells (inhibition ratio:90%), and the expression of BNIP3was obviously down-regulated as well (P<0.05).5. In Kyse-140cell line that was hypermethylation of BNIP3gene, the expression of BNIP3mRNA and protein were not increased under hypoxia than those under normoxia before treated with5-Aza-CdR (P>0.05). The expression of BNIP3mRNA and protein were increased under hypoxia than those under normoxia after treated with5-Aza-CdR. After HIF-1α-siRNA transfection, the HIF-1α was down-regulated efficiently in Kyse-140cells, and the expression of BNIP3was obviously down-regulated as well (P<0.05).Conclusion:1. The promoter hypermethylation may be one of the predominant inactivation mechanisms of the BNIP3gene in human esophageal cancer cell lines. Methytransferase inhibitor5-Aza-CdR can restore the expression of BNIP3by reversing methylation status of BNIP3gene promoter region.2. DNA methylation and histone H3-K9methylation in BNIP3gene promoter region play synergistic effect on the transcriptional repression of BNIP3gene. Methytransferase inhibitor5-Aza-CdR can restore the expression of BNIP3by reversing DNA hypermethylation status and reducing histone H3-K9methylation in the promoter of BNIP3gene.3. Hypoxia can increase the protein level of HIF-1α and up-regulate the expression of BNIP3. Silencing HIF-1α can reduce the expression of BNIP3which is induced by hypoxia.4. DNA methylation can inhibit the expression of BNIP3induced by hypoxia. Methytransferase inhibitor can restore the expression of BNIP3induced by hypoxia.