SIRT6 Overexpression Potentiates Apoptosis Evasion In Hepatocellular Carcinoma Via BCL2-Associated X Protein-Dependent Apoptotic Pathway

Objective Through the study of the activation of the BCL2-associated X protein (Bax) signaling pathway regulated by SIRT6 via its deacetylase activity to discuss the mechanisms of hepatoma carcinoma.Methods1.The expression level of SIRT6 in 101 pairs of hepatoma carcinoma(HCC) and adjacent nontumoral liver was analyzed by Western Blot and qRT-PCR.The expression level of SIRT6 in primary hepatocyte(PHH),immortalized human liver cell line MIHA and 6 HCC cell lines was determined by Western Blot and qRT-PCR.2.The level of SIRT6 in HCC cell lines was infected with the lentivirus expression shSIRT6.The proliferation number of HCC cells was performed by trypan blue exclusion assay,DNA synthesis was analyzed by EdU incorporation assay.The ability of colony was determined by colony formation assays.The apoptosis of HCC cells was tested by flow cytometry and Western Blot.3. SIRT6 was overexpressed in immortalized human liver cell line MIHA by transfection of vector expressing SIRT6. The proliferation number of MIHA was performed by trypan blue exclusion assay,DNA synthesis was analyzed by EdU incorporation assay.The ability of anchorage-independent growth was determined by soft agar assays.The apoptosis of MIHA cells was tested by flow cytometry and Western Blot.4. The mRNA level of genes related to apoptosis were detected by qRT-PCR.The protein level of screening genes were furthered tested by Western Blot. The localization of Bax was observed by Immunofluorescence and Western Blot. The proliferation number of HCC cells transfected with Bax siRNA and shSIRT6 was performed by trypan blue exclusion assay.The apoptosis of HCC cells transfected with Bax siRNA and shSIRT6 was tested by flow cytometry and Western Blot.5. The changes of core sequence of Bax promoter in SIRT6 silencing cells was analysed by Dual-Luciferase Reporter Assay System.The histone protein acetylation level of core sequence of Bax promoter was tested by Chromatin Immunoprecipitation. The transcription factors to Bax promoter were tested by Chromatin Immunoprecipitation.6. The localization of SIRT6 and Ku70 were observed by Western Blot.The interaction of Ku70-Bax was tested by Co-immunoprecipitation.The protein level of Ku70 in SIRT6-depleted cells was analyzed by Western Blot. The acetylation level of Ku70 in SIRT6-depleted cells was determined by Co-immunoprecipitation. The interaction of Ku70-Bax in SIRT6 cells was investigated by Co-immunoprecipitation.7. The cell mode results were tested by xenograft tumor model in nude mice.Results1. Western blot analysis indicated that SIRT6 overexpression was detected in 65/101 HCCs (64%,p<0.001). Patients with high SIRT6 expression levels in tissues had significantly shorter overall survival rates than those with low SIRT6 expression (p=0.024). SIRT6 was almost undetectable in PHHs, whereas high-level expression of SIRT6 was observed in the 6 HCC cell lines at both the mRNA and protein levels.2. The loss of SIRT6 significantly decreased the proliferation rate of all HCC cell lines. EdU staining also indicated that the loss of SIRT6 dramatically inhibited DNA synthesis.SIRT6 knockdown reduced the numbers and sizes of cell colonies as determined by a colony formation assay. SIRT6 depletion induced HCC cell lines apoptosis.3. A stable liver cell line MIHA overexpressing SIRT6 exhibited increased growth rates and enhanced DNA synthesis. SIRT6 overexpression in MIHA cells promoted the anchorage-dependent growth of these cells, SIRT6 overexpression significantly enhanced MIHA cell resistance to apoptosis in response to doxorubicin treatment.4. Gene expression profiling indicated that SIRT6 silencing resulted in significant alterations in the Bax-mediated proapoptotic pathway. SIRT6 knockdown resulted in the marked redistribution of Bax to mitochondria. Bax silencing significantly reduced apoptosis that was initiated by silencing SIRT6.5. SIRT6 was recruited to the promoter of Bax, where it deacetylated histone 3 lysine 9 (H3K9) and suppressed Bax promoter activity. Binding of transcription factors (p53 and E2F-1) to Bax promoter was also generally increased in SIRT6-depleted cells.6. SIRT6 also disrupted the Ku70-Bax interaction by deacetylating Ku70 and sequestered Bax from mitochondria. Importantly, silencing of Bax expression abolished the proapoptotic effects exerted by SIRT6 suppression in HCC cells.7.1n mouse xenografts, SIRT6 suppression inhibited the growth rate and size of tumors. Conclusion SIRT6 is an important protumorigenie factor in liver carcinogenesis. Thus, the therapeutic targeting of SIRT6 may offer options for HCC treatment.