The Regulatory Mechanism Of SIRT6 On The Chemosensitivity Of Human Hepatoma Cells

ObjectiveTo investigate the underlying mechanism of SIRT6 on chemosensitivity of human hepatocellular carcinoma cells,we further revealed the role of SIRT6 in the chemotherapy of human hepatoma cells,thus providing new clues for clinical chemotherapy of HCC.Methods1.q RT-PCR and Western blot were used to detect the expression levels of Sirtuin family members in Huh-7 cells under the treatment of chemotherapeutic agents(Doxorubicin,Cisplatin,Sorafenib).2.qRT-PCR and Western blot were used to detect the expression levels of SIRT6 in Huh-7 and SK-Hep-1 cells under the treatment of chemotherapeutic agents.Western blot was used to detect the expression levels of SIRT6 in Huh-7 and SK-Hep-1 cells under the treatment of various concentration of chemotherapeutic drugs.3.Western blot was used to detect SIRT6-depleted efficiency in Huh-7 and SK-Hep-1 cells.Further,MTS assay and Flow Cytometry were used to detect the cell viability and apoptosis in SIRT6-depleted Huh-7 and SK-Hep-1 cells under the treatment of chemotherapeutic drugs,respectively.4.MTS assay and Flow Cytometry were used to detect the cell viability and apoptosis in SIRT6-overexpressing SK-Hep-1 cells with the treatment of chemotherapeutic drugs,respectively.5.qRT-PCR was used to screen multidrug resistance-related genes and apoptosis-related genes in SIRT6-depleted HCC cells under the treatment of chemotherapeutic drugs.Western blot was further applied to verify the expression level of MDR1 in HCC cells.qRT-PCR and Western blot were used to detect the expression level of MDR1 in SIRT6-overexpressing SK-Hep-1 cells with the treatment of chemotherapeutic drugs.qRT-PCR and Western blot were used to detect the expression level of MDR1 in HCC cells under the treatment of chemotherapeutic drugs.q RT-PCR was used to detect SIRT6 and MDR1 mRNA levels in 24 patients with HCC and the correlation between SIRT6 and MDR1 was analyzed.6.Dual luciferase reporter system was used to detect the promoter activity of MDR1 in SIRT6-depleted HCC cells under the treatment of chemotherapeutic drugs.Ch IP assay was used to detect the acetylation of H3K9 in MDR1 promoter in SIRT6-depleted HCC cells under the treatment of chemotherapeutic drugs.Databases(GENECARDS and PROMO)predicted transcription factors and q RT-PCR were used to screen the transcription factor by which SIRT6 regulated MDR1 promoter activity.qRT-PCR and Western blot were used to verify the expression level of C/EBPβ.Further,co-immunoprecipitation assay was used to detect the interaction between SIRT6 and C/EBPβ in HCC cells under the treatment of doxorubicin.Dual luciferase reporter system was used to detect the promoter activity of MDR1 in SIRT6-depleted and C/EBPβ-overexpressing HCC cells under the treatment of doxorubicin.Western blot was used to detect the protein level of MDR1 in SIRT6-depleted and C/EBPβ-overexpressing HCC cells under the treatment of doxorubicin.7.Flow Cytometry was used to examine the apoptosis in SIRT6-depleted and MDR1-overexpressing HCC cells under the treatment of chemotherapeutic drugs.Results1.Chemotherapeutic agents treatment increased the mRNA and protein expression of SIRT1,SIRT2,SIRT5,SIRT6,and SIRT7.In particular,the most significant increase has been observed in SIRT6.In contrast,SIRT3 expression decreased.2.Chemotherapeutic agent treatment enhanced SIRT6 expression in a dose-dependent manner.3.SIRT6 depletion resulted in decreased cell viability and increased apoptosis in HCC cells under the treatment of chemotherapeutic agents.4.SIRT6 overexpression increased cell viability and reduced apoptosis in HCC cells under the treatment of chemotherapeutic agents.5.SIRT6 depletion reduced MDR1 expression in HCC cells under the treatment of chemotherapeutic agents.Further,SIRT6 overexpression upregulated MDR1 expression in SK-Hep-1 cells under the treatment of chemotherapeutic drugs.Chemotherapeutic agents treatment increased the mRNA and protein expression of MDR1.The correlation between SIRT6 and MDR1 expression was positive in HCC tissues.6.SIRT6 depletion inhibited MDR1 promoter activity in HCC cells under the treatment of chemotherapeutic agents.However,the acetylation of H3K9 in MDR1 promoter was not changed in response to SIRT6 depletion.SIRT6 depletion reduced the mRNA and protein levels of C/EBPβ in HCC cells under the treatment of chemotherapeutic drugs,thereby inhibiting the promoter activity and protein level of MDR1.7.MDR1 overexpression restored the effect of SIRT6 depletion on apoptosis in HCC cells under the treatment of chemotherapeutic drugs.ConclusionSIRT6 depletion enhanced chemosensitivity of human hepatoma cells by downregulating MDR1 expression through suppressing C/EBPβ.SIRT6 may serve as a novel target to enhance chemosensitivity in HCC cells.