Histone Deacetylase Inhibitor Downregulate The Bmi-1by Repressing C-Myc

Background and ObjectLiver cancer is the fifth most common cancer world wide and the third leading causeof cancer death. Primary liver cancer consists predominantly of hepatocellular carcinoma(HCC) and intrahepatic cholangiocarcinoma (ICC). Recent studies have shown that livercancer stem cells play an important role in the occurrence, metastasis and recurrence ofliver cancer. It is important understanding the mechanism of self-renewal anddifferentiation of liver cancer stem cells and hepatic progenitor cells in the establishmentof therapeutic approach by targeting liver cancer stem cell therapy.In our previous work, we found histone deacetylase inhibitor (HDACi) sodiumbutyrate (NaB) can downregulate Bmi-1(B lymphoma murine leukemia virus insertionregion1homolog) expression in Liver Epithelial Progenitor Cells (LEPCs), and inducedLEPCs to differentate into mature hepatocytes. Bmi-1is a member of Polycomb group(PcG) family, PcG play an important role in maintaining a variety of tissue stem cellself-renewal and are highly expressed in many tumors. Recent studies also show thatBmi-1is one of the hallmarks of liver cancer stem cells. This study was to investigate therole of Bmi-1in liver cancer cell and liver progenitor cell proliferation; study the HDACiinduced down-regulation of Bmi-1and the underlying mechanisms; and observe thetreatment effects of HDACi on liver tumors.MethodsIn this study, we first infected liver progenitor cell line LEPCs with Bmi-1shRNAmediated by lentivirus. The knockdown efficiency of Bmi-1was analyzed in both mRNAand protein levels. Cell cycle was analyzed by FACS assay. Cell proliferation wasrevealed by immunostaining of Ki-67. Then, mouse hepatoma carcinomaline (Hepa1-6and H22cells) were treated by three small molecules HDACi (Sodium butyrate, NaB),Valproic Acid (VPA) and Vorinostat (SAHA), the expression of Bmi-1after treatmentwas analyzed by quantitative RT-PCR and Western blot assays. Cell proliferation wasdetermined Ki-67immunostaining. The effect of HDACi on cell proliferation of mousehepatocellular carcinoma cells with Bmi-1overexpression was also analyzed. Thepossible mechanisms of HDACi induced Bmi-1down-regulation were elucidated byanalyzing the upstream regulatory genes of Bmi-1by qRT-PCR in mouse hepatomacarcinoma. SAHA treated and SAHA untreated Hepa1-6cells were transplanted into C57BL/6mice in left and right flanks. Control groups were injected with PBS, and theexperimental groups were administered continuously with SAHA,(50mg/kg)3weeks.The tumor tissue was analyzed to check whether HDACi downregulate the expression ofBmi-1in tumor tissue, and inhibit the growth of tumor cells and tumor formation.Hepa1-6cells transfected with control plasmid and the Bmi-1overexpressing plasmidwere transplanted into the left and right flanks of C57BL/6mice. Control groups wereinjected with PBS, and the experimental groups were continuous administration withSAHA (50mg/kg)3weeks. The tumor sizes were measured. The tumor tissues were usedto analyze cell proliferation. At the same method using H22cells were transplanted intoKunming mice in vivo.Then, we examined whether the transcriptional downregulation of c-Myc by HDACiin Hepa1-6. Hepa1-6cells were transfected with c-Myc to detect whether c-Myc restoreBmi-1expression under HDACi treatment.ResultsThis study found that Bmi-1shRNA transduction significant downregulated theexpression of Bmi-1in LEPCs. Ki-67immunofluorescence staining showed that cellproliferation was significantly reduced. Cell cycle analysis showed that cells werearrested at G0/G1phase. At the same time, expression of mature hepatocytes markerswas upregulated in LEPCs. Further, we found that HDACi (including NaB, VPA andSAHA) can significantly reduce the expression of Bmi-1mRNA and protein in mousehepatoma cell lines (Hepa1-6and H22), and significantly inhibit the proliferation ofhepatocellular carcinoma cells. Hepa1-6and H22cells overexpression of exogenousBmi-1can tolerance HDACi inhibiton on cell proliferation.We found that HDACi couldsignificantly downregulate c-Myc expression by hepatoma cells. c-Myc is upstreamregulatory genes of Bmi-1; Hepa1-6transfected c-Myc plasmids can restore the HDACireduced Bmi-1expression. Our results show that HDACi reduced Bmi-1expressionthrough repressing c-Myc in hepatocellular carcinoma cells.In vivo tests found that mouse Hepa1-6and H22tumor weight and volume werelower than those in the control group after the SAHA treatment, while SAHA candownregulate the expression of Bmi-1in solid tumor, and the inhibition of cellproliferation. The tumor cells overexpressing exogenous Bmi-1significantly enhancetumorigenic ability. The cells were obviously tolerant of HDACi on growth inhibition. ConclusionBmi-1plays an important role in self-renewal and maintains LEPCs; HDACi caninhibit the growth of mouse hepatocellular carcinoma by down-regulating Bmi-1; HDACirepress Bmi-1expression by down-regulating the expression of c-Myc. This study showsthat targeting Bmi-1through modulating epigenetic mechanism could be new method forliver cancer treatment.