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  • br hTERT was required for the BPTF mediated proliferative pr


    3.5. hTERT was required for the BPTF-mediated proliferative promotion and stemness maintenance in HCC cells
    Given the role of BPTF in mediating chromatin remodeling and gene expression, we hypothesized one or more potential target genes tran-scriptionally regulated by BPTF might contribute to its function in HCC progression. Considering hTERT functionalizes as a pivotal factor in HCC tumorigenesis and stemness promotion [30,31], we then in-vestigated the expression regulation of hTERT by BPTF in HCC to ex-plore the molecular mechanism accounting for BPTF's tumor-promoting effect and stemness maintenance in HCC development. Firstly, we analyzed hTERT expression in tumor tissues from 9 cases of HCC pa-tients, the samples of which were the same as the ones used in Fig. 1 for BPTF expression determination. hTERT similarly displayed high ex-pression at mRNA and protein level in 5 cases, and its expression trend was positively correlated with BPTF expression (Fig. S3A, B). Con-sistently, in HepG2 and Hep3B cells, BPTF knockdown significantly down-regulated hTERT expression at the protein and mRNA levels, and hTERT promoter-driven luciferase expression when the promoter se-quence corresponds to −2063-+40, −1605-+ 40 and −902-+ 40, but not to other regions (Fig. 5A, B, C), suggesting the transcriptional regulation of hTERT by BPTF. These results also showed that the pos-sible regulatory site was located at hTERT promoter region −902-−321. ChIP assay further verified the binding of BPTF at this fragment of hTERT promoter (Fig. 5D). We further analyzed the binding of me-thylated histones at hTERT promoters upon BPTF silencing. As shown in Fig. 5D, BPTF knockdown caused nearly no change of their bindings at hTERT promoters. r> In order to clarify whether the elevated proliferative capacity and 
    stemness traits mediated by BPTF depend on its regulation on hTERT, we first investigated the effect of hTERT itself on stemness maintenance in HCC cells. As shown in Fig. S2, when hTERT expression was down-regulated, the expression of BPTF was nearly unchanged, while the expression of hallmarks related to stemness maintenance (EpCAM, CD44 and c-kit) were inhibited. We next overexpressed hTERT in HCC Vaborbactam following BPTF knockdown. Colony formation and tumorsphere formation analysis indicated that hTERT overexpression reversed BPTF silencing-caused inhibition of tumor cell growth and tumorsphere-forming ability (Fig. 5E, F). The down-regulated expression of hall-marks related to stemness maintenance mediated by BPTF silencing, including EpCAM, CD44 and c-kit, was also reversed under hTERT overexpression (Fig. 5G). Thus, the suppression of tumor cell growth and CSC-related features with BPTF silencing in HCC is at least partially mediated by the down-regulated hTERT level.
    3.6. BPTF knockdown suppressed tumor growth and CSC-associated characteristics in a HCC xenograft mouse model
    The effect of BPTF on HCC development was further validated in vivo based in a HCC xenograft mouse model. Hep3B cells with or without stably knocked down expression of BPTF were subcutaneously injected into the right flank of nude mice respectively. 14 days later, the sizes of the formed tumors were monitored once every two days for nearly two weeks. Then the mice were sacrificed and the tumor weight was measured. Compared with the control group, the tumor size, weight and volume were significantly diminished in BPTF knockdown group (Fig. 6A–D). Of note, the expression regulation of hTERT by BPTF was confirmed again in vivo. Consistent with the effect in vitro, RT-PCR, western blot and immunohistochemistry staining analysis in-dicated BPTF knockdown considerably suppressed the expression level of hTERT (Fig. 6E–G). In addition, the level of stemness-related hall-marks, CD44 and EpCAM in xenografts of HCC, was also impeded with BPTF knockdown (Fig. 6F, G). These findings demonstrated again that BPTF expression is required to maintain tumor cell growth in HCC progression by activating hTERT expression.
    3.7. HCC with BPTF knockdown exhibits low metastatic potential
    The maintenance of stemness is required for tumor cells to migrate and recolonize at new sites [32,33]. Given the regulation of CSC-related traits by BPTF in HCC in vitro, we then assessed the metastatic potential of HCC driven by BPTF using an in vivo lung metastasis mouse model. 3 × 106 Hep3B cells stably expressing BPTF shRNA fused with mCherry fluorescent protein were injected into the tail vein of nude mice. 7 weeks after injection, compared to the animals injected with cells ex-pressing control shRNA, considerably fewer lung tumor nodules and wrinkles were observed in animals injected with cells expressing BPTF shRNA (Fig. 7A, B). Western blot and IHC analyses showed that these tumors in lung tissues formed with BPTF knockdown cells exhibited low expression of hTERT, EpCAM, CD44 and c-kit (Fig. 7C, D). These results reveal that BPTF promote the metastatic potential of HCC and its in-hibition impedes survival and regrowth of HCC at a distant site.