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  • br Fig Knockdown of BPTF inhibited the proliferation and

    2020-08-18


    Fig. 2. Knockdown of BPTF inhibited the proliferation and invasion of HCC cells. (A) BPTF was knocked down in Bel7402, Hep3B and HepG2 1-Methoxy PMS by transfecting these cells using its specific shRNA plasmids. (B) Detection of relative cell viability in Bel7402, Hep3B and HepG2 cells by MTT assay upon suppressing BPTF. (C) Colony formation assay of Bel7402, Hep3B and HepG2 cells after BPTF was silenced and pictures were taken by the gel imaging. The number of the colonies was also shown. (D) Cell migration assay in Bel7402 and HepG2 cells following BPTF knockdown, and the migration rate was calculated. The percentages of migration cells were calculated relative to the original gap. (E) Cells with BPTF knockdown were staining after incubation of 48 h, observed and photographed, and the percentages of invasion cells were calculated.
    Fig. 3. BPTF knockdown inhibited the CSC properties in HCC cells. (A) BPTF was knocked down in HepG2, Hep3B and Bel 7402 cells and tumorsphere formation was observed by using inverted microscope after two weeks of culture. The number of spheres bigger than 50 µm was quantified and shown. Scale bars, 100 µm. (B) Flow cytometry was used to analyze CD24+ /CD44+ cells in Bel7402 cells by using PE-CD24, APC-CD44 antibody when BPTF was knocked down. (C) CD24+ cells were detected in Hep3B and HepG2 cells by Flow cytometry when BPTF was knocked down. (D) The protein expression of stemness-associated marker, including CD44, CD133, and c-Kit, upon BPTF silencing by western blot.
    Fig. 4. Downregulation of BPTF sensitized chemotherapeutic drugs-mediated anti-neoplastic effects in HCC cells. (A) Hep3B and HepG2 cells with stable knockdown of BPTF were treated with different concentrations of 5-FU or DDP and the cell viability was tested by MTT assay. (B) The IC50 value of 5-FU and DDP was calculated on the basis of Fig. 4A and shown. (C) Acridine orange/ethidium bromide double staining method was used to evaluate the degree of apoptosis caused by DDP upon BPTF silencing and the pictures were taken by Inverted fluorescence microscope. Scale bars, 20 µm. (D) FACS analysis was used to detect the apoptosis by FITC-AV/PI staining in HepG2 and Bel7402 cells mediated by DDP upon BPTF silencing. (E) The expression level of cleaved-caspase-7, cleaved-caspase-9, cleaved-PARP and Bcl-2 was determined by western blot in HepG2 and Bel7402 cells treated by DDP upon BPTF silencing.
    (caption on next page)
    Fig. 5. BPTF promoted cell growth and maintained the CSC traits in HCC cells by targeting hTERT. (A–B) The knockdown of BPTF down-regulated hTERT expression at the protein and mRNA levels in Hep3B and HepG2 cells. (C) Detection of the luciferase activity driven by different fragments of hTERT promoter in Hep3B and HepG2 cells when BPTF was stably knocked down. (D) The binding of BPTF, H3K4me3 and H3K27me3 at hTERT promoter(−902 to −321)was validated by chromatin immunoprecipitation assay. (E) Colony formation assay in Hep3B and HepG2 cells upon stable silencing of BPTF followed by hTERT overexpression. Number of colonies was presented on the right. (F) Tumorsphere formation assay in Hep3B and HepG2 cells upon stable silencing of BPTF followed by hTERT overexpression. The number of spheres bigger than 50 µm was quantified and shown. Scale bars, 100 µm. (G) The expression of stemness-associated markers, including EpCAM, CD44, and c-Kit, was shown by western blot in Hep3B and HepG2 cells upon stable silencing of BPTF followed by hTERT overexpression.
    3.4. BPTF knockdown sensitized HCC cells to chemotherapeutic drugs
    The acquisition of chemoresistance seems to be closely related to the intrinsic or required properties of CSCs [29]. Thus, CSC-focused therapy is destined to form the core of any effective anticancer strategy. Given the above findings of the stemness-promoting role for BPTF in HCC cells, we hypothesized that BPTF expression might modulate the sensitivity of HCC cells to chemotherapeutic drugs. In accordance to our hypothesis, BPTF shRNA-expressing HepG2 and Bel7402 cells were more sensitive to cisplatin (DDP) or 5-Fluorouracil (5-FU) treatment (Fig. 4A), displaying a significantly reduced IC50 of these two drugs, compared to the control shRNA-expressing cells (Fig. 4B), suggesting the improved anti-tumor function for chemotherapeutic treatment upon BPTF silencing.
    In order to further clarify the potential molecular mechanisms of BPTF in its regulation of sensitivity to chemotherapy drugs in HCC cells, we then examined the apoptosis induction by chemotherapeutic drugs with BPTF knockdown. The AO/EB staining was initially carried out to study such induction. The upregulation of apoptosis in cells induced by DDP treatment plus BPTF shRNA expression was observed (Fig. 4C). Consistently, BPTF silencing in HepG2 and Bel7402 cells significantly increased apoptosis induction under DDP treatment under PI/Annexin V staining (Fig. 4D) and was accompanied by the down-regulated anti-apoptotic protein Bcl-2 and the up-regulated pro-apoptotic protein cleaved caspase-7, cleaved caspase-9, and cleaved PARP (Fig. 4E). Thus, BPTF knockdown sensitized HCC cells to chemotherapy at least par-tially via inducing more apoptosis.