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  • Redox Biology br that the expression of BPTF was


     Redox Biology 20 (2019) 427–441
    that the expression of BPTF was higher in cirrhosis, hepatocellular carcinoma, liver cell dysplasia than in normal tissues, and was the highest in HCC samples (Fig. 1C). Consistently, TCGA data also showed that the BPTF DNA copy number in HCC samples were greatly more than that in normal tissues (Fig. 1D). Furthermore, we performed im-munohistochemistry (IHC) assay of BPTF expression based on tissue microarray of 81 HCC patients. The representative IHC staining from 6 different patients were shown in Fig. 1E. BPTF had a high expression level in HCC in most cases (high in 53 cases, and low in 28 cases) (Fig. 1F). In addition, the TNM-staging results indicated that patients displaying high BPTF expression were usually categorized in advanced malignancy stages (mostly at Ⅱ, Ⅲ and Ⅳ stage), while patients dis-playing low BPTF expression were categorized in preliminary stages of malignancy (mostly atⅠ,Ⅱ, and Ⅲ stage) (Fig. 1G). All these results to-gether demonstrate that BPTF was overexpressed in HCC cells.
    3.2. BPTF knockdown inhibited HCC cell proliferation and invasion
    To study the functional significance of BPTF in HCC, we knocked down its expression using its specific shRNAs in three different HCC cell lines (Bel7402, Hep3B2.1–7, HepG2). Among the designed three dif-ferent shRNAs targeting BPTF, two of them (sh2 and sh3) caused more effective silencing (Fig. 2A). Therefore, these two shRNAs were used in the following studies. MTT assay showed that BPTF knockdown led to decreased proliferative capacity in HCC CORM3 (Fig. 2B). Consistent with this, the inhibition of proliferation was also reflected by the decreased colony formation under BPTF silencing in HCC cells (Fig. 2C). We then examined whether knockdown of BPTF could weaken the cell migration and invasion viability in Bel7402 and HepG2 cells. Wound scratch assay revealed that BPTF knockdown reduced the cells migaration capacity (Fig. 2D). Similarly, cell invasion capacity was suppressed by BPTF knockdown (Fig. 2E).
    3.3. BPTF regulated stemness phenotypes of HCC cells
    Combined with the previous reports that BPTF is required for the establishment of the anterior-posterior axis of the mouse embryo during the earliest stages of development [11] and mediates pro-oncogenic role in cancer progression, including melanoma and lung cancer [14,15,25],we deduced BPTF might play a critical role in the stemness maintenance of HCC. Initially, we observed the effect of BPTF knock-down on the ability of tumorsphere formation. HCC cells with stable knockdown of BPTF by lentivirus-mediated shRNA transfection were cultured for two weeks in serum-free medium specific for CSCs, and the number and size of the formed tumorspheres were observed. Compared to the control group, BPTF silencing significantly suppressed the tu-morsphere formation ability in HCC cells (Fig. 3A). The three-dimen-tional images of the formed tumorspheres and the corresponding fluorescent expression of mCherry gene fused with shRNA targeting BPTF under inverted fluorescence microscope also indicated such at-tenuated trend of tumorsphere formation caused by BPTF knockdown (Fig. S1).
    CD44/CD24 has been reported to drive CSC progression in different cancer types [26–28]. We detected the influence of BPTF on their ex-pression level in HCC cells. We knocked down BPTF expression using CORM3 its specific shRNAs in Bel7402, Hep3B, and HepG2 cells and determined the CD44 or CD24 level by flowcytometry. Compared with the control group, BPTF-specific shRNAs-treated cells inhibited the levels of CD44 and CD24 (Fig. 3B, C). Only CD24 expression change was shown in Hep3B and HepG2 cells as the change for CD44 was subtle (Fig. 3C). Besides CD44 and CD24, the expression of other stemness marker in case of silencing BPTF, such as CD133 and c-Kit, was also significantly down-regulated in Bel7402 and Hep3B cells (Fig. 3D). All these findings collectively demonstrate the crucial role of BPTF in maintaining the stemness of HCC stem cells.
    Fig. 1. The high expression of BPTF in HCC cells and tissues. (A) The expression of BPTF was detected in normal hepatic cell line L-O2 and HCC cells (HepG2, Hep3B, SNU449, Bel7402) by Western blot analysis. Percentages of BPTF expression relative to β-actin expression were depicted aside. (B) The expression of BPTF in carcinoma tissue and adjacent tissues was determined by western blot from 9 cases of patients with HCC. (C) Box plots comparing BPTF mRNA levels in normal liver (10), Cirrhosis (13), hepatocellular carcinoma (35), and liver cell dysplasia (17) in 75 samples sets from Oncomine. (D) TCGA data showing BPTF DNA copy numbers in normal liver tissues (115) and hepatocellular carcinoma (97). (E) IHC analysis was performed based on tissue microarray, and the representative staining results are shown. (F) The number of patients with high or low expression of BPTF according to IHC staining. (G) The number of patients at different clinical stages with the high or low expression of BPTF by means of TNM stages.