br Results br BPTF was highly expressed
3.1. BPTF was highly expressed in HCC STM2457 and tumor tissues and positively correlated with patients’ advanced stages
We firstly detected the expression of BPTF in human HCC cells and tumor tissues. Western blot analysis indicated that BPTF was highly expressed in five different HCC cell lines and one immortalized liver cell line (L-O2) (Fig. 1A), as well as tumor tissues from 9 cases of patients with HCC, by comparison to the levels of BPTF in the corresponding adjacent non-cancer tissues (Fig. 1B). These results were further con-firmed by the obtained data from Oncomine database. The BPTF mRNA level of clinical tissue samples collected in Wurmbach Liver showed 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 cells (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  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).