br Fig BPTF accelerated tumor growth and maintained stemness
Fig. 6. BPTF accelerated tumor growth and maintained stemness of HCC cells in vivo by HCC xenograft model. The Balb/c nude mice aged 4–6 weeks were divided into three groups (BPTF-control, BPTF- sh2, BPTF- sh3, n = 4 for each group) and 4 × 106 Hep3B cells stably expressing BPTF shRNAs or control shRNAs were respectively injected into the right flank of each mouse. (A) The tumor was dissected at 4 weeks after injection and the images were presented. (B) The tumor weight was measured. (C) The tumor volume was measured. (D) The tumor volume was calculated within two weeks after tumors were formed. (E) Total RNA was extracted from tumor tissue and the expression of BPTF and hTERT was detected by RT-PCR with their specific primers. (F) The tumor tissues were lysed and the expression of BPTF, hTERT and stemness-associated markers were detected by western blot. (G) Immunohistochemistry (IHC) analysis of BPTF, hTERT, EpCAM, CD44 and hematoxylin-eosin (HE) staining from tumor xenografts.
Fig. 7. BPTF promoted the lung metastasis of HCC cells in nude mice. Mice was divided into 4 groups, PBS (No Hep3B) or 4 × 106 cells stably expressing BPTF shRNAs (Hep3B-sh2 or Hep3B-sh3) or control shRNAs (Hep3B-control) respectively fused with mcherry gene were injected into tail vein in each mouse. 45 days after injection: (A) The 210826-40-7 images of the mouse body are shown. Excitation: 523 nm, Emmission: 560–660 nm, Filter: 560 nm. (B) The lung morphology and nodules are presented. (C) The lung tissue was taken and lysated for western blot to determine the expression of BPTF, hTERT and stemness-associated markers. (D) HE staining and IHC staining of BPTF, hTERT, EpCAM, and CD44 in metastatic lung tissues.
Fig. 8. BPTF expression was correlated positively with hTERT expression and poor survival in patients with HCC. (A) The relation between the level of BPTF or hTERT expression and clinicopathologic characteristics. (B) The numbers of patients with high or low expression of BPTF or hTERT and their expression relationship. (C) The percentage of hTERT expression level in patients with high or low BPTF expression. (D) Kaplan Meier analysis revealing the correlation between different clinicopathological parameter and 5-year overall survival. (E) The overall survival curve reflecting the relation between overall survival and expression of BPTF (P = 0.025) or hTERT (P = 0.007) by Kaplan Meier analysis. (F) The multivariate Cox proportional hazards model analysis of risk factors showing that TNM stage and BPTF expression were independent prognostic risk factors in HCC.
3.8. BPTF expression was positively correlated with hTERT expression and poor prognosis in patients with HCC
We next evaluated the clinical significance and correlation of BPTF with hTERT in HCC. IHC staining was used to detect the expression of BPTF and hTERT in tumor tissue samples of HCC patients based on tissue microarray from 81 HCC cases. The level of gene expression was divided into 2 classes (high and low). The relationship between BPTF or hTERT expression and the clinicopathologic characteristics of patients was analyzed firstly. As shown in Fig. 8A, BPTF and hTERT expression was tightly relevant to the TNM stage (P = 0.025, P = 0.007 respec-tively), but not to sex, age and differentiation. Among all these 81 pa-tients tested, 27 cases (about 35%) showed high expression of BPTF and hTERT and 18(22%) showed their low expression simultaneously (Fig. 8B). Furthermore, among patients with high expression of BPTF, nearly half of them displayed high expression of hTERT, and among patients with low expression of BPTF, nearly 64% showed low expres-sion of hTERT (Fig. 8C), suggesting a potential correlation between BPTF and hTERT level in HCC. In addition, Kaplan Meier analysis showed that BPTF and hTERT expression was remarkably associated with 5-year survival of patients (Fig. 8D). Consistent with the tumor-promoting effect of BPTF in vitro and in vivo, the overall survival analysis indicated patients with high expression of BPTF or hTERT owned significantly lower survival rate (P = 0.025 or P = 0.007) compared to the ones with low expression (Fig. 8E). Also, the multi-variate Cox proportional hazards model analysis indicated BPTF ex-pression was an independent prognostic risk factor in HCC (HR=0.1947, 95%CI: 1.009–3.756, P = 0.047) (Fig. 8F). Thus, con-sistent with the results from in vitro and animal study, BPTF expression is positively correlated with hTERT expression clinically, and BPTF expression is predictive of the short survival and is an independent predictor of survival in HCC patients.
Although BPTF has been previously reported to be implicated in chromatin remodeling, embryonic development and thymocyte ma-turation , little is known about its effect in tumorigenesis and de-velopment. Here, integrating all the experimental data in our study, we reported for the first time the functional role of BPTF and the associated molecular mechanisms in HCC progression. The silencing of BPTF in-hibited HCC cell proliferation and colony formation, weakened the CSC characteristics and sensitized chemotherapeutic drugs in vitro. Simi-larly, BPTF knockdown led to the inhibited tumor growth and the de-creased metastatic potential in vivo, confirming the tumor-promoting role of BPTF in HCC again. Moreover, the further mechanistic studies revealed hTERT functioned as the pivotal downstream factor regulated by BPTF to mediate its oncogenic function, including stemness main-tenance.