ZRANB1 expression is increased in human HCC tissues and HCC cells
To determine the expression levels of ZRANB1 in HCC tissues, we performed quantitative real-time PCR on 72 HCC specimens and their corresponding non-tumor tissues. Upregulation of ZRANB1 mRNA level was markedly observed in 55 of the 72 pairs (Fig. 1a). Consistently, we detected the protein expression of ZRANB1 in these 72 pairs of HCC tissues and corresponding normal tissues. Quantitative analyses showed that ZRANB1 was significantly overexpressed in HCC tissues compared with non-tumor tissues (Fig. 1b). Consistently, we analyzed the relationship between ZRANB1 expression and clinicopathological features in 72 HCC patients. The results showed that ZRANB1 overexpression was significantly correlated with advanced tumor size, TNM stage, vascular invasion and intrahepatic metastasis (Table 1). Furthermore, we performed immunohistochemical staining to examine ZRANB1 expression in 132 pairs of HCC tissues (including the 72 fresh HCC specimens) and found that ZRANB1 expression was markedly upregulated in HCC tissues compared with paired adjacent normal tissues (Fig. 1c, d). Kaplan–Meier analyses of the overall survival (OS) rate revealed that the high ZRANB1 expression group (i.e., IHC staining with strong and moderate expression) had a worse prognosis than the low ZRANB1 group (i.e., IHC staining with weak and negative expression) (Fig. 1e). Then, we explored ZRANB1 expression in HCC cell lines and two normal human hepatocyte lines (HL7702 and THLE-3). As expected, the expression level of ZRANB1 was significantly higher in HCC cell lines (Huh7, MHCC97H, HepG2, and HCCLM3) than that in HL7702 and THLE-3 cells at both the protein and mRNA levels (Fig. 1f, g). Furthermore, univariate and multivariate logistic regression analyses indicated that ZRANB1 was an independent predictor of poor prognosis for patients with HCC (Table 2). Our results revealed that ZRANB1, which is overexpressed in HCC, might play an essential role in HCC carcinogenesis and prompted us to explore the precise function of ZRANB1 in HCC.
ZRANB1 facilitates HCC cell proliferation and metastasis in vitro
To investigate how aberrant ZRANB1 levels affect HCC carcinogenesis, we inhibited ZRANB1 expression with shRNA (small hairpin RNA). Then, the ZRANB1 knockdown efficiency was confirmed by western blot and qRT-PCR analysis (Supplementary Fig. 1a, b). To examine the proliferation ability of HCC cells transfected with control shRNA or shRNA targeting ZRANB1, we performed EdU and colony formation assays. The EdU assay results suggested that ZRANB1 knockdown markedly inhibited HCCLM3 cell and Huh7 cell growth (Fig. 2a, b). Similarly, the colony formation assay results also showed that decreased ZRANB1 expression reduced the colony-forming capacity of HCC cells (Fig. 2c, d). Furthermore, we investigated the proliferation ability of ZRANB1-overexpressing HCC cells. The results of both the EdU and colony formation assays suggested that ZRANB1 overexpression enhanced HCC cell growth (Supplementary Fig. 2a, b).
To further verify the impact of dysregulated ZRANB1 expression on HCC cell metastasis, we performed transwell migration and invasion assays and the scratching assay. The transwell migration and invasion assay results showed that ZRANB1 knockdown significantly suppressed HCCLM3 and Huh7 cell migration and invasion (Fig. 2e, f). In addition, the scratching assay results suggested that ZRANB1 suppression can inhibit HCC cell migration (Fig. 2g, h). Consistent with these results, ZRANB1 upregulation in HCCLM3 and Huh7 cells contributed to the opposite effects, as evaluated by transwell migration, invasion and the scratching assays (Supplementary Fig. 2c, d). These results indicated that ZRANB1 can facilitate HCC cell proliferation, migration and invasion in vitro.
ZRANB1 drives HCC carcinogenesis in vivo
To further understand the biological effects of ZRANB1 on HCC carcinogenesis, we performed tumorigenicity assays and lung metastasis experiments in animal subjects. In the tumorigenicity assay, nude mice were injected subcutaneously with shNC or shZRANB1 HCCLM3 cells expressing a luciferase reporter. Using IVIS to monitor tumor growth in mice, imaging analysis showed that ZRANB1 stable knockdown decreased tumor growth significantly (Fig. 3a). Tumor weights and volumes of the nude mice were recorded throughout the experimental period. Decreased ZRANB1 expression significantly inhibited HCC growth compared with that in the control group (Fig. 3b, c). Additionally, the Ki-67 level was markedly decreased in tumors with ZRANB1 suppression, as assessed through immunofluorescence experiments (Fig. 3d). In contrast, ZRANB1 upregulation promoted HCC progression in nude mice compared with that in the control group (Supplementary Fig. 2e, f).
To further evaluate the contribution of ZRANB1 to HCC metastasis in vivo, we established a lung metastasis model via tail vein injection. Luciferase-labelled HCCLM3 cells transfected with shNC or shZRANB1 were injected into the tail vein of nude mice. Luciferase fluorescence intensity of the progressive HCC in nude mice was detected by IVIS. The results showed a striking reduction of the metastatic capacity of ZRANB1 knockdown HCCLM3 cells to the lung (Fig. 3e). Additionally, H&E staining and the number of lymph node metastases results showed that the incidence of lung metastasis was significantly reduced in the ZRANB1 inhibition group compared with the control group (Fig. 3f, g). Furthermore, we demonstrated that ZRANB1 upregulation promoted HCC lung metastasis (Supplementary Fig. 2g). Collectively, these results showed that ZRANB1 can enhance HCC growth and metastasis in vivo.
LOXL2 expression is highly correlated with ZRANB1 in HCC
To obtain a more robust understanding of the mechanism by which ZRANB1 regulates HCC carcinogenesis, we performed RNA-seq on shNC and shZRANB1 HCCLM3 cells to identify the potential downstream targets of ZRANB1. Our data demonstrated that LOXL2 was the gene with the most marked decrease in expression after ZRANB1 suppression (Fig. 4a). Consistent with this finding, we detected the expression level of LOXL2 in shNC and shZRANB1 HCCLM3 cells and found that LOXL2 expression was reduced at both the protein and mRNA levels after ZRANB1 inhibition (Fig. 4b, c). In contrast, LOXL2 expression showed a significant increase in HepG2 cells with ZRANB1 upregulation (Fig. 4d, e). Importantly, we detected the mRNA and protein levels of ZRANB1 and LOXL2 in 72 HCC samples, representative images of ZRANB1 and LOXL2 protein expression in HCC tissues and adjacent non-tumor tissues are shown in Figure 4f. Additionally, the scatter plots suggested that LOXL2 expression was significantly correlated with ZRANB1 expression at both the mRNA and protein levels (Fig. 4g, h). These results revealed a significant relationship between ZRANB1 and LOXL2 in HCC.
LOXL2 is critical for ZRANB1-mediated HCC progression
Considering the correlation between ZRANB1 and LOXL2, we further investigated whether LOXL2 plays an essential role in ZRANB1-mediated HCC progression. After upregulating LOXL2 expression in ZRANB1 knockdown HCCLM3 cells, we assessed ZRANB1 and LOXL2 protein expression by western blot. Our results showed that although LOXL2 overexpression increased the LOXL2 protein level in HCCLM3 cells, ZRANB1 suppression markedly abated the increased LOXL2 (Fig. 5a). We then performed EdU assay and transwell migration and invasion assays to explore the impact on HCC cell growth and metastasis. Similarly, LOXL2 upregulation significantly enhanced HCCLM3 cell proliferation, migration and invasion; however, ZRANB1 inhibition effectively decreased the enhanced proliferation and metastasis capacities of HCCLM3 cells caused by LOXL2 overexpression (Fig. 5b, c). We further performed rescue experiments in vivo and assessed tumor growth, tumor weight and lung metastasis in nude mice. Consistent with the observations in vitro, our results showed that ZRANB1 knockdown also significantly suppressed the enhancement of tumor growth and HCC cells lung metastatic ability induced by LOXL2 upregulation (Fig. 5d, e, f).
In comparison, we also transfected LOXL2 shRNA into ZRANB1-overexpressing HepG2 cells and performed western blot and a series of functional assays. Consistent with the results of the previous experiments, ZRANB1 upregulation markedly reversed the decreased LOXL2 expression in LOXL2 knockdown HepG2 cells (Supplementary Fig. 3a). Furthermore, the EdU and transwell assays results showed that LOXL2 suppression significantly inhibited the increased proliferation, migration and invasion capacities of HepG2 cell induced by ZRANB1 overexpression (Supplementary Fig. 3b, c).
ZRANB1 promotes LOXL2-mediated HCC carcinogenesis through SP1
We next investigated how ZRANB1 regulates LOXL2 in HCC cells. Our co-IP results showed that ZRANB1 cannot interact with LOXL2 directly (Supplementary Fig. 4a, b). A previous study showed that SP1 can bind to the LOXL2 promoter and regulate its expression directly in breast cancer . In addition, it has reported that SP1 can be regulated by the deubiquitinase USP33 . We thus hypothesized that ZRANB1 might regulate LOXL2 through SP1. We evaluated the protein expression of SP1 and LOXL2 in HepG2 cells transfected with shZRANB1 RNA or His-ZRANB1 overexpression plasmids and, as expected, found that the protein expression levels of SP1 and LOXL2 were significantly correlated with the ZRANB1 expression level (Fig. 6a). Importantly, ZRANB1 knockdown decreased the transcriptional activity of the LOXL2 luciferase reporter, whereas upregulation of ZRANB1 showed the opposite effects (Fig. 6b). However, neither up- nor downregulation of ZRANB1 had a significant effect on SP1 mRNA expression (Fig. 6c). Furthermore, we performed rescue experiments to explore the relationships among ZRANB1, SP1 and LOXL2 in HCC cells. The western blot results showed that ZRANB1 downregulation decreased the upregulation of SP1 and LOXL2 in SP1-overexpressing HCCLM3 cells (Fig. 6d). Moreover, the SP1 upregulation-induced enhancement of LOXL2 luciferase reporter transcriptional activity was alleviated by ZRANB1 knockdown (Fig. 6e). Through RTCA and transwell migration and invasion assays, ZRANB1 knockdown was found to reverse the increases in the proliferation and metastasis capacities of HCCLM3 cells induced by SP1 overexpression (Fig. 6f, g). Taken together, these results provide further support for functional associations among ZRANB1, SP1 and LOXL2 in HCC carcinogenesis.
ZRANB1 can deubiquitinate and stabilize SP1
Having confirmed the correlation between ZRANB1 and SP1, we aimed to explore the interaction of them. It has been reported that ZRANB1 can deubiquitinate EZH2 and significantly increase its stability in breast cancer . Additionally, our results in Fig 6 a and c show that ZRANB1 expression had a notable impact on SP1 expression only at the protein level. Moreover, another study reported that SP1 can be degraded through the ubiquitin-proteasome pathway and can be deubiquitinated by USP33 . We thus hypothesized that ZRANB1 might deubiquitinate SP1 and stabilize it. As expected, the results of co-IP experiments showed that ZRANB1 can bind SP1 directly in HCCLM3 cells (Fig. 7a). We also found that ZRANB1 can bind with SP1 directly in HCCLM3 cells cotransfected with His-ZRANB1 and Flag-SP1 (Fig. 7b). To evaluate whether ZRANB1 can regulate SP1 degradation through the ubiquitin-proteasome pathway, we treated HepG2 cells transfected with ZRANB1 shRNA or His-ZRANB1 plasmids with or without 15 μM MG132. Our results showed that neither knockdown nor upregulation of ZRANB1 had a notable effect on the SP1 protein level in HepG2 cells treated with MG132 compared with those not treated with MG132 (Fig. 7c). Furthermore, we used a 20 μM dose of the translation inhibitor cycloheximide (CHX), and at the indicated times, we detected the SP1 protein level in HepG2 cells transfected with ZRANB1 shRNA or His-ZRANB1 plasmids. Our data demonstrated that ZRANB1 overexpression significantly decreased the degradation rate of SP1, while decreased ZRANB1 levels had the opposite effect (Fig. 7d, e).
Additionally, we further investigated the dose-dependent impact of ZRANB1 on SP1. Our data showed that a dose increase in the ectopic ZRANB1 level contributed to SP1 protein accumulation in HCC cells (Fig. 7f). Next, an equal dose of Flag-SP1 and an enhancing dose of His-ZRANB1 were cotransfected into HCCLM3 and Huh7 cells, and a dose-dependent effect of ZRANB1 overexpression on SP1 protein levels was observed (Fig. 7g). Finally, our results revealed that ZRANB1 suppression markedly increased the ubiquitination level of SP1, while ZRANB1 upregulation showed the opposite impact on SP1 (Fig. 7h, i). Thus, our data indicated that ZRANB1 can act as a deubiquitinase of SP1 and stabilize it.
Clinical significance of ZRANB1, SP1 and LOXL2 in HCC tissue
To extend our findings to the clinical setting, we analyzed the expression patterns of ZRANB1, SP1 and LOXL2 in 72 paired human HCC specimens. Our results showed that ZRANB1, SP1 and LOXL2 were upregulated in HCC tissues, whereas in adjacent normal tissues, the opposite results were observed (Fig. 8a). Notably, there was a marked correlation between ZRANB1 and SP1 protein expression but no significant correlation at the mRNA level (Fig. 8b, c). For SP1 and LOXL2, significant correlations were observed at both the mRNA and protein levels (Fig. 8d, e).
In summary, our study highlights the novel role and molecular mechanism of ZRANB1 in HCC carcinogenesis. ZRANB1 promotes HCC carcinogenesis by directly deubiquitinating SP1 and stabilizing it via the SP1-LOXL2 axis (Figure 9). Our findings may provide a promising alternative therapeutic strategy for HCC.