KLF7 silencing suppressed cell proliferation and invasion, and induced cell cycle arrest and apoptosis in HCC cell lines
To reveal the role of KLF7 in the proliferative capacity, we examined knockdown efficiency of KLF7 medicated by siRNA technology, in Huh-7 and SKHEP1 cell lines (Fig. 1a). After KLF7 knockdown, CCK-8 assay confirmed that knockdown of KLF7 obviously inhibited cell proliferation in both cell lines (Fig. 1b and Fig. 1c). Furthermore, the colony formation assay implied that the rates of colony formation of KLF7-silencing cells were much lower than those transfected with si-NC (Fig. 1d and Fig. 1e). These results suggested that KLF7 silencing repressed cell growth in HCC cells.
In addition, flow cytometry assay was performed to identify the stages of cell cycle progression in both HCC cell lines transfected with si-KLF7 or si-NC. The results indicated that Huh7 and SKHEP1 cells transfected with si-KLF7 both result in an obvious increase in the percentages of cells at G0/G1 phase, a marked decrease in S phase in Huh7 cells, while an increase in S phase and a remarkable decrease at G2/M phase in SEHEP1 cells (Fig. 1g and Fig. 1h, All p<0.01). There was no obvious alteration of G2/M phase in Huh7 cells. These results indicated that knocking down KLF7 could lead to Go/G1 arrest. To clarify the potential molecular mechanism underlying the inhibition of cell growth after KLF7 knockdown, FITC-Annexin Ⅴ and PI doubling staining assay was conducted to determine the effect of KLF7 downregulation on HCC cell apoptosis. As Fig. 1f revealed, the percentage of apoptosis cells in both Huh7 and SKHEP1 cells transfected with si-KLF7 were higher than those transfected with Huh-siNC (si-KLF7-1, p<0.01; si-KLF7-2, p<0.05) or SKHEP1-siNC (si-KLF7-1, p<0.05; si-KLF7-2, p<0.01). These data indicated that downregulation of KLF7 promote cell apoptosis in HCC cell lines, which revealed that KLF7 contributed to tumor progression through suppressing apoptosis.
Since metastasis process of cancer cell is recognized as an important indicator of tumor progression, we explored the capacity of cell invasion regarding transfection of siKLF7 or siNC into HCC cells. By Transwell assay in Huh-7 and SKHEP1 cell lines, cell invasion was clearly repressed upon KLF7 knockdown (Fig. 1i and Fig. 1j, all p<0.01). Collectively, these data suggested that KLF7 could regulate cell process, including cell proliferation, invasion, cell cycle and cell apoptosis of HCC cell lines.
Overexpression of KLF7 aggravates HCC cell progression
Correspondingly, to further unravel the oncogene roles of KLF7 in HCC development, KLF7 was remarkably upregulated in Huh-7 and SKHEP1 cells by pcDNA3.1-KLF7 plasmid transfection (Fig. 2a). CCK-8 assay and colony formation assay results showed that KLF7 overexpression enhanced cell proliferation as well as colony formation ability in both cell lines (Fig. 2b-e). In addition, flow cytometry assay in Huh-7 and SKHEP1 cells showed that overexpressing of KLF7 effectively promoted cell cycle progression (Fig. 2f-2h), while inhibited cell apoptosis (Fig. 2i-j, all p<0.01). Cell migration was also increased in Huh-7 and SKHEP1 cell lines, transfected with KLF7 overexpressing vectors (Fig. 2k-m, p<0.01 for Huh-7 cells, p<0.001 for SKHEP1 cells). These results indicated that KLF7 overexpression obviously promoted cell proliferation, invasion, and contributed to cell cycle and suppressed cell apoptosis of HCC cell lines.
KLF7 expression affects the growth of HCC transplanted tumors in vivo
To further investigate whether ectopic expression of KLF7 affects the growth of HCC xenograft tumors in vivo, a xenograft tumor model was established by subcutaneously injected shNC, sh-KLF7, empty vector or KLF7-overexpressing MHCC97H cells into the front flank of nude mice. As Fig. 3 suggested, the xenograft tumors transfected with sh-KLF7 grew much slower and with smaller tumor volume and weight than those transfected with shNC-MHCC97H cells (Fig. 3a-c, p<0.01 for tumor weight). Correspondingly, the tumor size and weight were larger in mice injected with KLF7-overexpressing MHCC97H cells than tumors injected with empty vector cells (Fig. 3d-f, p<0.001 for tumor weight). These results revealed that KLF7 expression affected tumor growth in vivo, which was consistent within in vitro study.
KLF7 contributes to tumor progression by regulating VPS35 expression
KLF7 functions as a critical transcription factor involved in the regulating of target gene expression in multiple biological events. We hypothesized that KLF7 medicated its effects on HCC tumor growth and invasion by regulating target gene expression. Western blots showed that knockdown of KLF7 suppressed VPS35 in SHEP1 and Huh-7 cell lines (Fig. 4a). Based on TCGA database analysis, there was a strong positive correlation between KLF7 expression and VPS35 expression (Fig. 4b). To confirm the activity of the binding sites, we performed luciferase reporter assay and found that the reporter activity was increased by overexpression of KLF7 (Fig. 4c), which revealed that KLF7 has a transcriptional activation effect on VPS35 expression.
CHIP-qPCR assay suggested that KLF7 could interact with the promoter of VPS35 (Fig. 4d). To further confirm that the observed phenotypes were medicated by the dysregulation of VPS35/KLF7 axis, several functional rescue assays were conducted. We found that upregulation of KLF7 promoted cell viability and colony formation were rescued by VPS35 knockdown, respectively (Fig. 4e-h, p<0.01). In addition, silencing of VPS35 partially reverted the promotion of HCC cell invasion induced by the upregulation of KLF7 (Fig. 4j-l, p<0.01). Collectively, our data demonstrated that KLF7 promotes HCC tumor growth and invasion by stimulating the expression of VPS35.
KLF7/VPS35 axis exerts its role on the HCC progression via enhancing the β-catenin signaling
Previous studies have revealed that the β-catenin signaling pathway plays a crucial role in cancer cell progression. Therefore, we examined whether KLF7/VPS35 axis medicates the canonical β-catenin signaling. As shown in Fig. 5a, compared with control cells, the protein level of β-catenin was reduced in KLF7-silencing Huh-7 and SKHEP1 cells. Conversely, KLF7 overexpression had opposite effect (Fig. 5b). Additionally, downregulation of VPS35 reduced the expression of β-catenin, while VPS35 overexpression increased β-catenin expression in Huh-7 and SKHEP1 cells (Fig. 5a-b). Subsequently, we found that β-catenin downregulation medicated by si-KLF7, which could be rescued by VPS35 overexpression in Huh-7 and SKHEP1 cells (Fig. 5c). Opposite results were obtained in HCC cells transfected with KLF7 overexpression and VPS35 knockdown (Fig. 5d). These data suggested that KLF7/VPS35 axis regulated β-catenin expression. Next, the clinical relation between expression of VPS35 and β-catenin was detected by western blot in HCC tissues. As expected, western blot results showed that β-catenin expression was highly when VPS35 was overexpressed, while β-catenin levels were lower in VPS35 downregulated tissues (Fig. 5e), which revealed a strong positive correlation between expression of VPS35 and β-catenin in HCC samples (Fig. 5f). Thus, we examined cell survival of HCC cells transfected with VPS35-overexpressing or empty vectors, following treatment with β-catenin inhibitor, GK974. As Fig. 5g and Fig. 5h suggested, upregulation of VPS35 effectively improved chemosensitivity to β-catenin inhibitor, GK974, in HCC cells. While VPS35 overexpression increased cell colony formation, GK974 significantly suppressed colony formation ability in Huh-7 and SKHEP1 cells (Fig. 5i-k, all p<0.001). In collusion, KLF7/VPS35 axis contributed to HCC cell growth via activating β-catenin signaling.
VPS35 interacts with Ccdc85c to participate in HCC progression
Given that KLF7 regulated VPS35 expression in HCC cells, we subsequently explored the underlying mechanisms of KLF7/VPS35 axis contributed to HCC progression by co-IP assay. As Fig. 6 revealed, VPS35 could interact with Ccdc85c in VPS35-overexpressing HCC cell lines, which was confirmed by mass spectrometry and western blot analysis (Fig. 6a-b). Additionally, the clinical relevance between expression of Ccdc85c and HCC patients suggested that Ccdc85c was high expressed in HCC patients, which was strongly associated with poor overall survival (p=0.036) or disease-free survival (p<0.027) of HCC patients (Fig. 6c-e). Further, rescue biological experiments, including cell viability, colony formation, transwell assay and cell apoptosis assay, were performed to investigate whether VPS35 regulated HCC progression via Ccdc85c. As western blot results suggested, Ccdc85c was obviously reduced by siRNA, which was upregulated by VPS35 overexpression (Fig. 6f). CCK-8 and colony formation assay showed that VPS35-overexpressing medicated cell proliferation was partly reverted by Ccdc85c knockdown in HCC cells (Fig. 6g-i), whereas overexpression of Ccdc85c effectively enhanced cell growth of HCC. By transwell assay, downregulation of Ccdc85c would rescue the invasion ability affected by VPS35 upregulation (Fig. 6j-k). These data clarified that VPS35 interacted with Ccdc85c to participate in HCC progression.