KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway

Abstract

Objective: Dysregulation of KLF7 participates in the development of various cancers, but it is unclear whether there is a link between HCC and aberrant expression of KLF7. The aim of this study was to investigate the role of KLF7 in proliferation and migration of hepatocellular carcinoma (HCC) cells.

Methods: CCK8, colony growth, transwell, cell cycle analysis and apoptosis detection were performed to explore the effect of KLF7, VPS35 and Ccdc85c on cell function in vitro. Xenografted tumor growth was used to assess in vivo role of KLF7. Chip-qPCR and luciferase reporter assays were applied to check whether KLF7 regulated VPS35 at transcriptional manner. Co-IP assay was performed to detect the interaction between VPS35 and Ccdc85c. Immunohistochemical staining and qRT-PCR analysis were performed in human HCC sampels to study the clinical significance of KLF7, VPS35 and β-catenin.

Results: Firstly, KLF7 was highly expressed in human HCC samples and correlated with patients' differentiation and metastasis status. KLF7 overexpression contributed to cell proliferation and invasion of HCC cells in vitro and in vivo. KLF7 transcriptional activation of VPS35 was necessary for HCC tumor growth and metastasis. Further, co-IP studies revealed that VPS35 could interact with Ccdc85c in HCC cells. Rescue assay confirmed that overexpression of VPS35 and knockdown of Ccdc85c abolished the VPS35-medicated promotion effect on cell proliferation and invasion. Finally, KLF7/VPS35 axis regulated Ccdc85c, which involved in activation of β-catenin signaling pathway, confirmed using β-catenin inhibitor, GK974. Functional studies suggested that downregulation of Ccdc85c partly reversed the capacity of cell proliferation and invasion in HCC cells, which was regulated by VPS35 upregulation. Lastly, there was a positive correlation among KLF7, VPS35 and active-β-catenin in human HCC patients.

Conclusion: We demonstrated that KLF7/VPS35 axis promoted HCC cell progression by activating Ccdc85c-medicated β-catenin pathway. Targeting this signal axis might be a potential treatment strategy for HCC.

Keywords: Ccdc85c; Hepatocellular carcinoma; KLF7; LGK974; VPS35; Β-catenin.

Fig. 1

KLF7 was overexpressed in human HCC samples. a qRT-PCR assay examined KLF7 expression in 20 pairs of human HCC and adjacent normal samples. p = 0.001. b qRT-PCR assay examined KLF7 expression in HCC tissues with or without metastasis. p = 0.0072. c qRT-PCR assay examined KLF7 expression in HCC tissues of high and low differentiation status. p = 0.0259. d IHC staining examined KLF7 expression in HCC and normal samples. e Overall survival was analyzed in HCC patients who were divided into KLF7 high and low expression group (n = 90 per group). p = 0.28

Fig. 2

Downregulation of KLF7 inhibited HCC cell proliferation, invasion and induced cell cycle arrest and apoptosis. a Western blot assay examined knockdown of KLF7 expression mediated by siRNA in two HCC cell lines (Huh-7 and SKHEP1). b, c CCK-8 assay was performed to determine the proliferation capacity of KLF7-silencing Huh-7 (b) and SKHEP1 cells (c) after transfection with siRNA or siCtrl. d, e Colony formation assay was applied to measure the proliferation of si-KLF7-transfected Huh-7 and SKHEP1 cells. f Flow cytometry assay was used to detect cell apoptosis rate in Huh-7 and SKHEP1 cells. g, h Flow cytometry assay was conducted to identify the stages of cell cycle progression in Huh-7 and SKHEP1 cells. i, j Transwell assay was used to examine cell invasion in Huh-7 and SKHEP1 cells transfected with si-KLF7 and siCtrl. Each experiment was independently repeated at three times. *p < 0.05, **p < 0.01

Fig. 3

Overexpression of KLF7 aggravates cell progression in HCC cell lines. a Overexpression efficiency of KLF7 in Huh-7 and SHEP1 cells was confirmed by western blot. b, c Cell viability of KLF7-overexpressing Huh-7 and SHEP1 cells was determined by CCK-8 assay. d, e Colony formation of KLF7-overexpressing Huh-7 (p < 0.001) and SHEP1 (p < 0.01) cells were detected by colony formation assay. f Flow cytometry was preformed to detect the progression stages of cell cycle in KLF7-overexpressing Huh-7 and SHEP1 cells. g, h Statistical results of cell cycle in Huh-7 (p < 0.01) and SHEP1 cells (p < 0.01). i, j Cell apoptosis was determined after the transfection of pcDNA3.1-KLF7 or empty vector into Huh-7 and SHEP1 cells. k Representative images of transwell assay. l, m Transwell invasion assay of Huh-7 (p < 0.01) and SHEP1 (p < 0.001) cells transfected with KLF7-overexpressing vectors or empty vectors. *p < 0.05, **p < 0.01, ***P < 0.001

Fig. 4

KLF7 expression affect tumor growth of HCC cells in vivo. a MHCC97H cells stably transfected with shKLF7 or shNC were subcutaneously injected into nude mice (n = 5). Four weeks later, mice were killed and photographed. b Tumor volume curves were drawn according to the tumor diameter. c Tumor weight of KLF7-knockdown nude mice was measured. d MHCC97H cells stably transfected with KLF7-overexpressing or empty lentivirus were subcutaneously injected into nude mice (n = 5). Representative images of tumors that striped from the nude mice four weeks later. e Tumor growth curve for the xenograft tumors. f Tumor weight of KLF7- overexpressing MHCC97H cells was presented. **p < 0.01, ***P < 0.001

Fig. 5

KLF7 contributes to tumor progression by regulating VPS35 expression. a Western blot analysis of VPS35 and cyclin D1 in si-KLF7 and siCtrl HCC cells. b A relationship between VPS35 and KLF7 was identified by TCGA database. d Luciferase reporter assay was used to determine the luciferase activity in HCC cells transfected with empty plasmids or plasmids containing VPS35. e ChIP-qPCR analysis of the interaction between KLF7 and VPS35 promoter sequence. f Western blot analysis of VPS35 in VPS35 knockdown HCC cells. g Cell viability of VPS35 knockdown HCC cells determined by CCK-8 assay. h Western blot analysis of VPS35 in KLF7 overexpression and VPS35 knockdown HCC cells. i Cell viability of KLF7 overexpression and VPS35 knockdown HCC cells determined by CCK-8 assay. j, k Representative images and statistical data of colony formation assay after simultaneously transfected with KLF7-overexpressing and VPS35-silencing SKHEP1 cells. l, m Representative images and statistical data of cell invasion assay after simultaneously transfected with KLF7-overexpressing and VPS35-silencing SKHEP1 cells. **p < 0.01

Fig. 6

KLF7/VPS35 axis contributed to HCC progression via enhancing the β-catenin signaling. a Knockdown of KLF7 and VPS35 affected active-β-catenin and GS expression in HCC cells detected by western blot, respectively. b Overexpression of KLF7 and VPS35 affected active-β-catenin expression in HCC cells detected by western blot, respectively. c KLF7-silencing and VPS35-overexpressing affected active-β-catenin expression in Huh-7 and SKHEP1 cells. d KLF7-overexpressing and VPS35-silencing affected active-β-catenin level in Huh-7 and SKHEP1 cells. e The correlation between VPS35 and β-catenin expression in HCC and normal tissues. f, g Cell viability of VPS35-overexpressing HCC cells treated with GK974 in Huh-7 and SKHEP1 cells. h Representative images of colony formation assay in VPS35-overexpressing HCC cells treated with GK974. i, j Colony formation of VPS35-overexpressing cells treated with GK974 in Huh-7 and SKHEP1 cells

Fig. 7

VPS35 interacts with Ccdc85c to participate in HCC progression. a Mass spectrometry analysis of VPS35-interacted proteins after co-IP. b Western blot was performed to confirm co-IP results. c The expression level of Ccdc85c in HCC patients and normal tissues by analyzed TCGA data. d The overall survival of HCC patients using KM-plotter by TCGA database analysis. e The disease-free survival of HCC patients predicted using KM-plotter. f Western blot determined Ccdc85c expression following siRNA or VPS35 overexpression. g CCK-8 assay was performed to examine cell viability following alteration of Ccdc85c and VPS35 expression in HCC cells. h colony formation and statistical data was performed to examine cell proliferation following alteration of Ccdc85c and VPS35 expression in HCC cells. i Statistical analysis of colony formation assay results. j Transwell assay was performed to examine cell invasion following alteration of Ccdc85c and VPS35 expression in HCC cells. k Statistical analysis of transwell assay results. **p < 0.01, ***P < 0.001

Fig. 8

KLF7/VPS35 axis promoted hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway. A schematic graph of how KLF7/VPS35/CCDC85C/β-catenin axis promoted HCC growth