MCM7 promotes cancer progression through cyclin D1-dependent signaling and serves as a prognostic marker for patients with hepatocellular carcinoma

Abstract

DNA replication is a central procedure of cell proliferation, whereas aberrant DNA replication is indicated to be a driving force of oncogenesis. Minichromosome maintenance complex component 7 (MCM7) plays an essential role in initiating DNA replication. To investigate the potential oncogenic properties and prognostic value of MCM7 in hepatocellular carcinoma (HCC), we conducted immunohistochemistry staining of MCM7 in 153 HCC samples and found that MCM7 high expression level was associated with worse overall survival (OS) of HCC patients. Mechanistically, knockdown of MCM7 significantly inhibited cellular proliferation in vitro and HCC tumorigenicity in vivo. Cyclin D1 was proved to be regulated by MCM7-MAPK signaling pathway. Clinically, high expression of both MCM7 and cyclin D1 exhibited a relatively high sensitivity and specificity to predict worse outcome of HCC patients. Taken together, our results suggest that MCM7-cyclin D1 pathway may participate in cancer progression and serve as a biomarker for prognosis in HCC.

Figure 1

MCM7 was overexpressed in HCC and association with patients' survival. (a) Representative IHC staining images of MCM7 expression in human HCC and nontumor tissues, bar=100 μm. (b) The IHC index of MCM7 in HCC and nontumor tissues. (c) Association between MCM7 expression and clinicopathological variables in HCC patients. (d and e) The cumulative overall survival differences between patients with high and low MCM7 expression in all patients (d) and different subgroups (e). *P<0.05

Figure 2

Knockdown of MCM7 inhibited proliferation of HCC cells. (a) MTT assay of HCC cells transfected by Lv-shRNA-MCM7 or Lv-shRNA-Control. (b and c) Colony-forming ability of HCC cells transfected by Lv-shRNA-MCM7 or Lv-shRNA-Control. (d and e) Flow cytometry analysis for apoptosis in HCC cells after 72 h of transduction. Data are represented as mean±S.E.M. from three independent experiments. *P<0.05; **P<0.01; ***P<0.001

Figure 3

Downregulation of MCM7 restrains cell cycle progression by suppressing cyclin D1 expression. (a and b) Cell cycle analysis of HCC cells transfected by Lv-shRNA-MCM7 or Lv-shRNA-Control. (c) A total of 16 cell cycle-related genes were screened after lentiviral vector transduction. (d) Western blot analysis of cyclin D1, CDK4, p27, p21, total RB and p-RB in HCC cells after lentiviral vector transduction. Data are represented as mean±S.E.M. from three independent experiments. *P<0.05; **P<0.01

Figure 4

MAPK signaling participated in regulating the MCM7–cyclin D1 signaling in HCC cells. (a) Correlation of MCM7 with CCND1 gene expression in three HCC cell lines. (b) Correlation of MCM7 with 12 MAPK pathway member gene expression. (c) Heat map of changes in the expression of 12 MAPK pathway member genes after lentiviral vector transduction. Western blot analysis (d) and corresponding bar graphs (e) validated the alteration in the activity of three major MAPK members (ERK, JNK and p38) in HCC cells. (f) Correlation of CCND1 with 12 MAPK pathway member gene expression. (g) Heat map of changes in the expression of CCND1 after pretreatment with SB203580 (p38 inhibitor) or U0126 (ERK inhibitor) at different time points (3, 6, 12 and 24 h).; (h) Western blot analysis for cyclin D1 expression level in HCC cells pretreated with SB203580 (for 6 h) or U0126 (for 24 h). Data are represented as mean±S.E.M. from three independent experiments. *P<0.05, **P<0.01, ***P<0.001

Figure 5

Knockdown of MCM7 inhibited tumor growth in vivo. (a) Tumor nodules after subcutaneously inoculation of HCC cells transfected with Lv-shRNA-MCM7 or Lv-shRNA-Control for 4 weeks (n=5 for each group). (b) Xenograft tumor volume of different groups bearing HCC cells transfected with Lv-shRNA-MCM7 or Lv-shRNA-Control. (c) Representative IHC staining of MCM7 and Cyclin D1 in xenograft tumors of different groups. Bar=100 μm. *P<0.05, **P<0.01

Figure 6

MCM7 and cyclin D1 served as potential biomarkers for prognosis of HCC. (a) Representative IHC staining of MCM7 and cyclin D1 in the same HCC tissue, bar=100 μm. (b) Linear correlation analysis of MCM7 and cyclin D1 expression in HCC tumor tissues. (c) Diagnostic test evaluations of either MCM7 or cyclin D1 alone or in combination in predicting the risk for patients' death. Prognostic value of combination of MCM7 and cyclin D1 in overall HCC patients (d) and different subgroups (e)