Centromere Protein N Participates in Cellular Proliferation of Human Oral Cancer by Cell-Cycle Enhancement

Abstract

Centromere protein N (CENP-N), an important member of the centromere protein family, is essential for kinetochore assembly and chromosome segregation; however, the relevance of CENP-N in cancers remains unknown. The aim of this study was to investigate CENP-N expression and its functional mechanisms in oral squamous cell carcinoma (OSCC). CENP-N expression was up-regulated significantly in vitro and in vivo in OSCCs. Overexpressed CENP-N was closely (p < 0.05) correlated with tumor growth using quantitative reverse transcriptase-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. CENP-N knockdown (shCENP-N) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with up-regulation of p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, CDK2, and CDK4. Interestingly, we newly discovered that calcitriol (1, 25-dihydroxyvitamin D3) controlled the CENP-N expression level, leading to inhibition of tumor growth similar to shCENP-N cells. These results suggested that CENP-N plays a critical role in determining proliferation of OSCCs and that calcitriol might be a novel therapeutic drug for OSCCs by regulating CENP-N.

Keywords: Calcitriol; Cell-cycle arrest at G1 phase; Cellular proliferation; Centromere protein N; Oral squamous cell carcinoma.

Fig 1

Increased CENP-N mRNA and protein expression levels in OSCCs. (A) Significant (*p < 0.05, Student's t-test) up-regulation of CENP-N mRNA is seen in 10 OSCC cells compared with the HNOKs by RT-qPCR. The data are expressed as the mean ± standard error of the mean (SEM) from three assays. (B) The expression of CENP-N protein is up-regulated in OSCC cells compared with the HNOKs by immunoblot analysis. (C) Representative IHC results for CENP-N protein in normal oral tissue and primary OSCC tissue. Scale bars = 50 μm. (D) The status of CENP-N protein expression in primary OSCC (n = 100) and its normal counterparts by the IHC scoring system.

Fig 2

shRNA knockdown cells of CENP-N in OSCC (Ca9-22 and SAS-derived transfectants). (A) CENP-N mRNA expression in shCENP-N cells is significantly (*p < 0.05, Student's t-test) lower than that in shMock cells. (B) Immunoblot analysis shows that the CENP-N protein levels in the shCENP-N cells are also markedly lower than that in shMock cells. (C) The shCENP-N cells and shMock cells were counted on 7 consecutive days. The results are expressed as the mean ± standard error of the mean (SEM) of values from three assays. Cellular growth is inhibited significantly (*p < 0.05, Student's t-test) after 144 h culture in the shCENP-N cells. (D) Flow cytometric analysis shows that the percentage of the G1 phase in the shCENP-N cells is increased compared with the shMock cells. (E) Immunoblot analysis shows up-regulation of p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, CDK2, and CDK4 in the shCENP-N cells compared with shMock cells.

Fig 3

CENP-N mRNA and protein levels in calcitriol-treated cells. (A, B) Calcitriol significantly inhibits CENP-N mRNA and protein expression levels in the Ca9-22 and SAS cell lines in a dose-dependent manner (*p < 0.05, Student's t-test). (C) The expression of CENP-N mRNA after treatment with calcitriol (optimal concentration, 20 µM in the Ca9-22 cell line and 10 µM in the SAS cell line). The expression of CENP-N mRNA in the calcitriol-treated cells is lower than in the control cells (*p < 0.05, Student's t-test). (D) The expression of CENP-N protein in the calcitriol-treated cells is lower than in the control cells.

Fig 4

Effect of calcitriol treatment. (A) To assess the effect of calcitriol on cellular proliferation, the calcitriol-treated cells and control cells were counted on 7 consecutive days. The results are expressed as the mean ± standard error of the mean (SEM) of values from three assays. Cellular growth is inhibited significantly (*p < 0.05, Student's t-test) after 144 h culture in the calcitriol-treated cells. (B) Flow cytometric analysis shows the percentage of G1 phase in calcitriol-treated cells is increased compared with control cells. (C) Immunoblot analysis shows up-regulation of p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, CDK2, and CDK4 in the calcitriol-treated cells compared with the control cells. (D) Schematic representation of the cellular proliferation pathway by CENP-N. CENP-N silencing and calcitriol-treatment are involved in G1 phase-related genes, leading to delay of cellular proliferation.