Upregulation of CENPM promotes hepatocarcinogenesis through mutiple mechanisms

Abstract

Background: Hepatocellular carcinoma (HCC) still remains a dominating medical challenge in early diagnosis and clinical therapy. Centromere protein M (CENPM) has been proved to be over-expressed in HCC tissues, but carcinogenic mechanism of CENPM contributing to liver cancer is poorly understood.

Methods: In this study, we first explored mRNA and protein levels of CENPM in HCC samples, matching adjacent non-tumor tissues and six hepatoma cell lines by polymerase chain reaction (PCR), western blotting and immunohistochemistry (IHC). Clinical data of HCC patients downloaded from The Cancer Genome Atlas (TCGA) were also analyzed. The character of CENPM concerned with HCC progression through several functional experimentations in vitro and in vivo was researched. Bioinformatics was carried out to further discover biological functions of CENPM.

Results: CENPM was positively up-regulated in HCC and connected with a poor prognosis. Silencing CENPM repressed cell proliferation in vivo and in vitro, and knock-down CENPM inhibited cell migration and invasion. Additionally, depletion of CENPM can promote cell apoptosis and arrested cell cycle. Furthermore, single-gene gene set enrichment analysis (GSEA) analysis indicated that CENPM was linked to the P53 signaling pathway and cell cycle pathway, and our research supported this prediction. Finally, we also found that miR-1270 was a negative regulator and participated in post-transcriptional regulation of CENPM, and hepatitis B virus X protein (HBx) can promote hepatocellular carcinoma by suppressing miR1270.

Conclusion: CENPM was closely associated with HCC progression and it could be considered as a new possible biomarker along with a therapeutic target for HCC.

Keywords: Bioinformatics; Centromere protein M (CENPM); Hepatocellular carcinoma (HCC); P53; The Cancer genome atlas (TCGA); miR-1270.

Fig. 1

The expression of CENPM in different GEO and TCGA datasets. a The mRNA level of CENPM in 24 kinds of tumor types from TCGA. b CENPM expression in 50 paired HCC group downloaded from TCGA RNA-seq datasets. c, d Heatmaps of top 20 differential expressed mRNAs identified from GSE45436 and GSE55092

Fig. 2

CENPM was overexpressed in HCC and significantly associated with poor prognosis. a, b CENPM was upregulated in tumor compared with normal liver tissues in HCC samples from GSE45436 and GSE55092. c Expression levels of CENPM in HCC tissues were higher than corresponding non-tumor tissues via qRT-PCR of 68 pairs of HCC samples. d Differential expression of CENPM in tumor grades. e CENPM protein level in 8 pairs of human clinical HCC tumor samples and adajacent non-tumor specimens. f CENPM relative expression in HCC cell lines (SMMC7721, Hep3B, HepG2, HCCLM3, Huh7 and SK-Hep1) compared with the immortalized normal human hepatic cell (L02). g Typical images of immunohistochemistry (IHC) in 18 pairs of HCC tissues showing the protein expression of CENPM in HCC and adjacent nontumor tissues. h, i Kaplan-Meier analysis of overall survival and disease free survival in 364 HCC patients from the TCGA dataset. ***P < 0.001,T-Tumor; NT-Nontumor

Fig. 3

Knock-down CENPM inhibited HCC proliferation, cell invasion and cell migration in vitro. a, b CENPM knockdown efficiency and overexpression efficiency were confirmed by qPCR and western blotting in HepG2, Huh7 and HCCLM3 cell lines. c Colony formation assay showed that CENPM suppressed or promoted cell proliferation both in CENPM-knockdown HepG2 and Huh7 cell lines or CENPM overexpressing HCCLM3 cell line. d, e Downregulation of CENPM in HepG2 and Huh7 cell lines inhibited cell invasion and migration and CENPM overexpression in HCCLM3 reversed these results. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Depletion of CENPM arrested cell cycle and induced cell apoptosis. a FACS analysis showing significant increased or decreased in the proportion of cells in G2/M phase, respectively, when CENPM was knockdown in Huh7 and HepG2 cell or overexpressed in HCCLM3 cells. b Silencing CENPM promoted cell apoptosis in Huh7 and HepG2 cell lines or inhibited cell apoptosis in HCCLM3 cells. c Representative signal pathways of CENPM single-gene GSEA analysis. d Immunofluorescence images showed that CENPM downregulation promoted expression level of nuclear phosphorylated P53. e Western blotting analysis of C-myc, Bcl2, CyclinD1, Bax, P21, cleaved-caspase3 in CENPM-depleted Huh7 and HepG2 cell lines or CENPM-overexpressed HCCLM3 cells. *P < 0.05, **P < 0.01

Fig. 5

The carcinogenic effect of CENPM in vivo. a Representative images of tumor volume and tumors removed from nude mice implanted with shRNA-CENPM transfected HCCLM3 cells (n = 5) or control cells (n = 5). b, c Characteristic images of an orthotopic transplantation tumor model of HCC in nude mice,HE staining and PET/CT. d Xenografts were fixed, embedded in paraffin and stained with CENPM, Bax, cleaved caspase 3, Ki67 antibodies for analysis of tumor growth and apoptosis. **P < 0.01

Fig. 6

HBx upregulated CENPM by targeting miR1270. a The heatmaps of partial differential expressed genes in GSE96851 and GSE38941. b CENPM was up-regulated in HBV-related liver tissues compared with normal tissues in GSE96851 and GSE38941. c miR-1270 was recognized as a post-transcriptional regulator of CENPM by microRNA target prediction programs. d Western blotting showed HBx stably expressed in Huh7 and HepG2 cell lines. e miR-1270 was downregulated by RT-qPCR in HBx stably expressing Huh7 and HepG2 cell lines. f Transfection of HBx resulted in upregulation of CENPM through miR-1270, as determined by PCR. *P < 0.05, **P < 0.01

Fig. 7

miR-1270 negatively regulated CENPM expression in human HCC. a Overexpression of miR-1270 declined CENPM mRNA and protein expression level in Huh7 and HepG2 cells. b CENPM mRNA and protein level was increased after inhibition of miR-1270. c miR-1270 was downregulated in HCC in GSE36915. d IHC of CENPM in HCC samples showed overexpression of miR-1270 reduced CENPM protein levels. e Luciferase reporter assay demonstrated that upregulation of miR-1270 decreased luciferase activity of wild-type CENPM 3′UTR but not that of the mutant. *P < 0.05, **P < 0.01, ***P < 0.001