SPC25 overexpression promotes tumor proliferation and is prognostic of poor survival in hepatocellular carcinoma

Abstract

Background: The nuclear division cycle 80 (NDC80) complex assures proper chromosome segregation during the cell cycle progression. SPC25 is a crucial component of NDC80, and its role in hepatocellular carcinoma (HCC) has been explored recently. This study characterized the differential expression of SPC25 in HCC patients of different races and HBV infection status.

Methods: Expression patterns of SPC25 were evaluated in TCGA and Chinese HCC patients. Kaplan-Meier analysis was applied to examine the predictive value of SPC25. In vitro and in vivo functional assays were conducted to explore the role of SPC25 in HCC. Bioinformatics methods were applied to investigate the regulatory mechanisms of SPC25.

Findings: The mRNA levels of SPC25 were up-regulated in HCC. SPC25 has a significantly higher transcriptional level in Asian patients than Caucasian patients. SPC25 promoted HCC cell proliferation in vitro and tumor growth in vivo by accelerating the cell cycle. We identified transcription factors, miRNAs, and immune cells that may interact with SPC25.

Interpretation: The findings suggest that increased expression of SPC25 is associated with poor prognosis of HCC and enhances the proliferative capacity of HCC cells. SPC25 could serve as a valuable prognostic marker and a novel treatment target for HCC.

Keywords: HCC prognosis; HCC progression; SPC25.

Figure 1

SPC25 expression in normal human tissue, human tumors, and in HCC. (A) SPC25 mRNA expression in normal human tissues. GTEx: The Genotype-Tissue Expression project; FPKM: Fragments Per Kilobase per Million. (B) SPC25 mRNA expression in multiple human cancers is higher compared to corresponding normal tissues. RPKM: Reads Per Kilobase per Million. (C) SPC25 mRNA is significantly overexpressed in HCC samples compared to normal controls, according to UALCAN. (D) Both male and female HCC patients showed a higher SPC25 transcriptional level than normal controls, according to UALCAN. (E) UALCAN analysis showed that SPC25 mRNA levels are significantly higher among Caucasian, African-American, and Asian HCC patients than normal controls. Besides, SPC25 showed a relatively higher transcriptional level in Asian patients in contrast with Caucasian patients. (F) SPC25 mRNA expression in Asians HCC patients is significantly higher than that in the Caucasian HCC patients based on TCGA HCC samples screened by race. (G) SPC25 mRNA is significantly overexpressed in Chinese HCC tumor specimens vs. corresponding adjacent normal liver tissue. (H) Representative images of SPC25 protein expression in normal liver and HCC samples from Chinese patients. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

Prognostic role of SPC25 mRNA in HCC patients. (A) Effect of SPC25 expression level on HCC patient survival (UALCAN). (B) Effect of SPC25 expression level and gender on HCC patient survival (UALCAN). (C) Effect of SPC25 expression level and Race on HCC patient survival (UALCAN). (D) Effect of SPC25 expression level and Race on HCC patient survival (TCGA samples). (E) Subgroup overall survival analysis of SPC25 mRNA level in Asian or Caucasian HCC patients. (F) Subgroup overall survival analysis of SPC25 mRNA level in HCC patients with or without hepatitis b. (G) Subgroup overall survival analysis of SPC25 mRNA level in Asian HCC patients with or without hepatitis b.

Figure 3

Univariate regression analysis of clinical information in TCGA HCC samples.

Figure 4

Functional analysis of SPC25 overexpression in HCC cells. (A) The protein levels of SPC25 were detected by western blot analysis in SPC25 or vector-transfected cells. β-actin was used as an endogenous control. (B) CCK-8 assays, (C) colony formation, and (D) non-adherent colony formation assays demonstrated that overexpression of SPC25 promoted proliferation of Huh7 and PLC8024. (E) Images of xenograft tumors derived from SPC25-transfected cells and their vectors in nude mice. Tumor sizes were compared in the right chart. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

DNA methylation modification and transcription factors associated with SPC25 in HCC. (A) SPC25 DNA methylation modification in HCC. (B) Correlation between SPC25 mRNA expression and DNA methyltransferase (DNMT) expression. (C) Top 20 transcription factors (TFs) that potentially regulate SPC25 in HCC. (D) TFs with high regulatory potential in HepG2 cell lines (10k distance to transcription start site, TSS). (E) POLR2A mRNA expression is significantly higher in HCC samples than in normal samples. (F) Correlation between SPC25 and POLR2A mRNA expression. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

miRNAs that down-regulated SPC25 expression in HCC. (A) Two miRNA prediction datasets, starBase, and Targetscan were used to predict miRNAs that may bind to SPC25 mRNA. (B) miRNAs negatively correlated with SPC25 mRNA expression (data from Linkomics). (C) Correlation between SPC25 mRNA and miR-451 expression. (D) Dual-luciferase reporter assay for confirmation of the targeting relationship between miR-451a and SPC25; *P < 0.05; statistical data were presented as mean ± standard deviation; data between two groups were analyzed by paired t-test; one-way analysis of variance was used for multi-group comparisons; the experiment was repeated three times independently, NC negative control.

Figure 7

Mechanistic investigation into how SPC25 promotes HCCs growth. (A) Gene Set Enrichment Analysis (GSEA) shows statistically significant different biological function between subgroups of high SPC25 level and low SPC25 level. (B) STRING analysis of interactions between SPC25 and other proteins. (C) Correlation between SPC25 and cell cycle-regulation related genes, including CDK1, cdc25A, Cyclin A2, Cyclin B1, Cyclin D1, Cyclin E1. (D) Two siRNAs (si#1 and si#2) against SPC25 effectively silenced SPC25 expression, as determined by qRT-PCR. Negative control siRNA (siNC) and β-actin were used as negative and endogenous controls, respectively. The data are represented as the mean ± s.d. of three independent experiments. **P < 0.01. (E) Silencing SPC25 decreased the protein level of SPC25, CDK1, cdc25A, Cyclin A2, Cyclin B1, Cyclin D1, and Cyclin E1.

Figure 8

(A) Correlations between SPC25 expression and six types of immune cells infiltrated in HCC tissues. CPM: Counts of exon model per million mapped reads. (B) The infiltration level of six types of immune cells classified by the copy number variance (CNV) of the SPC25 gene.