Abnormal Expression of Centromere Protein U Is Associated with Hepatocellular Cancer Progression

Background Hepatocellular carcinoma (HCC) is one of the most common malignancies globally, but its molecular mechanism is unclear. Abnormal expression of centromere protein U (CENPU) is closely related to diverse human cancers. The purpose of this article was to evaluate the function and potential mechanisms of CENPU in HCC development. Methods We performed bioinformatics analysis of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Gene Expression Profiling Interactive Analysis (GEPIA), and Kaplan-Meier plotter databases to investigate the clinical significance and prognostic value of CENPU in HCC. Western blotting and immunohistochemical staining were used to measure protein expression, while reverse transcription-quantitative polymerase chain reaction (qRT-PCR) was used to determine mRNA expression. Cell Counting Kit8 (CCK-8) and colony formation assays were conducted to examine cell proliferation. Transwell and wound healing assays were used to assess cell migration and invasion. Gene set enrichment analysis (GSEA) was used to explore the potential signaling pathways of CENPU involved in HCC. Results High expression of CENPU in HCC was predicted by public database analysis and indicated a poor prognosis. CENPU expression was significantly higher in HCC tissues and cells than in normal tissues and cell. In vitro, CENPU promoted the proliferation, migration, and invasion of HCC cells. GSEA results indicated that CENPU was linked to the Notch signaling pathway, and our research supported this prediction. Conclusion CENPU promotes the malignant biological process of HCC and may be a promising target for HCC treatment.


Introduction
Hepatocellular carcinoma (HCC) is a common malignancy with high morbidity and mortality rates worldwide [1][2][3]. Currently, the standard treatments for HCC patients mainly involve liver resection, liver transplantation, radiofrequency ablation, transarterial chemoembolization, and chemotherapy [4][5][6][7]. Although considerable effort has been devoted to the study of surgical procedures or chemical therapies, the 5-year survival rates of patients with HCC in China have been reported to be as low as 12% [8]. Therefore, exploring new effective treatment modalities for HCC has crucial clinical significance.
Notch signaling is a crucial pathway in tumorigenesis through the regulation of cell proliferation, apoptosis, and differentiation [22]. Villanueva et al. reported that Notch signaling is activated in human HCC samples and promotes formation of liver tumors in mice [23]. However, the relationship between CENPU and Notch signaling pathway in HCC remains unexplored.
In this study, we first explored the expression and clinical significance of CENPU through bioinformatics analysis, which suggested that CENPU is overexpressed in HCC tis-sues and that high CENPU expression is correlated with poor prognosis among patients with HCC. Subsequently, our data indicated that CENPU is highly expressed in human HCC tissues and cell lines, which was consistent with the results of the public database analysis. We also explored the biological role of CENPU in human HCC cells and  Figure 1: CENPU expression in public databases. (a) TCGA and GTEx dataset analysis showed that CENPU was upregulated in digestive system cancers. (b-d) CENPU was elevated in HCC tissues compared with normal liver tissues from the GSE84402, GSE14520, and GSE46408 based on GEO datasets. (e) CENPU expression was significantly higher in HCC tissues than normal liver tissues according to TCGA HCC database (374 tumor and 50 normal tissues). (f) CENPU expression was significantly higher in 50 cases HCC tissues than in the matched normal liver tissues. * * P < 0:01 and * * * P < 0:001.    2.6. Immunohistochemistry (IHC) Staining. HCC tissues and adjacent tissues were fixed, embedded in paraffin, and cut into 4 μm sections. Then, the samples were heated at 100°C for 15 min incitric acid buffer for antigen retrieval. CENPU antibody (Affinity, 1 : 200) was added as the primary antibody and incubated at 4°C overnight. The following day, the sections were incubated with fluorescence-labeled secondary antibodies (Affinity, China) and finally stained with the diaminobenzidine (DAB) method (Beyotime, China). The degree of IHC staining was scored by two independent pathologists.

CCK-8 Assay.
A total of 2 × 10 3 cells per well were cultured and seeded into 96-well plates for 0, 24, 48, and 72 hours. Ten microliters of CCK-8 reagent (BestBio, China) was added to each well, and the cells were cultured for 2 hours. The absorbance at 450 nm was recorded using an automated microplate reader (BioTek Instruments, USA).

Colony Formation Assay.
A total of 1 × 10 3 cells were added to a 6-well plate and incubated for two weeks. Then, the colonies on the plates were fixed using 4% paraformaldehyde and stained with 0.1% crystal violet solution, and the colonies were imaged and quantified.
2.9. Wound Healing Assay. Cells were seeded into a 6-well plate and allowed to reach approximately 90% confluence. A linear wound was scratched across the cell monolayer using a 200 μL tip. The cells were observed and photographed at 0 hours and 48 hours using an inverted microscope.
2.10. Transwell Assay. Transwell chambers (Corning, USA) were used to determine cell invasion and migration ability. A total of 5 × 10 4 cells/well were plated into the upper chamber, and the lower chamber was maintained with 10% FBS medium. After 24 hours of culture, invading and migrating cells were stained with 0.1% crystal violet and imaged under an inverted microscope.
2.11. Statistical Analysis. All data were analyzed using Graph-Pad Prism and expressed as the mean ± SD. Comparisons between two groups were assessed by Student's t-test or χ 2 test. Univariate and multivariate Cox regression analyses

CENPU Expression in Public Datasets.
We first analyzed the expression of CENPU in common digestive cancers in TCGA and GTEx datasets. The results showed that CENPU expression was upregulated in esophageal cancer, stomach cancer, pancreatic cancer, HCC, colon cancer, and rectal cancer tissues compared with normal tissues (Figure 1(a)). CENPU expression was elevated in HCC tissues relative to normal liver tissues in GEO datasets (Figures 1(b)-1(d)). By analyzing the mRNA sequencing datasets from TCGA HCC cohort, we identified that CENPU was elevated in human HCC tissues versus normal liver tissues (Figures 1(e) and 1(f)). As shown in Table 1, overexpression of CENPU was significantly correlated with histologic grade (P = 0:002), tumor-node-metastasis (TMN) stage (P = 0:001), and tumor depth (P < 0:001), while overexpression of CENPU was not connected with age, sex, M, and N classification (Table 1).

CENPU Expression Status in HCC Tissues and Cell
Lines. As mentioned previously, CENPU expression was elevated in HCC tissues compared with normal liver tissues in public datasets. We initially investigated the expression of CENPU in our fresh HCC tissues and adjacent normal liver tissues, and the results suggested that CENPU expression was elevated in HCC tissues relative to adjacent normal liver tissues (Figures 3(a)-3(c)). Consistently, we detected the expression of CENPU in LO2 normal liver cells and HCC cell lines including HepG2, Huh7, SMCC7721, Bel7402, and HCCLM3. The results showed that the expression of CENPU was elevated in HCC cell lines compared with the LO2 cell line, especially in Bel7402 and SMCC7721 cells (Figures 3(d) and 3(e)). Hence, these two cell lines were used for subsequent experiments.

CENPU Knockdown Suppresses HCC Cell Proliferation,
Migration, and Invasion. As presented above, CENPU expression was significantly associated with the prognosis of HCC patients. Accordingly, we hypothesized that downregulation of CENPU would suppress the malignant behaviors of HCC cells. To explore the potential biological function of CENPU, we selected Bel7402 and SMCC7721 cells as CENPU knockdown models. Knockdown effectiveness was evaluated by western blotting and fluorescence microscopy (Figures 4(a) and 4(b)). CCK-8 assay (Figures 4(c) and 4(d)) and colony formation assay (Figure 4(e)) indicated that knockdown of CENPU significantly inhibited the proliferation of the Bel7402 and SMCC7721 cells. Subsequently, Transwell assays (Figure 4(f)) and wound healing assays (Figure 4(g)) proved that CENPU downregulation suppressed the migration and invasive capability of Bel7402 and SMCC7721 cells.  Figure 5(c)). Colony formation assays indicated that CENPU overexpression promoted Huh7 cell proliferation ( Figure 5(d)). Transwell assays revealed that CENPU overexpression promoted Huh7 cell migration and invasion ( Figure 5(e)). Then, a wound healing assay showed that CENPU overexpression markedly promoted Huh7 cell migration ( Figure 5(f)).
3.6. CENPU Modulates the Notch Signaling Pathway. To evaluate the possible underlying molecular mechanism of CENPU in the progression of HCC, GSEA was conducted to identify the signaling pathway of CENPU in HCC. GSEA results showed that CENPU overexpression was positively associated with Notch signaling pathway (Figure 6(a)). Therefore, we examined the major proteins such as Notch1, Hes1, and Hey1 in the Notch pathway by western blotting. Inhibition of CENPU expression in Bel7402 and SMCC7721 cells decreased the expression of these proteins in the Notch signaling pathway (Figure 6(b)). These results revealed that high expression of CENPU in HCC may activate the Notch signaling pathway.

Discussion
Hanissian et al. [9] first reported that CENPU was correlated with myeloid leukemia factor 1 (MLF1) and located in both the nuclei and cytoplasm of cells. CENPU localizes to human chromosome 4q35.1 and encodes a 46 kDa protein [10]. More recently, previous studies suggested that CENPU was expressed in various tissues such as a fetal liver, bone marrow, and thymus and testis [11][12][13][14][15]. Accumulating evidence has indicated that CENPU is upregulated in several human cancers and may play a key role in cancer progression [16][17][18][19][20]. Studies have revealed that CENPU expression is highly expressed in NSCLC tissues and that CENPU knockdown represses tumor proliferation and metastasis in NSCLC cells by regulating the Wnt/β-catenin signaling pathway [16]. Additionally, Wang et al. [19] reported that CENPU is upregulated in NSCLC tissues and facilitates lung cancer cell proliferation by targeting the transcription factor FOXM1. CENPU is elevated in human bladder cancer tissues compared with surrounding tissues, and high CENPU expression is obviously associated with tumor size, TNM stage, and poor prognosis [18]. Recent researchers revealed that CENPU highly expressed in ovarian cancer samples and that overexpression of CENPU augments ovarian cancer aggressiveness by regulating high-mobility group box 2 (HMGB2) [17]. Pan et al. [20] demonstrated that CENPU promotes tumor angiogenesis through the cyclooxygenase-2-(COX-2) mediated signaling pathway in triple-negative breast cancer. Nevertheless, the functional role of CENPU in HCC remains unclear.
In this study, we first revealed that CENPU is upregulated in common digestive cancer tissues compared to normal tissue through bioinformatics analysis. Therefore, the oncogenic value of CENPU in HCC was explored for a further study. Bioinformatics analysis confirmed that high CENPU expression is related to a poor prognosis in HCC patients. Additionally, univariate and multivariate analyses showed that TMN stage, tumor depth, distant metastasis, and CENPU expression can be used as independent risk factors for HCC. These results indicated a possible function of CENPU in HCC pathogenesis.
To the best of our knowledge, this is one of the first studies to determine CENPU expression in HCC cells and explore its clinical value among patients with HCC. Consistent with public databases, our data indicated that CENPU is highly expressed in HCC tissue, whereas in adjacent noncancerous tissues CENPU expression is low. Moreover, HCC cell lines showed higher expression of CENPU than a normal liver cell line. In the current study, we conducted in vitro assays to investigate the effect of CENPU on tumor development. Furthermore, our results revealed that downregulation of CENPU restrained HCC cell proliferation, migration, and invasion, while upregulation of CENPU facilitated HCC cell proliferation, migration, and invasion. Our findings indicated that CENPU has a tumorigenic role in the development of HCC.
To further study the molecular mechanism of CENPU in HCC, we performed GSEA to investigate potential related signaling pathways. The GSEA results revealed that CENPU expression was associated with the Notch signaling pathway. Some studies have found that the Notch signaling pathway regulates cell differentiation in addition to cell proliferation and metastasis in a variety of cancers [24][25][26][27]. We demonstrated that the expression of Notch signaling pathway proteins including Notch1, Hes1, and Hey1 was significantly decreased after CENPU knockdown in HCC cells. Together, these findings indicated that CENPU might be an oncogene that promotes HCC progression through the Notch signaling pathway. Hence, further research on this topic is recommended in the future.

Conclusions
In summary, our findings indicate that CENPU is upregulated in HCC and is an unfavorable predictor of prognosis in HCC patients. Furthermore, CENPU may promote HCC cell proliferation, migration, and invasion through the Notch signaling pathway. Therefore, targeting CENPU might represent a novel therapeutic strategy for HCC patients.