High mRNA Expression of CENPL and Its Significance in Prognosis of Hepatocellular Carcinoma Patients

Centromere proteins (CENPs) are the main constituent proteins of kinetochore, which are essential for cell division. In recent years, several studies have revealed that several CENPs were aberrantly expressed in hepatocellular carcinoma (HCC). However, numerous centromere proteins have not been studied in HCC. In this study, we used databases of Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), the Kaplan-Meier Plotter, cBioPortal, the Human Protein Atlas (HPA), and TIMER (Tumor Immune Estimation Resource) and immunohistochemical staining of clinical specimens to investigate the expression of 15 major centromere proteins in HCC to evaluate their potential prognostic value. We found that the mRNA levels of 4 out of 15 centromere proteins (CENPL, CENPQ, CENPR, and CENPU) were significantly higher in HCC than in normal tissues, and their mRNA levels were associated with the tumor stages (p values < 0.01). Patients with higher mRNA levels of CENPL had poorer overall survival, progression-free survival, relapse-free survival, and disease-specific survival (p values < 0.05). Furthermore, the higher levels of CENPL mRNA were associated with worse overall survival in males without hepatitis virus infection (p values < 0.05). The protein expression level of CENPL in human HCC tissue was higher than that in normal liver tissue. In addition, the expression of CENPL was positively correlated with the levels of the tumor-infiltrating lymphocytes. The results suggest that the high mRNA expression of CENPL may be a potential predictor of prognosis in HCC patients.


Introduction
Hepatocellular carcinoma (HCC) is the fourth major cause of cancer-related death globally [1]. Patients with early-stage HCC are eligible to local ablation, surgical resection, or liver transplantation. Unfortunately, due to the lack of satisfactory biomarkers in diagnosis, treatment, and prognosis, morbidity and mortality continue to increase [2]. Therefore, it is necessary to explore novel diagnostic, therapeutic, and prognostic biomarkers for HCC.
The centromere proteins (CENPs) are assembled in repetitive noncoding DNA regions (centromere DNA). They are named in alphabetical order, according to the date of identifi-cation [3]. To date, more than 100 centromere proteins have been identified. These proteins are attached to chromosomes and spindle microtubules and are involved in precise regulation and guidance of chromosome separation during mitosis or meiosis [3,4] A growing number of studies have revealed that several CENPs were abnormally expressed in lung, breast, ovarian, bladder, esophageal, and colorectal cancers and were correlated with the prognosis of patients [5][6][7][8][9][10][11][12][13][14].

Methods
2.1. Oncomine Database Analysis. We used Oncomine (http://www.oncomine.org), an online cancer microarray database [33], to analyze the transcription levels of 15 CENPs in tumors and normal tissues from HCC and other cancers. Datasets of clinical tumor and normal control specimens were compared using Student's t -test. For all cancers and genes, the threshold was set as the p value of 0.0001 and the fold change of 2.   Figure 1: The transcription levels of CENP in different types of cancers. The mRNA levels of CENPL, CENPQ, CENPR, and CENPU were significantly higher in HCC than in normal tissues (p < 0:01) (Oncomine).  [34]. It was used to analyze mRNA differential expression in HCC and liver tissues, as well as the correlation between expression levels and pathological stages. Differential threshold of | log 2 FC| was 1 and the p value cutoff of 0.01.

Disease Markers
based proteomics, transcriptomics, and systems biology (https://www.proteinatlas.org/). The Human Pathology Atlas is based on a systems-based analysis of the transcriptome of 17 main cancer types using data from 8000 patients [36]. In this database, we verified the protein expression of CENPs with potential prognostic value in normal liver and HCC tissues.

Immunohistochemical Staining.
Five μm sections were obtained from paraffin-embedded tumor and nontumor specimens from 28 patients with clinically diagnosed HCC. All these sections were dewaxed in xylene and rehydrated in alcohol followed by wet autoclave pretreatment in citrate buffer for antigen retrieval (10 minutes at 120°C, pH = 6:0). Then, the sections were rinsed with phosphate buffer saline. Immunohistochemical staining for antibody to CENPL (rabbit: bs13836R, Beijing Biosynthesis Biotechnology, China) was performed using the avidin-biotin-peroxidase complex method. The primary antibody was applied to the sections and allowed to react for 1 hour at room temperature. The sections were then incubated with biotinylated antimouse/rabbit antibody (1 : 200 dilution for CENPL) for 30 min and avidin-biotin-peroxidase reagent for 25 min. After color development with diaminobenzidine, the sections were counterstained with hematoxylin.

Relationship between the mRNA Levels of CENPs and
Pathological Stages of HCC. Then, we selected 4 CENPs (CENPL, CENPQ, CENPR, and CENPU) with high expression in HCC as shown in both the Oncomine and GEPIA databases to further analyze the relationship between mRNA levels and tumor stages. The analysis showed that high mRNA levels of CENPL, CENPQ, CENPR, and CENPU were significantly correlated with tumor stages (p < 0:01) (Figure 4).

Correlation between the Increased mRNA Levels and the Prognosis of CENPs in Patients with HCC.
To evaluate the prognostic value of 4 CENPs (CENPL, CENPQ, CENPR, and CENPU) with high mRNA expression in HCC patients, we used the Kaplan-Meier Plotter to analyze their overall survival (OS), progression-free survival (PFS), relapse-free survival (RFS), and disease-specific survival (DSS). It was found that patients with higher mRNA levels of CENPL were associated with significantly poor OS, PFS, RFS, and DSS   Table 2). Patients with higher CENPQ mRNA expression had poorer OS, PFS, and DSS (p < 0:05), and patients with higher CENPR mRNA levels had poorer PFS and RFS (p < 0:05). Patients with increased CENPU mRNA levels had lower PFS and RFS (p < 0:05) ( Figure 5, Table 2). The level of CENPL mRNA showed a potential value in predicting the prognosis of patients with HCC. Therefore, we further explored the relationship between the mRNA expression of CENPL and the OS of patients in subgroups as divided by parameters including gender, race, alcohol consumption, and hepatitis virus infection. The results showed that males with high expression of CENPL mRNA had poorer OS (Figure 6(a), Table 3). Asians with higher mRNA levels of CENPL had worse OS (Figure 6(b), Table 3). Patients with higher CENPL expression had worse OS regardless of alcohol consumption (Figure 6(c), Table 3). Among patients without hepatitis virus infection, patients with higher expression of CENPL had poorer OS ( Figure 6(d), Table 3). Furthermore, we found that higher  Table 3).

Mutations and Copy Number Alterations of CENPL in HCC.
We used the cBioPortal database to investigate the mutations and copy number alterations of CENPL. Gene alterations occurred in 4% of the patients in the 5 datasets (Figure 7(a)). There was a significant correlation between the gene variation and patients' OS and disease-free survival (p < 0:05) (Figures 7(b) and 7(c)).

Relationship between the Expression of CENPL and
Immune Cells in HCC. To further understand the relationship between CENPL mRNA level and immune infiltration cells in an HCC microenvironment, we used TIMER to evaluate immune cell infiltration data from The Cancer Genome Atlas (TCGA). The results illuminated that the expression of CENPL was positively correlated with infiltrating levels of B cell, CD4+ T cell, CD8+ T cell, neutrophil, macrophage, and dendritic cell in HCC, respectively (p < 0:01) (Figure 8). Similarly, the expression of the CENPL gene was positively associated with the tumor purity (p < 0:05) (Figure 8).

The Expression of CENPL Protein in Tumor and Normal
Tissues of HCC Patients. In order to learn the expression of CENPL protein in HCC specimens, we firstly studied it in the HPA database. The immunohistochemical staining results indicated that the expression of CENPL protein was significantly higher in HCC tissues than in normal liver tissues, and the protein staining intensity score was modest in HCC tissues and low in normal liver tissues (Figure 9(a)). The CENPL protein was mainly localized in the cytoplasm and membrane (Figure 9). Our further clinical specimens confirmed similar results. In 28 clinical specimens, the expression level of CENPL protein was higher in HCC tissues (16/28) than in noncancer tissues (12/28) (Figure 9(b)).

Discussion
In this study, the transcriptions of 15 CENPs in HCC were investigated by using the Oncomine and GEPIA databases. Results showed that the mRNA levels of CENPL, CENPQ, CENPR, and CENPU in HCC tissues were significantly higher compared with those in normal tissues. Their mRNA levels were significantly correlated with the pathological stages of HCC. Patients with higher mRNA levels of CENPL had poorer OS, PFS, RFS, and DSS. Then, the prognostic value of CENPL was further studied under different clinical parameters. We found that higher levels of CENPL mRNA were associated with poorer OS in white and Asian male patients without hepatitis virus infection. Mutation analysis showed that CENPL was rarely mutated, suggesting that the pathophysiological role of CENPL in HCC was not mediated by gene mutation. In addition, we found that the expression of CENPL was positively correlated with infiltrating levels of B cell, CD4+ T cell, CD8+ T cell, neutrophil, macrophage, and dendritic cell in HCC. Finally, HPA data and immunohistochemical staining of our clinical specimens showed that CENPL was overexpressed in HCC tissues.
CENPs play an important physiological role in the cell cycle, and their expression levels are strictly regulated; therefore, no pathological effect was shown. However, many CENPs with an abnormal expression were found in tumor cells. To date, it is not clear whether this result is an initiating factor or a result of tumor development. On the one hand, the abnormal expression of CENPs may interact with other    [40]. On the other hand, chromosomal and genetic instability are now recognized as the important cause of cancer, and the abnormal expression of CENPs is likely to play important roles through this mechanism [5,[41][42][43]. These results illustrate the complex mechanism between CENPs and HCC. Furthermore, the expression of CENPs in patients with cirrhosis has not been reported, which may help to reveal the mechanism of CENPs and HCC. CENPL is a subunit of the CENPH-CENPI-associated centromeric complex that targets CENPA to centromeres and is required for proper kinetochore function and mitotic progression [44]. Diseases associated with CENPL include the chromosome 22Q11.    In this study, we found that CENPL was overexpressed in HCC, and the mRNA level was closely related to various clinicopathological parameters of patients. These results suggest that CENPL may play an important role in HCC and that CENPL is a potential target for future immunotherapy and a prognostic predictor. However, there are several limitations to this study. We have not yet verified the association between CENPL and tumor-infiltrating lymphocytes in human or mouse specimens. The effects of CENPL on the proliferation, apoptosis, and metastasis of HCC cells have not been further studied. Therefore, in-depth studies are needed in the future.
In conclusion, the present study suggests that CENPL mRNA may be a potential prognostic biomarker of HCC patients.