Introduction

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, constituting for approximately 3 % of adult malignancies and 90 % of all neoplasms arising from the kidney (Rini et al. 2009). Clear cell RCC (ccRCC), which accounts for approximately 80–90 % of all RCC cases, is the most common histological subtype (Vincenzi et al. 2006). RCC may act out clinically occult for most of its disease development. Only 6–10 % of patients accompany by the classic triad of hematuria, flank pain, and flank mass (Lee et al. 2002). The mortality of metastatic RCC at 15 months is 50 % and the 5-year survival rate is less than 10 % (Volpe and Patard 2010).

Surgical resection and ablative therapy remain known few effective treatments for localised RCC, and they are also used for removing the tumor burden in metastatic disease (Ljungberg et al. 2015). RCC is relatively resistant to radiation therapy and chemotherapy. Chemotherapy is used only occasionally, in certain tumor types. Although targeted therapy and immunomodulatory agents are considered standard of care in patients with metastatic disease (Rini et al. 2009), nearly all patients die from untreatable metastatic disease. It is necessary to identify more effective RCC-specific biomarkers that can improve early diagnosis, prediction of prognosis and targeted therapy (Ngo et al. 2014; Shi et al. 2014; Yu et al. 2013).

Centromere protein H (CENPH) is an essential component of the active kinetochore complex, localized in the inner plate of the kinetochore with CENPA and CENPC (Fukagawa et al. 2001; Sugata et al. 2000). Spindle microtubules attach on kinetochore to pull apart the sister chromatids. Normal expression of kinetochore formation proteins is vital to mitosis (Bakhoum and Compton 2012); however, disruption of core kinetochore proteins may induce an increase in chromosomal instability, aneuploidy and oncogenesis (Duensing and Duensing 2010; Kramer et al. 2002). The CENPH somatic frameshift mutation contributes the development of gastric cancer and colorectal cancer (Kim et al. 2010). A study in zebrafish uncovered an essential role of CENPH in mitosis, embryonic development and tumorgenesis (Zhao et al. 2010).

Recently, CENPH has attracted considerable interest for its novel prognostic value in various malignant tumors. Previous studies have suggested that CENPH was upregulated and may be a promising prognostic marker in human oral carcinomas, tongue cancer, nasopharyngeal carcinoma, hypopharyngeal cancer, lung cancer, breast cancer, esophageal carcinoma, gastric carcinoma, colorectal cancer, and hepatocellular carcinoma (Guo et al. 2008; He et al. 2013; Liao et al. 2011, 2007, 2009a, b; Lu et al. 2013; Shigeishi et al. 2006; Tomonaga et al. 2005; Wang et al. 2012). Our transcriptome sequencing data of RCC also showed CENPH expression was remarkable upregulated in carcinoma tissue (unpublished results).

In the present study, we aimed to explore the expression level, clinical significance, and biological functions of CENPH in RCC. We first examined the expression level of CENPH in ccRCC tissues and RCC cell lines by using qRT-PCR and Western blot. Next, we analyzed its correlations with clinicopathological characters in order to determine the clinical significance of CENPH in ccRCC. Additionally, we conducted in vitro assays to demonstrate the biological functions of CENPH in RCC development and progression. Our research revealed a novel molecule involved in the progression of RCC.

Methods

Patients and surgical specimens

Twenty-one pairs of tumor tissues and matched adjacent nontumor tissues obtained from ccRCC patients in Zhujiang Hospital of Southern Medical Universty (Guangzhou, China) were frozen and stored in liquid nitrogen for qRT-PCR analysis. In addition, 109 formalin fixed and paraffin-embedded ccRCC samples were collected from the First Affiliated Hospital of Jinan University (Guangzhou, China) for immunohistochemical assays use. All 109 patients underwent curative surgical resection from May 1, 2005 to December 31, 2009 at the hospital and were clinically and histopathologically diagnosed. The clinical stage of these enrolled patients was classified according to the 2010 American Joint Committee on Cancer (AJCC) staging system. Before surgery, none of the patients received molecular targeting therapy. Clinicopathological characteristics of the patients are summarized in Table 1. This study complied with the local ethics committee guidelines and written informed consent was obtained from each patient. All 109 ccRCC patients had been followed up until December 2014 or death with a median observation time of 54 months.

Table 1 Relationship between CENPH expression level and clinicopathologic characteristics
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Cell culture

Immortalized normal human proximal tubule epithelial cell line HK-2 was purchased from the American Type Culture Collection (ATCC, USA). Human RCC cell lines A704, A498, ACHN and 786-O were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (CCCAS, China). HK-2 cells were cultured in DMEM/F12 medium (Gibco). Other cells were maintained in RPMI 1640 medium (Gibco). All mediums were supplemented with 10 % fetal bovine serum (FBS) (Gibco) and 1 % penicillin–streptomycin solution (Gibco). The culture plates were incubated at 37° with 5 % CO2 in a sterile incubator.

RNA extraction and reverse transcription-PCR

Total RNA from surgically obtained tissues and cultured RCC cell lines were isolated using RNA isolation kit (GenStar Biosolutions). RNA was reverse transcribed into cDNA using PrimeScript RT reagent Kit (Takara Bio) according to instructions provided. The cDNA was then used for the evaluation of the relative mRNA levels of the CENPH, running in an ABI 7300 Analyzer (Applied Biosystems). SYBR Green I (Tiangen Biotech) was used for the fluorescent detection. The relative expression levels of CENPH gene were internally normalized against that of the housekeeping gene GAPDH. The primer sequences of CENPH and GAPDH (Sangon Biotech, Shanghai, China) were as follows: CENPH, forward primer, 5′-CCTTATTTTGGGGAGTAAAGTCAAT-3′ and reverse primer, 5′-ACAAATGCACAGAAGTATTCCAAAT-3′; GAPDH, forward primer, 5′-GGAGTCCACTGGCGTCTT-3′ and reverse primer, 5′-CTTGAGGCTGTTGTCATACTTC-3′. The PCR amplification were performed for 95 °C for 15 m, then 45 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 32 s in 20 μl reaction volume. The relative quantitative value of CENPH mRNA was expressed by the 2−ΔΔCt method. Each experiment was completed in triplicates and repeated independently three times.

Western blot assay

Culture cells in 6-well plates were subjected to standard Western blot assay as described previously (Lai et al. 2014). Mouse monoclonal anti-CENPH antibody (1:200, Santa Cruz), mouse monoclonal anti-Ki-67 antibody (1:1000, GeneTex), and mouse monoclonal anti-survivin antibody (1:150, Santa Cruz) was used to detect the expression level of target proteins. Mouse monoclonal anti-GAPDH antibody (1:250, Santa Cruz) was used to detect the loading control protein GAPDH.

Immunohistochemistry (IHC)

All samples were previously embedded in paraffin blocks, cut in 4-μm-thick sections, deparaffinized and dehydrated. After antigen retrieval by boiling in citrate buffer (10 mmol/L, pH 6.0), these sections were treated with 3 % H2O2 solution, blocked with 4 % bovine serum albumin for 30 min and incubated overnight with primary mouse monoclonal anti-CENPH antibody (1:120, Santa Cruz) at 4 °C. Then sections were incubated with corresponding secondary antibody, and subsequently stained with DAB kit (ZSGB Bio) to achieve visualization. The negative control was treated in an identical manner except that the primary antibody was omitted. The overall staining for CENPH was measured by the multiplication of staining percentage (0–100 %) and staining intensity on a 4 point scale (none = 1, weak = 2, moderate = 3, strong = 4), resulting in an overall product score. The immunohistochemical staining evaluation was performed independently by two authors without knowledge of the clinic outcome information.

Transfection

Small interfering RNA that targeted CENPH (si-CENPH) and a scrambled negative control (si-NC) were generously provided by GenePharma (Shanghai, China). The sequences of si-CENPH were 5′-UUGAAAGUCUCAUCCUGUCTT-3′. Human RCC ACHN and 786-O cells were transfected with either 50 nmol si-CENPH or si-NC using Lipofectamine 3000 transfection reagent (Life Technologies) according to the manufacturer’s instruction. After 48 h, cells transfected with small-interfering RNA (siRNA) were harvested for qRT-PCR and Western blot to determine the transfection efficiency.

Cell proliferation assay

ACHN and 786-O cells that transfected with siRNA were seeded into 96-well plates. Cell density was adjusted to 5 × 103/well, and the final volume was 150 μL/well. CKK-8 solution (10 μL) was added to the plates 24, 48, 72, and 96 h later. Then, the cells were oscillated for 2 h at 37 °C. The absorbance was detected at 570 nm with a Microplate Reader (Bio-Rad). Each assay was performed in triplicate wells and repeated three times.

Colony formation assay

Cells transfected with si-CENPH and si-NC were trypsinized, counted and plated in 6-well plates at the density of 200 cells per well in triplicate. After cultured for 12 days, the colonies were fixed with absolute ethyl alcohol for 20 min and then stained with 0.01 % crystal violet for 15 min. Then the number of visible colonies which contained more than 50 cells was manually counted.

Apoptosis assay using flow cytometric

Annexin V-kFluor488/PI Apoptosis Detection Kit (KeyGEN Bio) was used to detect apoptosis. ACHN and 786-O cells were seeded into 6-well plates, then they were transfected with siRNA for 2 days. The cells were washed with PBS twice after digested and harvested by no EDTA trypsin, and resuspended in 500 μL binding buffer. Then 5 μL Annexin V-keyFluor488 and 5 μL propidium iodide (PI) were added to this cell suspension. Stained cells were instantly analyzed by flow cytometry (BD FACSAria III). Apoptosis assay was performed in triplicate, and the cell apoptosis rate was calculated as follows: (early apoptotic cell number + apoptotic cell number/total cell number) × 100 %.

Statistical analysis

Statistical analysis was performed using SPSS 20 statistical software (IBM, Chicago, IL, USA). Continuous data statistics were performed with two-tailed paired Student’s t test. Categorical data were analyzed using the two-side Chi square test. Overall survival (OS) curves were estimated by the Kaplan–Meier method and compared by using the log-rank test. Univariate and multivariate analyses were conducted according to the Cox proportional hazards regression model to assess associations between the clinicopathologic variables and survival. In all analyses, the differences were considered statistically significant whenever P < 0.05.

Results

Increased expression of CENPH in ccRCC tissues and cell lines

To determine the CENPH mRNA expression, qRT-PCR analysis was conducted on 21 cases of paired ccRCC tissue and adjacent nontumor tissue. Our results exposed a statistically significant rise of CENPH mRNA in ccRCC tissues, as compared to the matched adjacent non-tumor tissues (P < 0.01, Fig. 1a).

Fig. 1
figure 1

The CENPH expression levels in ccRCC tissues and RCC cell lines. The CENPH expression levels were established by using qRT-PCR and Western blot. a Expression of CENPH mRNA in 21 matched pairs of ccRCC tissues and adjacent normal tissues was examined by qRT-PCR. b, c Expression of CENPH mRNA and protein in RCC cell lines (A704, A498, 786-O and ACHN) and HK-2 was examined by qRT-PCR (b) and Western blot (c). Results are expressed as mean ± SD for three replicate determination. All data analyzed using Student’s t test. *P < 0.05; **P < 0.01

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Next, we determined whether CENPH was also upregulated in the human ccRCC cell lines; we used qRT-PCR and Western blot. The expression of CENPH mRNA and protein were determined for four RCC cell lines (ACHN, 786-O, A704 and A498) and compared with their expression in human proximal tubule epithelial cell line HK-2. CENPH mRNA and protein were highly expressed in three ccRCC cell lines (ACHN, 786-O and A704) compared to HK-2 cells (P < 0.05, Fig. 1b, c).

Immunohistochemical analysis of CENPH expression in ccRCC tissues and its relationship to clinicopathological features

We investigated the status of CENPH expression in 109 paraffin-embedded archived ccRCC tissues by immunohistochemical staining. CENPH protein expression in tumors was usually increased compared with that in adjacent normal tissues. Histological assessment revealed that 87 of 109 (79.8 %) ccRCC tissues showed positive CENPH staining. CENPH stained mainly in the nucleus of the cells, while the cytoplasm also lightly stained in some patients (Fig. 2). Fifty cases showed low CENPH expression (CENPH− or CENPH+), and 59 cases exhibited high CENPH expression (CENPH++ or CENPH+++). IHC was employed to investigate the association between CENPH expression and clinicopathological features in the 109 ccRCC specimens. The expression level of CENPH was significantly associated with histological grade (P = 0.001), tumor stage (P = 0.014), and distant metastasis (P = 0.024). There was no significant association between CENPH expression and patients’ gender, age, tumor size and lymph nodes metastasis. Detailed data is shown in Table 1.

Fig. 2
figure 2

Expressions CENPH in human ccRCC tissues and matched non-cancer tissues. a Negative or low-level staining of CENPH in paired normal tissue. b Low-level staining of CENPH in tumor tissue. c Moderate staining of CENPH in tumor tissue. d Strong staining of CENPH in tumor tissue. Scale bars, 25 μm

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High CENPH expression predicts poor prognosis in patients with ccRCC

Kaplan–Meier analysis showed that patients with high CENPH expression had significantly shorter overall survival time than those with low CENPH expression (log-rank test, P = 0.002) (Fig. 3). As in Table 2, CENPH expression level (P < 0.001) and clinical stage (P = 0.005) were significantly correlated with overall survival rate of patients with ccRCC. A multivariate analysis also showed that relative expression of CENPH (P < 0.001) and clinical stage (P = 0.018) were independent prognostic factors for the overall survival of ccRCC patients (Table 2). These results revealed that CENPH expression level can be developed as a powerful independent prognostic factor in ccRCC patients.

Fig. 3
figure 3

The survival analysis of CENPH. Patients with higher CENPH expression in tumor were closely correlated with poorer overall survival than patients with lower CENPH expression in tumor

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Table 2 Prognostic factors in Cox proportional hazards model
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CENPH downregulation decreases cell growth

We chose two CENPH high expression cell lines, ACHN and 786-O, to further investigate the role of CENPH in human RCC cells. After transfection with si-CENPH, cells showed a down-regulated mRNA and protein expression of CENPH compared to negative control (si-NC) group (P < 0.05, Fig. 4a, b). Our result indicated that we successfully down-regulated the CENPH expression in human RCC cell line ANCH and 786-O.

Fig. 4
figure 4

Knockdown of CENPH inhibited cell growth in ACHN and 786-O cells. a The mRNA level of CENPH in ACHN and 786-O cells was significantly decreased by si-CENPH compared with si-NC group. b Western blot analysis shows that the protein expressions of CENPH, Ki-67, and survivin in ACHN and 786-O cells were significantly decreased by si-CENPH compared with si-NC group. c After transfection of si-CENPH, CCK-8 assays were conducted to determine the viability of ACHN and 786-O cells in 96-well plates for 72 h. d Silencing CENPH inhibited cells growth which was determined by colony formation assay after the cells plated onto 6-well plates for 12 days. e Si-CENPH induced apoptosis of ACHN and 786-O cells. *P < 0.05; **P < 0.01; ***P < 0.001

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Next, we conducted CCK8 and colony formation assay to determine whether knockdown of CENPH inhibited the proliferation of ACHN and 786-O cell lines. As shown in Fig. 4c, CENPH siRNA significantly decreased cell viability of ACHN and 786-O cells compared to control siRNA group, respectively. Furthermore, we found that the colony-forming ability of these two cell lines was lowered by transfection with CENPH siRNA (Fig. 4d). Taken together, these results indicated that CENPH is essential for the proliferation of RCC cell lines and may be involved in the tumorigenesis of RCC.

CENPH siRNA induces apoptosis of RCC cell lines

ACNH and 786-O cell lines were transfected with si-CENPH and si-NC. Two days later, the cell apoptosis ratio was detected by flow cytometry. The results demonstrated that cells transfected with si-CENPH had a significantly higher apoptosis rate than cells transfected with si-NC (Fig. 4e). This results suggested that the knockdown of CENPH had the potential to induce apoptosis of RCC cells.

CENPH modulates Ki-67 and survivin expression

As CENPH expression affected cell proliferation, we checked out the proliferation markers that could be modulated by CENPH. A significant decrease of Ki-67 expression was found in CENPH knockdown cells compared with cells transfected with control siRNA. Moreover, we also found CENPH silencing inhibited expression of an inhibitor of apoptosis protein survivin in RCC cell lines (Fig. 4b).

Discussion

In the current research, a comprehensive knowledge about the pathogenic characteristics of CENPH with respect to RCC oncogenesis was gathered, and our results indicate that overexpression of CENPH plays an important role in RCC oncogenesis and progression. Consistent with previous studies in other types of malignant tumor, we demonstrated that CENPH expression was higher in the RCC than that in adjacent non-tumor tissues, suggesting that CENPH may be involved in the malignancy of RCC.

CENPH was first reported as a novel mouse kinetochore protein in 1999 (Sugata et al. 1999), then it was reported in human HeLa cells in 2000 (Sugata et al. 2000). It was present outside centromeric heterochromatin and inside the kinetochore corona, constituted CENP multimers with other CENPs, localized constitutively to the inner kinetochore plate and played an important fundamental role in organization and function of the active human centromere-kinetochore complex (Sugata et al. 2000). High CENPH expression is associated with progression in many cancer types, such as oral carcinomas, tongue cancer, nasopharyngeal carcinoma, hypopharyngeal cancer, lung cancer, breast cancer, esophageal carcinoma, gastric carcinoma, colorectal cancer, and hepatocellular carcinoma (Guo et al. 2008; He et al. 2013; Liao et al. 2011, 2007, 2009a, b; Lu et al. 2013; Shigeishi et al. 2006; Tomonaga et al. 2005; Wang et al. 2012). Nevertheless, the function of CENPH in RCC has not been studied.

In this research, we tested CENPH mRNA expression in twenty-one pairs of ccRCC tissue and matched nontumor tissue by qRT-PCR. It was found that CENPH was overexpressed in ccRCC tissue. Besides, CENPH expression was upregulated in three of the four tested RCC cell lines compared with the human proximal tubule epithelial cell line HK-2 by qRT-PCR and western blot. These three high CENPH cell lines include both VHL mutated cell lines (A704 and 786-O) and no VHL mutation cell line ACHN. And the low CENPH cell line A498 has mutated VHL. This suggests that CENPH expression in human RCC does not depend on the VHL status. Consistent with these observations, our immunohistochemical study also confirmed that CENPH was overexpressed in 87 of 109 ccRCC specimens while negative staining was identified in the adjacent normal renal tissues. Furthermore, our immunohistochemical analysis showed that high CENPH expression was correlated significantly with the aggressive and invasive characteristics of ccRCCs, including Fuhrman grade, distant metastasis, and clinical stage. These results indicate that CENPH is overexpressed in ccRCC tissues and most RCC cell lines, and the CENPH expression may be closely related to clinical pathological features of ccRCC patients.

High CENPH expression has previously been suggested as a poor prognostic indicator in different cancers. In this study, the clinical significance of high CENPH expression in nuclear was further validated by its correlation with shorter OS time in ccRCC patients. Univariate survival analyses showed that high CENPH expression was associated with the death risk from ccRCC patients. Furthermore, multivariate survival analysis also showed that CENPH expression was an independent prognostic factor for OS. The above results suggested that CENPH overexpression might be important for the acquisition of high malignant potential in ccRCC. In addition, we may utilize the examination of CENPH expression by IHC as a useful prognostic tool for ccRCC patients.

In order to explore the biological role of CENPH in RCC cells and attempt the possibility of CENPH as a therapeutic target, we effectively downregulated CENPH expression in human renal adenocarcinoma cell line ACHN and human ccRCC cell line 786-O by CENPH siRNA in vitro. Our data of CCK8 and colony formation assays indicated that CENPH siRNA caused significant growth inhibition. This down-regulation of CENPH was associated with decreased expression of Ki-67, a marker of cellular proliferation. The cell growth suppressing function of CENPH siRNA has been validated in other cancer cell lines (He et al. 2013; Liao et al. 2009b; Wang et al. 2012). The knockdown of CENPH also increased apoptosis rate of RCC cells and reduced the expression of survivin, an inhibitor of apoptosis protein (Johnson and Howerth 2004). CENPH siRNA transfection can reduce survivin expression in gastric carcinoma and tongue cancer cells (He et al. 2013; Liao et al. 2009b). Our result in RCC is in agreement with them. Survivin is overexpressed in RCC and its high expression closely links with tumor aggressiveness and poor prognosis (Byun et al. 2007; Crispen et al. 2008; Krambeck et al. 2007; Ljungberg 2007; Zamparese et al. 2008). CENPH may play a role of oncogenes in RCC though influencing survivin expression. Chromosome instability (CIN) has been reported to play an important role in cancer by accelerating the accumulation of mutations by different mechanisms (Rajagopalan and Lengauer 2004). CENPH overexpression has been showed to induce aneuploidy in cancer cells (Tomonaga et al. 2005). And it is widely acknowledged that there is an intimate link between aneuploidy and chromosome instability (Nicholson and Cimini 2015). This may be the primary oncogenic mechanism of CENPH overexpression.

In conclusion, our data for the first time showed that CENPH is upregulated in RCC, overexpression of CENPH in RCC tissue is closely associated with the development and progression of tumor, and CENPH expression detected through IHC could be an independent prognostic indicator for patients with ccRCC. Moreover, we validated the anti-tumor ability of si-CENPH in vitro. CENPH may thus serve as a potential prognostic marker and a novel therapeutic target for RCC patients. Further, larger-scale studies are needed to clarify the prognostic value and the functional properties of CENPH for RCC.