Abstract
Background/Aim: Ring finger protein 126 (RNF126) belongs to the family of RING E3 ubiquitin ligases. Although RNF126 has been reported to be overexpressed in several cancers, the role of RNF126 in gastric cancer remains unclear. Materials and Methods: We investigated the RNF126 expression in 170 primary gastric cancer tissues by immunohistochemistry, and explored its prognostic impact. The effect of the RNF126 expression on the proliferation of cancer cells was evaluated in vitro. Results: The RNF126 expression was significantly associated with tumor depth and presence of venous invasion. The RNF126 status was identified as an independent prognostic factor (p<0.001). RNF126 gene silencing significantly inhibited the proliferation of gastric cancer cells, induced G1 phase arrest and increased the p21 protein level. Conclusion: RNF126 expression has a significant prognostic value in gastric cancer. RNF126 may play an important role in tumor progression of gastric cancer.
Gastric cancer (GC) is one of the most common causes of cancer-related death in the world (1, 2). Despite several advancements in the diagnosis, surgical techniques and chemotherapy, a number of patients, particularly those with advanced disease, present with recurrence following curative resection (3-5). Thus, novel therapeutic strategies for GC are required. In addition, identifying potential biomarkers to predict the outcomes of patients is important to improve the prognosis of GC patients.
The ubiquitin-proteasome system plays an important role in controlling protein turnover and regulating various signaling pathways and cellular processes (6), and is cascaded by three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) (7, 8). Dysfunction of the ubiquitin system has been suggested to cause or contribute to the development and progression of cancer (9). To date, the upregulation or down regulation of E3 ubiquitin ligases (e.g., Skp2, RBX1 and FBXW7) has been reported in GC (10-13).
Ring finger protein 126 (RNF126) belongs to the family of RING (really interesting new gene) E3 ubiquitin ligases, and has been identified as a ligase that promotes cancer cell proliferation through functional small interfering RNA (siRNA) library screening (14). RNF126 has been suggested to be involved in diverse biological phenomena, including the cell cycle, DNA repair, sorting of the cell surface membrane and protein quality homeostasis control (14-17). The overexpression of RNF126 has been identified in various cancer cell lines (e.g., breast cancer, tongue cancer, lung cancer and prostate cancer) and human primary cancer tissues (e.g., breast cancer and ovarian cancer) (14, 18-21). However, the functions and role of RNF126 in cancer still remain unclear. Moreover, there are only limited data on the expression and clinical relevance of RNF126 in cancer. In the present study, we investigated the expression, clinical significance and prognostic impact of RNF126 in GC. Furthermore, we evaluated the effect of the RNF126 expression on the proliferation of GC cells using an RNA interference method in vitro.
Materials and Methods
Clinical samples. One hundred seventy GC samples were obtained from the resected specimens, and then fixed in 10% phosphate-buffered formalin and embedded in paraffin. All patients underwent curative gastrectomy in the Department of Surgery, Nara Medical University, between January 2004 and December 2008. These patients had not undergone preoperative chemotherapy or radiotherapy. The tumor stage was determined according to the TNM classification system (22). Follow-up was continued until death or January 2016. The median follow-up period was 61.2 months. Written informed consent was obtained from all patients before the operation, according to our institutional guidelines. This study was approved by the Local Ethics Committee on Clinical Investigation of Nara Medical University Hospital (no. 1193-2).
Immunohistochemistry. The sections were stained using a DAKO EnVision system (Dako Cytomation, Kyoto, Japan), as described previously (13). As primary antibodies, a mouse monoclonal anti-RNF126 antibody (sc-376005, 1:50 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and a mouse monoclonal anti-human Ki67 antibody (M7240, 1:100 dilution; DAKO) were employed. Antigen retrieval was carried out by heating tissue sections using a Target Retrieval Solution at pH 9.0 (DAKO). To block endogenous peroxidase, sections were immersed in a 3% solution of hydrogen peroxide in absolute methanol for 20 min at room temperature. To evaluate the RNF126 and Ki67 expression, ≥1,000 tumor cells from the invasive front of tumors were scored at per field ×400 magnification, and the percentage of tumor cells showing positive staining was calculated. A cutoff point for RNF126 was selected to give the optimal separation between low-risk and high-risk patients in terms of overall survival (OS).
GC cell lines. MKN45 (poorly differentiated adenocarcinoma) and MKN74 (moderately differentiated adenocarcinoma) human GC cell lines were purchased from the RIKEN BioResource Center and cultured in RPMI 1640 supplemented with 10% FBS.
Real-time reverse transcriptase PCR. Total RNA was isolated, and cDNA synthesis was carried out. The real-time reverse transcriptase PCR analysis was then performed, as described previously (13). The ratio of the mRNA level of RNF126 was calculated as follows: (absolute copy number of RNF126)/(absolute copy number of β2-microglobulin).
Western blotting. Cell lysates were prepared, and subjected to immunoblotting analysis using antibodies against RNF126 (ab183102, 1:250 dilution; Abcam, Burlingame, CA USA), β-actin (8H10D10, 1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), p21 (sc-397, 1:100 dilution; Santa Cruz Biotechnology) and p27 (sc-528, 1:100 dilution; Santa Cruz Biotechnology).
Small interfering RNA transfection of RNF126. MKN45 and MKN74 cells were transfected with nonspecific control RNA (QIAGEN, Valencia, CA, USA) or 20 nmol/l of RNF126 siRNA (QIAGEN). Transfection was performed using the Lipofectamine system (Invitrogen), as described previously (13). The siRNA against RNF126 was selected to target the following DNA sequence: 5’-CCGGATTATATCTGTCCAAGA-3’.
Cell viability assay and cell cycle analysis. Cell viability was determined using the CellTiter-Blue® Cell Viability Assay kit (Promega Corp., Madison, WI, USA), as described previously (23). Briefly, aliquots of 5×103 of MKN45 and 3×103 of MKN74 cells per well were cultured in 96-well plates at 37°C for 24 h, then the cells were transfected with control or RNF126 siRNA. Following 72 h of incubation at 37°C, CellTiter-Blue® reagent was added to each well. Each experiment was performed at least three times. The cell-cycle analysis was performed using a Cycletest™ Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA), as described previously (23).
Statistical analysis. Results were expressed as the means and standard deviations or as numbers and percentages, and Student's t-test, a one-way ANOVA, the Chi-squared test, or Fisher's exact test was used for evaluating statistical significance. OS was defined as the duration from surgery to death. Survival curves were estimated according to the Kaplan–Meier method, and the log-rank test was carried out to determine differences in survival rate. A Cox proportional hazards model was used for univariate and multivariate survival analyses. All significant variables in the univariate analysis were used in a multivariate analysis. A Pearson correlation analysis was performed to examine the correlation between the expression levels of RNF126 and Ki67. A p-value less than 0.05 was considered statistically significant and confidence intervals (CI) were calculated at the 95% level. All statistical analyses were performed using the SPSS® software program, version 19.0 (SPSS, Chicago, IL, USA).
Results
In 161 (94.7%) GC tissue specimens, RNF126 was expressed in the cytoplasm of GC cells. The mean percentage of RNF126-positive cells in GC tissues was 43.5±26.7%.
We evaluated the relationships between RNF126 expression and clinicopathological characteristics. The positive rate of RNF126 was significantly associated with the tumor depth (p=0.001), pathological stage (p=0.008) and venous invasion (p=0.001; Table I).
The HR for high RNF126 for OS was highest when the cutoff value for the RNF126 expression was 63% (HR 2.865). Thus, the cutoff value for the RNF126 expression was set at 63%. Then, 51 patients (30%) with RNF126 positivity of ≥63% and 119 patients (70%) with RNF126 positivity of <63% were classified as the RNF126-high and RNF126-low groups, respectively (Figure 1a). The five-year OS rate of the RNF126-high group was significantly lower than that of the RNF126-low group (45.4% vs. 78.6%, p<0.001; Figure 1b). When comparing the survival rate according to tumor stage (Figure 1c, d and e), the RNF126-high group had a significantly lower OS rate than the RNF126-low group among patients with stage III disease (20.9% vs. 64.1%, p=0.002). The multivariate analysis showed that the RNF126 status, as well as tumor depth and lymph node metastasis, was an independent prognostic factor for OS (p<0.001; Table II).
At the time of analysis, 45 patients (26.5%) had postoperative recurrence. Overall, the recurrence rate was higher in RNF126-high tumors than in RNF126-low tumors (Table III). Hematogenous recurrence occurred more frequently in RNF126-high tumors than in RNF126-low tumors (p=0.008).
The involvement of RNF126 in the proliferation of GC cells was investigated, since RNF126 has been suggested to be required for the proliferation of cancer cells (14, 20, 24). A Ki67-positive rate was significantly higher in RNF126-high tumors than in RNF126-low tumors (28.4±12.8% vs. 19.4±11.6%, p<0.001; Figure 1f). Furthermore, we found a significant correlation between the expression levels of RNF126 and Ki67 (p<0.001, r=0.34).
We further examined the effects of RNF126 down-regulation using an RNA interference method. When transfected with RNF126 siRNA for up to 72 h, both the mRNA and protein expression levels of RNF126 in human GC cell lines MKN45 and MKN74 were substantially reduced (Figure 2a and b). As a result, the cell proliferation was significantly inhibited by RNF126 gene silencing in these cells (Figure 2c).
To precisely investigate the effect of RNF126 knockdown, we performed cell-cycle analysis. The percentage of cells in the G1 phase was significantly increased by RNF126 gene silencing in both cell lines [MKN45, control vs. RNF126 siRNA, 56.8±0.6% vs. 65.6±0.7% (p<0.001); MKN74, control vs. RNF126 siRNA, 48.5±0.1% vs. 56.5±0.1% (p<0.001); Figure 3a]. Western blotting showed that the protein level of p21 was increased by RNF126 knockdown (Figure 3b).
Discussion
Recently, increasing attention has been paid to the involvement of dysfunction of the ubiquitin systems in cancer progression (23, 25, 26). In GC, several studies have investigated the expression of E3 ubiquitin ligases, such as Skp2, RBX1 and FBXW7, and reported that their expression levels were independently associated with survival (10-13). In the present study, we evaluated the RNF126 expression, an E3 ubiquitin ligase, and demonstrated its clinical importance in GC. Some researchers have investigated the prognostic impact of RNF126 in cancer patients. Yang et al. evaluated the expression of RNF126, and identified the high expression of RNF126 as an independent predictor of a poor prognosis in breast cancer patients (21). Wang et al. reported that the decreased expression of RNF126 was correlated with improved disease-free and OS in patients with ovarian serous cystadenocarcinoma (19). In the present study, RNF126 was found to be abundant in GC tissues, and the higher expression of RNF126 was significantly associated with poor OS. Importantly, the RNF126 expression was identified as an independent predictor of OS. Our studies suggest that RNF126 is a potential prognostic biomarker for GC.
It has been suggested that the aberrant expression of E3 ubiquitin ligases may have clinical relevance in cancer patients (11-13). However, there have been only limited reports on the expression of RNF126 in cancer tissue samples. Yang et al. evaluated the clinical significance of RNF126 in breast cancer, and reported no significant associations between the RNF126 expression level and clinicopathological factors such as tumor stage, histologic grade and molecular subtype in breast cancer (21). In this study, we found significant correlations between the RNF126 expression and tumor-related factors, including tumor depth, tumor stage and venous invasion. These findings indicate that RNF126 may contribute to the progression of GC.
In the present study, we also revealed that the overexpression of RNF126 was associated with a higher risk of GC recurrence. Yoshino et al. showed that the higher expression of RNF126 was associated with decreased distant metastasis-free survival in breast cancer patients (18). In the present study, the higher expression of RNF126 was significantly correlated with poor OS in patients with stage III disease. In addition, RNF126-high tumors were associated with a significantly higher rate of postoperative recurrence in comparison to RNF126-low tumors. In particular, hematogenous recurrence occurred more frequently in cases involving RNF126-high tumors, and this higher rate of hematogenous recurrence was partly explained by the significant association between the expression level of RNF126 and venous invasion. These data indicate that RNF126 may be a useful predictor of postoperative recurrence in GC.
In addition, we evaluated the effect of the RNF126 expression level on the proliferation of GC cells. A number of studies have reported that dysfunction of E3 ubiquitin ligases contributes to stimulating cancer cell proliferation (9, 25, 27). Zhi et al. screened an E3 ubiquitin ligase siRNA library in breast cancer and prostate cancer cell lines, and identified RNF126 as a novel pro-proliferation target (14). The authors reported that knockdown of the RNF126 expression significantly inhibited cancer cell proliferation. The knockdown of the RNF126 expression was also reported to reduce the cell viability of tongue cancer cells and lung cancer cells (18, 20). In the present study, we found that a percentage of Ki67-positive tumor cells was significantly higher in RNF126-high tumors than in RNF126-low tumors. Furthermore, a positive correlation between the expression levels of RNF126 and Ki67 was observed. In addition, we examined the association between the RNF126 expression level and the cell viability of GC cells using an RNA interference method in vitro, and observed that RNF126 gene silencing significantly reduced the cell proliferation. These findings suggest that RNF126 may play an important role in the proliferation of GC cells.
We further investigated the mechanisms underlying the inhibition of cell proliferation observed with RNF126 gene silencing. E3 ubiquitin ligases are well known to ubiquitinate and degrade various cell cycle regulators (9, 25, 27). Zhi et al. reported that RNF126 silencing delayed G1-S progression and increased the protein level of cell cycle-dependent kinase inhibitor p21 in cancer cells (14). The authors also showed that the overexpression of RNF126 increased p21 protein ubiquitination. Consistent with these results, we showed that knockdown of the RNF126 expression resulted in G1 cell cycle arrest and increased the p21 protein level. These findings suggested that RNF126 might partly promote cell cycle progression through regulation of the level of p21 in GC. However, further investigations are needed to elucidate the precise role of RNF126 in the progression of GC.
Given its overexpression and prognostic value, RNF126 may represent a potential target for cancer treatment. Zhi et al. demonstrated that RNF126 gene silencing significantly inhibited tumor growth in vivo (14). Other studies also showed that knockdown of the RNF126 expression suppressed tumor growth in mouse models (18, 20). Recently, MLN4924, a small-molecule inhibitor of the cullin-RING E3 ubiquitin ligases, was developed (28). MLN4924 is currently under investigation for cancer treatment in clinical trials (29, 30). Taken together, RNF126 could be an attractive therapeutic target for cancer.
In conclusion, the present study demonstrated that the RNF126 expression has significant prognostic value in GC. Furthermore, our study suggests that RNF126 may play an important role in regulating the proliferation of GC cells. The results of the present study may provide the rationale for developing novel treatments targeting RNF126 in GC.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number JP16K10513.
Footnotes
Authors' Contributions
This study was designed and directed by KM and MS. Data were collected and analyzed by KM, SM, KW, TK and HN. KM, TK and HN performed and analyzed immunohistochemistry. KM, TK and SM performed and analyzed in vitro studies. The article was written by KM and commented on by all Authors.
Conflicts of Interest
All Authors have no conflicts of interest to disclose.
- Received January 15, 2020.
- Revision received January 25, 2020.
- Accepted January 28, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved