{'fraction_non_alphanumeric': 0.036409786950732353, 'fraction_numerical': 0.036118508655126495, 'mean_word_length': 5.05060422960725, 'pattern_counts': {'":': 0, '<': 16, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
RNF43 Inhibits Cancer Cell Proliferation and Could be a Potential Prognostic Factor for Human Gastric Carcinoma

Background/Aims: RNF43 is a member of transmembrane E3 ubiquitin ligases and plays important roles in tumor formation progression. In current study, we aimed to explore RNF43 expression and analyze its role in gastric carcinoma. Methods and Results: The level of RNF43 was detected in 77 cases of gastric carcinoma and matched normal tissues by real-time PCR, western blotting and immunohistochemistry. We found that the expression of RNF43 was significantly down-regulated in the gastric carcinoma tissues compared to the normal mucosae (all P<0.001). In addition, RNF43 was significantly correlated with histological differentiation (P = 0.001), T-stage cancer (P<0.001), depth of invasion (P<0.001), metastasis of regional lymph nodes (P<0.001), pTNM stage (P<0.001) and survival (P = 0.021). We further explored the biological functions of RNF43 in gastric carcinoma cell lines. Both gain- and loss-function assays show that RNF43 could suppress cell proliferation while promotes cell apoptosis. Further, we found that RNF43 was positively correlated with p53 and cleaved-caspase3 and negatively correlated with Ki67 and Lgr5. Concolusion: In conclusion, RNF43 might act as a tumor suppressor in gastric carcinoma and might be a potential indicator for the clinical assessment of gastric cancer prognosis


Introduction
As the fourth most common malignant tumor, the mortality rate of gastric cancer ranks second worldwide [1]. Currently, the poor prognoses of that gastric cancer are because this condition is normally diagnosed in the advanced stages. Furthermore, curative treatments, such as surgery, radiotherapy and chemotherapy, are not able to prevent recurrence and metastasis, which are the ultimately the leading causes of death in patients with gastric cancer. Tumor progression might be related to the alteration of genes and the over-activation of signal pathways that regulate abnormal cell renewal [2][3][4][5]. However, the exact mechanisms of gastric cancer are not yet fully understood. As a critical pathway in the regulation of proliferation, Wnt/β-catenin signaling is considered to play critical roles in embryonic development, adult tissue homeostasis [6] and aberrant activation in tumorigenesis in cancers [7].
Mutations in the downstream components of the Wnt pathway often cause cancer incidents. However, most mutations of the downstream Wnt signaling pathway cannot be inhibited by the Wnt inhibitors that are currently being developed. Recently, Hao et al [8]. and Koo et al [9]. simultaneously reported the unique transmembrane E3 ubiquitin ligase RNF43, which suppresses the initiation of Wnt/β-catenin and Wnt/PCP signaling by endogenous Wnt proteins, and this process is different from those of other negative regulators of Wnt signaling, such as APC and AXIN [10]. However, the expression level and function of RNF43 in gastric carcinoma have not been examined. Here, we found low levels of expression of RNF43 in gastric carcinoma tissues compared to adjacent normal gastric tissues. Our work further suggests that the over-expression of the RNF43 suppresses proliferation and induces apoptosis in gastric carcinoma cells. Moreover, the opposite results were achieved in the knockdown test. We conclude that RNF43 might also act as a tumor suppressor in gastric carcinoma.
The relative values of the expressions of the transcripts were calculated using the 2 -∆∆C (T) method [11,12]. The mRNA expression level of RNF43 was normalized to that of GAPDH. All experiments were performed in triplicate.

Gastric cancer tissue specimens
Paraffin-embedded tissue specimens of 77 primary gastric carcinomas and 77 samples of matched normal mucosal tissues adjacent to the carcinomas were obtained from the Department of Pathology of the Chinese People's Liberation Army (PLA) General Hospital (Beijing, China) from 2002 to 2007. An additional four pairs of tissue specimens were used for western blot examinations. Ethical approval for this study was not required by our institution because the experiments were not related to the patients' privacy, impairment, or treatment. Histological specimens from each patient were reviewed to confirm the diagnoses and clinic pathological parameters. Pathological TNM staging was evaluated according to the 2009 criteria of the International Union Against Cancer (UICC). All of the patients were followed up for survival analysis. The follow-up period was calculated from the date of surgery until October 31, 2012.

Immunohistochemistry
Immunohistochemical staining was performed on 3-to 4-µm sections of formalin-fixed, paraffinembedded tissues. The slides were dew axed in xylene and rehydrated. The slides were heated in 0.01 mol/L citrate buffer (pH 6.0) in a microwave oven for 2 min and 30 s at 100℃ for antigen retrieval. Next, the slides were incubated for 15 min with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. After a washing with phosphate-buffered saline (PBS) and blocking with 10% goat serum, the sections were treated with primary polyclonal rabbit antibody to human RNF43 (ab129401, Abcam, MA, USA) diluted 1:100 in blocking solution and incubated over-night at 4℃. After washing with PBS, the sections were incubated for 30 min with biotinylated secondary antibody (polyperoxidase-anti-mouse/rabbit IgG; Zymed Laboratories, South San Francisco, CA, USA), and peroxidase reactivity was then visualized using a 3,30-diaminobenzidine (DAB) substrate kit (Zymed). Finally, the slides were counterstained with hematoxylin. The primary antibody was replaced by PBS as a negative control.

Evaluation of immunohistochemistry
Antigen expression was independently evaluated using a light microscope by two investigators who were blind to all clinical pathology data. When there were discordant cases between the two investigators, the slides were reassessed on a double-headed microscope to establish a final result. For the RNF43 assessment, the immunohistochemical stainings of cells were evaluated according to a score that combined an intensity of staining scale with the percentage of cells stained [13]. The intensity of staining was scored as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; and 3+, intense staining. The percentage of stained cells was scored as follows: 0, no staining of any cells; 1+, positive staining in <10% of the cells; 2+, positive staining covering 10-50% of the cells; and 3+, positive staining of >50% of the cells. The final score was determined as the combination of these scores. Scores (extent + intensity) of ≤1 was considered negative, and scores between 2 and 6 were considered positive [14,15].

Protein extraction and western blotting
Protein extraction and western blotting were performed as previously described [16]. Tissue and cell lysates were prepared with lysis buffer. The protein content of the supernatant was measured using a standard method. The protein samples were loaded at 50μg/lane in 12% sodium dodecylsulfate (SDS), separated by polyacrylamide gel electrophoresis (PAGE), and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked with 5% non-fat milk in TBST (50 mmol/L Tris-HCl [pH 7.6], 150 mmol/L NaCl, 0.1% Tween 20) for 1 h at room temperature and then incubated with primary antibody (anti-RNF43, Abcam, USA, 1:200; GADPH, 1:1000, Santa Cruz Biotechnology) in blocking buffer at 4℃ overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase-coupled goat anti-rabbit secondary antibody (1:10000; Santa Cruz Bio-technology) for 2 h at room temperature. Enhanced chemiluminescence was used for detection. The β-actin bands were used as the loading controls. The protein quantity was analyzed with Quantity-One v4.4 software (Bio-Rad, Hercules, CA, USA). The target protein expression was evaluated as the relative intensity ratio of the target protein to the loading control.

Construction of recombinant adenovirus encoding RNF43 and lentivirus harboring RNF43 siRNA
For recombinant adenovirus construction, the RNF43 cDNA (Ad-RNF43) and the green fluorescence protein gene (Ad-GFP; control) cloned by PCR was inserted into pDC315-EGFP vector (purchased from Hanbio Co. Ltd, Shanghai, China) respectively, under the control of the mouse cytomegalovirus (CMV) promoter. The pDC315-X and pBHGlox E1,3Cre were co-transfected into HEK293 cells to generate the recombinant adenoviruses. Ad-RNF43 and Ad-GFP were propagated in HEK293 cells. The Propagated recombinant adenoviruses in the HEK293 cells were purified and the titer of virus was measured by plaque assays The stock solutions of Ad-RNF43 and Ad-GFP were 1×10 10 plaque formation unit (PFU)/ml, respectively.
For lentivirus construction, oligonucleotides with the following targeting sequences were used for the cloning of small hairpin RNA (shRNA)-encoding sequences in hU6-MCS-PGK-EGFP lentiviral RNAi vector (Hanbio, Shanghai, China): RNF43, 5'-GCAGGAGCTACGGGTCATTTC-3'. The recombinant lentivirus with shRNA-RNF43 was produced by co-transfection of 293T cells with plasmids PSPAX2 and PMD2G. Lentiviruscontaining supernatant were harvested 48 h after transfection and filtered through 0.22 μm cellulose acetate filters (Millipore, USA). Recombinant lentiviruses were concentrated by ultracentrifugation (2 h at 50,000 × g). buffer to a final concentration of 1×10 5 cells. Five microliters of Annexin V-FITC were added to 100μL of the cell suspension. After a 20-min incubation at room temperature in the dark, the cell suspension was centrifuged at 1,000g for5 min and then resuspended in 400µL binding buffer and 3µL PI on ice in the dark. Subsequently, the number of stained cells was assessed with a flow cytometer (BD FACS AriaII, NJ, USA). The Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) was used to measure the effects of RNF43 on tumor cell proliferation. The cells were plated at a density of 5,000 cells per well in 96-well plates in the presence of DMEM. The CCK-8 assays were performed at 0, 24, 48 and 72 h after infection. Ten micro liters of the cell proliferation reagent WST-8 were added to each well, and the cells were incubated for 1 h at 37 ℃. Viable cell counts were estimated by measuring the optical density at 450 nm. The amount of the dye generated by the dehydrogenase reaction in the cells is directly proportional to the number of living cells.

Cell apoptosis assay and cell proliferation assay
Statistical analyses SPSS V.17.0 (SPSS, Chicago, IL, USA) was used for the statistical analysis. The data regarding the relative expressions of RNF43 mRNA and protein are presented as the means ± the SDs. Paired-samples-t-tests were used to assess differences in the relative expressions of RNF43 mRNA and RNF43 protein between the gastric cancer cell lines and the normal mucosa cell lines. Univariate survival analysis was conducted according to the Kaplan-Meier method, and the difference between the survival curves was analyzed with the log-rank test. Multivariate survival analysis was performed using the Cox proportional hazard model. A positive coefficient indicates a worse prognosis, and a negative coefficient indicates a protective effect of the variable with which it is associated. Hazard ratios (HRs) and 95% confidence intervals (CIs) were obtained to assess the prognostic effects of each variable on the outcomes. Values of P<0.05 were considered statistically significant.

The level of RNF43 was significantly down-regulated in gastric carcinoma
The RNF43 mRNA and protein in the gastric carcinoma cell lines and the gastric epithelial cell line were respectively assessed by q-PCR and western blotting. As shown in Fig.  1 a and b, the RNF43 mRNA and protein expressions were significantly lower in the gastric carcinoma cell lines (BGC823, 7901, and MGC803) than in the normal epithelial cell line (GES-1; all P<0.005).The same results were also confirmed in 4 cases of gastric carcinomas and matched normal mucosal tissues (Fig. 1 c, d).
Clinicopathological characteristics of the patients The ages of the 77 patients ranged from 24 to 86 years (median 65 years; mean 59.6 years). Among these patients, the pathological diagnoses were well-differentiated adenocarcinoma in 4 cases, moderately differentiated adenocarcinoma in 14 cases, and poorly differentiated adenocarcinoma in 59 cases. According to the pathological TNM stage, 16 cases were stage I, 30 were stage II, 31 were stage III, and 0 were stage IV. During the follow-up period, 54 patients died, and 23 were alive at the end of follow up median survival time 44 months, mean survival time 46.4 ± 3.3 months).

The expression of RNF43 was correlated to patient's clinicopathological features and survival
We detected the lack of expression of RNF43 in the tumor tissues more frequently than that in the normal mucosal tissues (P<0.001). RNF43 expression was observed in the cytoplasm (Fig. 2). RNF43 expression was lower in the larger tumors (P < 0.001). A lack of expression of RNF43 was not found in the well-differentiated adenocarcinomas, in 31.25% (5 out of 16) of the moderately differentiated adenocarcinomas, and in 72.22% (39 out of 54) of the poorly differentiated adenocarcinomas. RNF43 expression was positively correlated with histological differentiation (P = 0.001). RNF43 expression was lower in advanced T-stage cancer (P<0.001). RNF43 expression was found to be positively correlated with depth of invasion and metastases of the regional lymph nodes (P<0.001). An apparent relationship was found between the frequency of RNF43 expression and the pTNM stage, and observations of the lack of expression of RNF43 gradually increased with the progression of pTNM stage (P<0.001; Table 1).
Survival curves were calculated according to Kaplan-Meier analysis, and the survival analysis with the log-rank test suggested that RNF43-negative patients exhibited a significantly shorter survival time (median 35 months, mean 37.78.53 ± 4.34 months) than did the RNF43-positive patients (median 85 months, mean 67.52 ± 5.32 months) (log-rank test, P<0.001) (Fig. 3). The 5-year survival rates were 18.2% for the RNF43-negative patients and 60.6% for the RNF43-positive patients. Multivariate analysis was performed using the Cox regression. RNF43 was found to significantly affect the outcome of gastric cancer and appeared to be an independent prognostic factor (P = 0.021) with a hazard ratio (HR) of 0.24 (95% confidence interval [CI] 0.072-0.804). Histological differentiation (HR 2.318, 95% CI 1.081-4.971, P = 0.031), the depth of invasion (HR 3.565, 95% CI 1.702-7.471, P =0.001), and lymph node metastasis (HR 2.201, 95% CI 1.207-4.013, P =0.01; Table 2) also affected the outcomes.  Immunohistochemical analysis of RNF43 in gastric carcinoma and adjacent normal tissues. Staining of RNF43 in adjacent normal tissue (strong positive, a ×100, b×200), highly differentiated gastric carcinoma tissue (moderately positive, c ×200, d×400), c moderately differentiated gastric carcinoma tissue (weakly positive, e ×200, f×400). poorly differentiated gastric carcinoma tissue (negative, g ×100, h×200).   Fig. 4a). Seventy-two hours after infection, cell apoptosis was determined by FACS. As shown in Fig. 4c, compared to the negative control groups, significant increases in cell apoptosis were observed in the MGC803 cells and GES-1 cells infected with adenovirus encoding RNF43 gene (P<0.05, Fig. 4e). Furthermore, BGC823 cells and GES-1 cells were infected with lentivirus harboring RNF43 siRNA. The cell growth curves revealed that the knockdown of RNF43 significantly promoted cell proliferation compared to the negative control group in a time-dependent manner (P<0.05, Fig. 4b). Seventy-two hours after infection, cell apoptosis was determined by FACS. As shown in Fig. 4d, compared to the negative control group, a trend toward the suppression of cell apoptosis was observed in the BGC823 cells and GES-1cells after knockdown of RNF43 (P>0.05, Fig. 4f). These results further support the potential anticancer activity of RNF43 in gastric carcinoma.
RNF43 positive correlated to p53 and cleaved-caspase3 but negative related to Ki67and Lgr5 MGC803 cells and GES-1 cells were infected with recombinant adenovirus encoding RNF43 or encoding EGFP as negative controls. Western blot tests revealed that the over-expression of RNF43 significantly upgulated the expression levels of p53 and cleaved-caspase3 in the MGC803 and GES-1 cell lines compared to the negative control groups (Fig. 5a). An interference test with shRNF43 revealed that the reduction of RNF43 resulted in significant decreases in the expressions of p53 and cleaved-caspase3 in the BGC823 and GES-1 cell lines (Fig. 5b). Additionally, we observed that the expressions of Ki67 and Lgr5 were inversely regulated by RNF43 in both the over-expression and interference tests (Fig. 5). These results demonstrate that RNF43 might have functions similar to those of p53 or cleaved-caspase3 and was inversely related to Ki67 and Lgr5.

Discussion
Ring finger protein 43 (RNF43) belongs to the family of E3 ubiquitin-protein ligases. Previous studies have identified RNF43 as a novel oncoprotein in colorectal cancer [17][18][19]. The current study demonstrated that RNF43 acts as an inhibitor of Wnt signaling by negatively regulating frizzled receptor surface expression [9]. Scholars also found that RNF43 is a tumor suppressor gene in mucinous ovarian tumors, and similar findings have been reported in studies of mucinous pancreatic precancerous cysts [20][21][22]. However, the function of RNF43 in human gastric carcinoma has never been reported.
In our study, we focused our attention on examining the expression level and function of RNF43 in gastric carcinomas. We hypothesized that RNF43 might also act as an inhibitor in gastric carcinoma cell proliferation as it has been reported to do in other types of cancer. Therefore, we investigated the mRNA and protein levels of RNF43 in three gastric carcinoma cell lines and one gastric epithelial cell line with qPCR and western blot analyses. Both test methods revealed that the expressions of RNF43 mRNA and protein in the gastric carcinoma cell lines were significantly down-regulated compared to those in the gastric epithelial cell line. We also investigated the above indicators in four pairs of gastric carcinoma tissues and found that the results were similar to those from the cell lines.
Our study investigated RNF43 in a certain number of gastric carcinoma samples with available follow-up data and showed that RNF43 expression was significantly reduced in the gastric carcinoma tissues compared to the distal normal mucosae. The decreased expression of RNF43 was significantly correlated with histological differentiation, tumor size, depth of invasion, lymph node metastasis, pTNM stage and survival. These results suggest that low expression levels of RNF43 are typically correlated with more malignant and metastatic tumors. RNF43 expression was found to be absent in 48.39% (15/31) of the stage II cancer tissues and 96.67% (29/30) of the stage III cancer tissues. We also found that low expression levels of RNF43 were closely related to the worst prognoses for gastric carcinoma. These results suggest that RNF43 might play a suppressive role in the development and progression of these tumors. Because the combination therapy of peptide vaccines and anti-cancer drugs for colorectal cancer is associated with increased expression of RNF43 [23,24], a novel therapeutic strategy based on RNF43 might be used in the future for patients with gastric carcinomas. Additionally, we analyzed the reason that we observed that there was no statistical association between pTNM and survival in the Cox proportional hazard model and found that the number of samples might have been insufficient.
In the in vitro experiments, we confirmed the anti-tumor effect of RNF43 in gastric carcinoma. Our results revealed that RNF43 had large and obvious effects on the proliferation and apoptosis of MGC803, BGC823 and GES-1 cells. These results revealed that, among three gastric carcinoma cell lines, the expression of RNF43 was highest in the BGC823 cells and lowest in the MGC803 cells. Therefore, we chose to over-express RNF43 in the MGC803 cell line with the pEGFP-RNF43 recombinant adenovirus and the BGC823 cell line to knock down RNF43 with the lentivirus shRNF43. The two methods were both performed in the normal epithelial cell line (GES-1). The CCk-8 and flow cytometric assays revealed that infection with pEGFP-RNF43 recombinant adenovirus significantly inhibited proliferation and promoted apoptosis in the MGC803 and GES-1 cells. In contrast, infection with shRNF43 significantly promoted the proliferation of BGC823 and GES-1 cells and limited the inhibition of apoptosis. We suggest that low levels of the expression of RNF43 might not inhibit the apoptosis but promote the proliferation of both carcinoma and normal cells. The classical tumor suppressor gene p53 is the gene that is most frequently mutated and inactivated [25,26]. Caspase3 is a type of photolytic enzyme that mediates cell apoptosis [27]. In our study, we observed positive relationships between RNF43, p53, and cleaved-caspase3. These data suggest that low levels of the expression of RNF43 might decrease apoptosis and promote proliferation of gastric carcinoma cells and might be one of conditions which result of the conversion to normal gastric epithelial cells to cancer. These data also suggest that RNF43 might act as a tumor suppressor in gastric carcinoma.
The leucine-rich repeat-containing G protein-coupled receptor Lgr5 has been identified as a novel stem cell marker in the intestinal epithelia, stomach, and hair follicles [28][29][30]. It has been confirmed RNF43 is specifically expressed in Lgr5-positive small intestinal crypt cell and inhibits cell proliferation. Researchers have found that Lgr5 participates in the degradation of rnf43/znrf3. Recently, scholars have analyzed the protein complex domain of Rspod1-Lgr5-rnf43/znrf3 and suggested thatLgr5 participates in the degradation of RNF43/ZNRF3 in vitro [31,32]. We conducted a test to determine whether RNF43 and Lgr5 exert similar effects in gastric carcinomas. We detected the Lgr5 protein levels following pEGFP-RNF43 recombinant adenovirus infection in both the gastric carcinoma cell line and the gastric epithelial cell line. Compared to the negative controls, the over-expression of RNF43 led to decreased expression of Lgr5. A similar result was also found in the interference test with shRNF43.These results indicate that RNF43 might be inversely related to the potential of the cancer stem cell marker Lgr5. The elucidation of the exact mechanism will require further effort in future studies. Our study also revealed that RNF43 expression was negatively correlated with Ki-67, which is recognized nuclear antigen-specific marker of cellular proliferation; this finding suggests that RNF43-negative cancer cells have higher proliferative activity.
In conclusion, our study used immunohistochemical methods to reveal that the expression of RNF43 was significantly lower in gastric cancer tissue than in normal mucosal tissue. This result was confirmed by western blot analysis. Furthermore, low levels of the expression of RNF43 in primary gastric carcinoma were significantly correlated with shorter survival, and low RNF43 expression was a significant predictor of poorer survival. RNF43 expression was also associated with gastric carcinoma cell proliferation. We believe that these findings can help to predicting clinical outcomes and contribute to the understanding of the inhibition of gastric carcinoma. Further investigations of RNF43 are required to identify its mechanism of action in gastric carcinoma and to develop new therapeutic agents.