The Role Of WWP1 In The Occurrence And Development Of Gastric Carcinoma And Its Preliminary Molecular Mechanisms

Background and ObjectiveGastric cancer (GC) is the most common gastrointestinal malignancy, and is the second leading cause of cancer related deaths. Although considerable advances has been made in therapy strategies and the study of molecular markers in GC, the prognosis and survival rates of the patients with advanced gastric carcinoma are quite poor, and the median survival time for the patients with GC is approximately 6-9 months. Therefore, it is urgent need to seek and identify the novel molecular biomarkers for diagnosis, prevention or therapy of the patients with GC.WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is localized in the region 8q21.3 in human chromosome. The human WWP1 protein contains 922 amino acid residues with the molecular weight of-110 kDa. It contains a C2 domain, four WW domains, and a HECT domain. WWP1 interacts with many signaling pathways, such as Notch signaling pathway, transforming growth factor (TGFβ) signaling pathway, mitogen-activated protein kinases (MAPKs) signaling pathway and Epithelial growth factor (EGF) signaling pathway, etc. These findings imply the diversity and complexity of WWP1 functions.More and more evidence has demonstrated that WWP1 is implicated in several diseases, such as infectious diseases, neurological diseases and aging, especially the processes of occurrence and development of cancers. In prostate and breast cancers, WWP1 mRNA and protein levels were frequently up-regulated. Subsequent investigation revealed that WWP1 level was significantly increased in oral cancer and hepacellular carcinoma. These studies indicate that WWP1 may play a pivotal role in the occurrence, development and progression of these tumors. However, to date, there were no reports about the role of WWP1 in the occurrence and development of GC and its preliminary molecular mechanisms worldwide。In the current study, expressions of WWP1 mRNA and protein were detected in GC tissues and normal gastric mucosa tissues by in situ hybridization, immunohistochemistry, real-time quantitative RT-PCR and Western blotting methods, the relationships between WWP1 expression and clinicopathological features of GC were investigated, associations of WWP1 expression with prognosis of the patients with GC were explored by follow-up using Kaplan-Meier survival curve, and independent prognostic factors of the patients with gastric carcinoma were further investigated by Cox proportional hazards model. Further investigation revealed expressions of WWP1 mRNA and protein in GC cells, meanwhile, effects of WWP1 downregulation on cell proliferation, cell cycle and apoptosis was analyzed by RNA interfering technology in GC cells, and cell cycle- and apoptosis-related protein expressions were analyzed. In addition, interactional proteins of WWP1 were investigated by bioinformatics, and Western blotting was employed to verify the possible regulation functions of WWP1 in GC. Finally, the model of GC implanted nude mice was established, effects of WWP1 downregulation on growth of GC implanted nude mice was investigated, and the associations of WWP1 expression with the survival rates of nude mice were studied by Kaplan-Meier survival curve.Part I:Expression of WWP1 in GC tissues and its associations with the prognosis of the patients with GCObjectiveTo investigate the expression of WWP1 in gastric carcinoma tissues and preliminarily explore its possible role in the occurrence and development of gastric carcinoma.Methods(1) In situ hybridization and immunohistochemistry were used to examine the expressions of WWP1 mRNA and protein in 131 cases of GC tissues and paired normal tissues, respectively. Meanwhile, Real-time qPCR and Western blotting were utilized to investigate WWP1 mRNA and protein expressions in GC tissues and paired normal gastric mucosa tissues, respectively.(2) The associations of the expressions of WWP1 mRNA and protein with the prognosis of the patients with GC were investigated using Kaplan-Meier survival curve and independent prognostic factors of the patients with gastric carcinoma were determined by Cox proportional hazards model.(3) Statistical analysis:Statistical analysis was carried out by SPSS 17.0 software, all data were expressed as x±s. The results of in situ hybridization and immunohistochemistry were evaluated by chi-square, the results of Real-time qPCR and Western blotting were performed by One-way ANOVA, and the associations of the expressions of WWP1 mRNA and protein with the prognosis of the patients with GC was analyzed by Kaplan-Meier survival curve. Independent prognostic factors of the patients with gastric carcinoma were investigated by Cox proportional hazards model. A P value less than 0.05 was considered to be statistical significance.Results(1) The results of in situ hybridization and immunohistochemistry showed that the positive expression ratios of WWP1 mRNA and protein in 131 cases of GC tissues were 66.41% and 61.83%, respectively, whereas 12.21% and 9.92% positive expression ratios for 131 cases of normal gastric tissues were found, and there were significant differences in WWP1 mRNA and protein expressions between GC tissues and normal tissues (X2=80.646 and 76.715, both P=0.000).(2) The results of Real-time qPCR and Western blotting revealed that relative levels of WWP1 mRNA and protein in randomly selected 10 cases of GC tissues were markedly higher than those in paired normal tissues, respectively, and the differences were statistical significance (P<0.05).(3) Expressions of WWP1 mRNA and protein were tightly associated with tumor differentiation, TNM staging, invasion depth and lymph node metastasis (all P<0.05), but not related to the patients’ age, gender and tumor size (all P>0.05).(4) As determined by the log-rank test (Mantel-Cox), the survival time of the patients with low WWP1 mRNA and protein levels was higher than those patients with high WWP1 mRNA and protein levels, and the differences were statistical significance (P=0.0134 and 0.0098, respectively).(5) The results of Cox proportional hazards model revealed that TNM stage, lymph node metastasis, WWP1 mRNA and protein could be independent prognostic factors of the patients with gastric carcinoma.Part Ⅱ:Effects of WWP1 downregulation on cell proliferation, cell cycle and apoptosis in GC cells and its possible molecular mechanismsObjectiveTo explore the effects of WWP1 downregulation on cell proliferation, apoptosis and cell cycle of gastric carcinoma and its regulation mechanisms.Methods(1) Expressions of WWP1 mRNA and protein were detected by Real-time qPCR and Western blotting in various GC cells and normal gastric mucosa epithelial cell GES-1。(2) WWP1 siRNA and control siRNA were transfected to GC MKN-45 and AGS cells by Lipofectamine 2000.(3) Expressions of WWP1, cyclin D1, CDK4, Bcl-2 and Bax proteins as well as several key proteins such as PTEN, p-Akt and total Akt in PTEN-Akt signaling pathway were detected by Western blotting after transfection with siRNA in gastric carcinoma cells, and Caspase-3 colorimetric assay kit was employed to examine the Caspase-3 activity in various treatment gastric carcinoma cells.(4) The effects of WWP1 downregulation on the proliferation of GC MKN-45 and AGS cells were investigated by CCK-8 proliferation experiment, and the effects of WWP1 downregulation on cell cycle and apoptosis of GC MKN-45 and AGS cells were investigated by Flow cytometry.(5) STRING 9.1 online software was employed to predict the interactional proteins of WWP1.(6) Statistical analysis:Statistical analysis was performed by SPSS 17.0 software, all data were expressed as x±s. The comparisons of mutiple sample tests were performed by One-way ANOVA, and a P value less than 0.05 was considered as statistical significance.Results(1) The relative WWP1 mRNA and protein levels in gastric carcinoma cells were significantly higher than those in normal gastric mucosa epithelial GES-1 cells, and the differences were statistical significance (P<0.05). Additionally, the relative WWP1 mRNA and protein levels in MKN-45 and AGS cells were markedly higher than those in other gastric carcinoma cells, and the differences were statistical significance (P<0.05).(2) Expression of WWP1 protein in WWP1 siRNA group was significantly lower than those in untreated group (MKN-45 and AGS) and control siRNA group, and the differences were statistical significance (P<0.05).(3) Compared with untreated group and control siRNA group, the proliferations of gastric carcinoma cells MKN-45 and AGS were obviously suppressed in WWP1 siRNA group, and the differences were statistical significance (P<0.05).(4) The percentages of G0/G1 phase in MKN-45 and AGS cells in WWP1 siRNA group (64.87±1.48% and 64.88±1.34%) were significantly higher than those in untreated group (50.19±1.46% and 50.25±3.02%) and control siRNA group (50.93±1.39% and 51.60±2.09%), and the differences were statistical significance (F=98.515 and 38.528, both P=0.000), whereas there was no difference in the percentage of G0/G1 phase between untreated group and control siRNA group (P>0.05). Besides, the percentages of S phase in MKN-45 and AGS cells in WWP1 siRNA group (20.32±1.25% and 23.47±2.26%) were significantly lower than those in untreated group (29.57±1.25% and 30.30±0.96%) and control siRNA group (30.36±1.17% and 30.23±1.34%), and the differences were statistical significance (F=62.386 and 17.692, P=0.000 and 0.003), whereas there was no difference in the percentage of S phase between untreated group and control siRNA group (P>0.05).(5) The results of Flow cytometry demonstrated that early apoptotic rates of MKN-45 and AGS cells in WWP1 siRNA group were 20.56±0.92% and 28.99±1.71%, which was significantly higher than those in untreated group (6.95±0.69% and 9.54±1.18%) and control siRNA group (8.35±1.87% and 10.06±1.46%), and the differences were statistical significance (F=04.561 and 171.636, both P=0.000), however, there was no difference in early apoptotic rate of MKN-45 and AGS cells between untreated group and control siRNA group (P>0.05). Besides, total apoptotic rates of MKN-45 and AGS cells in WWP1 siRNA group were 22.95±1.92% and 36.26±4.36%, which was significantly higher than those in untreated group (8.38±1.03% and 11.49±0.48%) and control siRNA group (9.92±1.29% and 11.48±0.88%), and the differences were statistical significance (F=90.441 and 91.844, both P=0.000), however, there was no difference in total apoptotic rate of MKN-45 and AGS cells between untreated group and control siRNA group (P>0.05).(6) Expression levels of cyclin D1, CDK4 and Bcl-2 proteins of MKN-45 and AGS cells in WWP1 siRNA group were significantly lower than those in untreated group and control siRNA group, whereas Bax expression and Caspase-3 activity in WWP1 siRNA group were markedly higher than those in untreated group and control siRNA group, and the differences were statistical significance (P<0.05).(7) The results of STRING 9.1 software prediction demonstrated that WWP1 interacted with many proteins in gastric carcinoma, such as PTEN, TGFβ1, STAT3 and SMAD7, etc.(8) Compared with untreated group and control siRNA group, PTEN expression was upregulated, whereas p-Akt level was downregulated, but total Akt level was not changed in MKN-45 and AGS cells in WWP1 siRNA group.Part III:The role of WWP1 in gastric carcinoma cells MKN-45 and AGS implanted nude miceObjectiveTo dissect the role of WWP1 downregulation in gastric carcinoma xenografted nude mice.Methods(1) Animal grouping:42 nude mice were randomly divided into MKN-45 implanted group and AGS cell implanted group, and each group contained 21 nude mice. The nude mice were randomly divided into three groups (7 nude mice per group):untreated group, control siRNA treatment group and WWP1 siRNA treatment group.(2) Subcutaneous inoculation of MKN-45 and AGS cells and treatment:2×106 MKN-45 cells and 3×106 AGS cells were subcutaneously inoculated into the right flank of nude mice in their backs. When the tumor volume reached around 55-80 mm3, no any treatment was performed in untreated group, and WWP1 siRNA and control siRNA (100ng per nude mice) were injected into nude mice in another 2 groups every three days and tumor volumes were measured by square caliper every three days. When the measurement was terminated, tumor tissues were harvested and tumor weights were determined. Meanwhile, associations of WWP1 expression with survival rate of nude mice were investigated by Kaplan-Meier survival curve.(3) WWP1 mRNA and protein levels were detected in gastric carcinoma MKN-45 and AGS implanted nude mice by Real-time qPCR and Western blotting.(4) Statistical analysis:Statistical analysis was performed by SPSS 17.0 software, all data were expressed as x±s. The comparisons of mutiple sample tests were performed by One-way ANOVA, and associations of WWP1 expression with survival rate of nude mice were investigated by Kaplan-Meier survival curve, and a P value less than 0.05 was considered as statistical significance.Results(1) The results of Real-time qPCR and Western blotting demonstrated that compared with untreated group (MKN-45 or AGS) and control siRNA treatment group, WWP1 mRNA and protein expressions of MKN-45 or AGS implanted tumors in WWP1 siRNA group were significantly decreased, and the differences were statistical significance (P<0.05), whereas there were no difference in WWP1 mRNA and protein relative levels between untreated group and control siRNA treatment group (P>0.05).(2) Compared with untreated MKN-45 or AGS group and control siRNA treatment group, the growths of nude mice tumors were evidently inhibited, and the differences were statistical significance (P<0.05).(3) The mean weights of MKN-45 and AGS implanted tumors in WWP1 siRNA group were 0.467±0.111 and 0.457±0.118, which was significantly lower than those in untreated group (1.823±0.102 and 1.838±0.095) and control siRNA treatment (1.714±0.119 and 1.950±0.153), and the differences were statistical significance (F=323.932 and 312.691, both P=0.000), however, there were no differences in the weights of MKN-45 and AGS implanted tumors between untreated group and control siRNA treatment (P>0.05).(4) The result of Kaplan-Meier survival curve showed that compared with untreated MKN-45 group and control siRNA treatment group, the survival time of nude mice was significantly prolonged in WWP1 siRNA group (P<0.05), and similar results were observed in AGS implanted nude mice.Conclusion(1) High WWP1 expression in GC tissues is tightly associated with tumor differentiation, TNM staging, invasion depth, lymph node metastasis and prognosis, and WWP1 may be an independent prognostic factor for the patients with GC, suggesting WWP1 plays a pivotal role in the occurrence and development of GC, suggesting WWP1 plays a pivotal role in the occurrence and development of GC.(2) WWP1 downregulation mediated cell proliferation proliferation, cell cycle distribution change and apoptosis may be closely correlated with the decreases of cyclin D1, CDK4 and Bcl-2 expressions as well as the increases of Bax expression and Caspase-3 activity.(3) The changes of vital biological functions mediated by WWP1 may be achieved via suppression of PTEN-Akt signaling pathway, and thus combiations of WWP1 and PTEN-Akt signaling pathway may be a novel direction for therapy of gastric carcinoma.(4) The suppressive role of WWP1 on GC implanted nude mice may provide new experimental basis for development of new anti-GC molecular target.