| Gastric cancer is one of the common malignant tumors in China, the mortality rate of which accounted for the first one of the digestive tract tumors. Although the early diagnosis, surgery and chemotherapy of gastric cancer have a lot of progress, the5-year survival rate is still very low. In-depth understanding of pathogenesis and developing new treatments are still central issues in research areas of gastric cancer.Ubiquitin-proteasome system(UPS), the main pathway of intracellular protein degradation, is the major target of anti-tumor drugs in recent years. Its malfunction may promote the genesis, development and metastasis of tumor. ROC1protein is a ring-type subunit of the SCF-E3ubiquitin ligase (SCF), whose main function is to promote SCF timely degrading a variety of substrates, including cell cycle regulators, transcription factors and signal transductants etc. Function disorders of ROC1-SCF E3ligase can lead to a range of diseases including tumor. Previous studies have shown ROC1is related with a variety of human tumors (including lung, liver, breast, colon and ovarian cancer) and related to death and growth of tumor cells, suggesting that ROC1can be used as a candidate of new anti-cancer target. But now there is not yet any report about ROC1and gastric cancer. This study is aimed to study the expression of ROC1in gastric cancer tissues and its clinical significance, and to observe its influence on the biological behavior of gastric cancer cells and tried to explain the potential molecular mechanism, so that we can gain in-depth understanding of biological functions of ROC1and explore a basis provided for its potential use in targeted therapy of gastric cancer.Purpose1. The expression of ROC1in gastric cancer and its clinical significance.1) The expression of ROC1in gastric cancer.2) The relationship between expression level of ROC1and clinicopathological parameters of gastric cancer.3) The relationship among expression levels of ROC1, anti-apoptotic gene Bcl-xl and proliferating cell nuclear antigen Ki67.4) The relationship between expression level of ROC1and survival prognosis of gastric cancer.2. The affect of ROC1silencing on the biological characteristics of gastric cancer cells.1) The interference effect of ROC1-siRNA on ROC1protein of gastric cancer cells.2) The effect of ROC1-RNAi on the growth, cell cycle and apoptosis of gastric cancer cells.3) The effect of ROC1-RNAi on the expression of cyclin dependent kinase inhibitor (CKI) P21and P27in gastric cancer cell.Methods1.1) We detected the expression of ROC1in59pairs of paired gastric cancer and adjacent formalin-fixed paraffin-embedded(FFPE) tissue by Immunohistochemical method to analysis the difference between expression of ROC1in gastric cancer tissue and adjacent tissues.2) We detected the expression of ROC1in83gastric cancer FFPE tissues by Immunohistochemical method to analysis the relationship between expression level of ROC1and clinicopathological parameters of gastric cancer.3) We detected the expression of ROC1, Bcl-xl and Ki67in83gastric cancer FFPE tissues by Immunohistochemical method to analysis the relationship among expression levels of ROC1, anti-apoptotic gene Bcl-xl and proliferating cell nuclear antigen Ki67.4) We detected the expression of ROC1in83gastric cancer FFPE tissues by Immunohistochemical method to analysis the relationship between expression level of ROC1and survival prognosis of gastric cancer.2.1) We detected expression of ROC1in MGC803cells of different groups by western blot method in48h,72h and96h after siRNA transfection to observe the interference effect of ROC1-siRNA on the expression of ROC1in gastric cancer cells.2) We detected the growth of MGC8O3cells in different groups by CCK8assay in48h,72h and96h after siRNA transfection, and detected the cell cycle and apoptosis of MGC803cells in all groups by PI staining-Flow CytoMeter method in Oh,24h,48h and72h after siRNA transfection.3) We detected expression of ROC1, P21and P27in MGC803cells of all groups by western blot method in48h,72h and96h after siRNA transfection to observe the effect of ROC1-RNAi on the expression of P21and P27in MGC803cells,Results11) Expression of ROC1in gastric cancer and adjacent tissues:ROC1were mainly localized in the nucleus and cytoplasm, with staining colors of pale yellow, brown to tan; the positive rate of ROC1in gastric cancer was100%, and it’s low expressed in32cases (55.2%) and highly expressed in26cases (44.8%); the positive rate of ROC1in adjacent tissues was93.2%, and it’s low expressed in55cases (93.2%) and highly expressed in0cases (0%). The expression of ROC1in gastric cancer was significantly higher than the adjacent tissue (chi-square=36.075, P=0.000).2) High expression of ROC1was highly correlated with TNM stage and5-year survival rates (both P<0.01). The rate of late-stage gastric cancer (III-IV period) in ROC1-high-expression patients was50.0%(20/40), while the rate of late-stage gastric cancer in ROC1-low-expression patients was20.9%(9/43), the difference was statistically significant (P<0.01); The rate of5-year survival in ROC1-high-expression patients was27.6%(8/29), while the rate of5-year survival in ROC1-low-expression patients was77.3%(17/22), the difference was statistically significant (P<0.01); The expression of ROC1was highly suspicious to be related to gender, distant metastases and degree of tumor infiltration.3) Expressions of ROC1and anti-apoptotic protein Bcl-xl showed a significant positive correlation (r=0.352, P<0.01, Spearman rank-related test); Expressions of ROC1and proliferating cell nuclear antigen Ki67showed a significant positive correlation (r=.244(P<0.05)).4) The rate of5-year survival in ROC1-high-expression patients was27.6%(8/29), while which was77.3%(17/22) in ROC1-low-expression patients, the difference was statistically significant (P=0.001). In univariate analysis, in addition to tumor size, lymph node metastasis, distant metastasis and TNM stage, expression of ROC1was correlated with prognosis of patients with gastric cancer. As of last follow-up time (May2011), survival rate of patients with low expression of ROC1was significantly higher than that of patients with high expression (x2value of11.742, P<0.01). Cox multivariate regression analysis revealed that tumor size, lymph node metastasis, distant metastasis, or expression of ROC1, Bcl-xl or Ki67were independent factors determining the prognosis of patients with gastric cancer. Patients with lymph node metastasis, distant metastasis, high expression of ROC1or Bcl-xl were at higher risk of death.2.1) The interference effect of ROC1-siRNA on ROC1was effective:the expression of ROC1in the ROC1-siRNA group was significantly lower than that of the negative control group (F=12.066, P=0,025), dropped to29%on average;2)The growth of gastric cancer cells in ROC1-siRNA group was inhibited and slowed down:we detected cell proliferation by CCK8method, compared with negative control group, cell proliferation of ROC1-siRNA group has slowed significantly and was time-dependent (minimum to60%)(F=31.099, P=0.000); flow cytometry analysis showed that compared with the negative control, the proportion of apoptotic cells increased (P<0.01). Comprehensively, growth of MGC803cells in vitro was significantly inhibited after ROC1was inhibited.3) Along with the expression of ROC1in ROC1-siRNA group was significantly inhibited, the expression of P21and P27were significantly increased (P<0.01).Conclusions1.1) The expression of ROC1in gastric cancer was significantly upregulated compared to adjacent tissues, suggesting that ROC1may play a role in the development of gastric cancer.2) High expression of ROC1was significantly associated with advanced gastric cancer and poor prognosis. The rate of advanced gastric cancer in patients with high expression of ROC1was higher and5-year survival rate in which was low; prompting that ROC1may play an important role in promoting the development of gastric cancer.3) expression of ROC1was significantly positively correlated with that of anti-apoptotic gene Bcl-xl or cell-proliferation associated nuclear antigen Ki67, suggesting that ROC1may promote the development of gastric cancer by promoting anti-apoptosis and leading to uncontrolled cell proliferation.4)5-year survival rate of patients with high expression of ROC1was significantly lower, suggesting that it may become a powerful marker to predict the prognosis of gastric cancer.2.1) ROC1-siRNA can significantly reduce expression of ROC1in gastric cancer cell line MGC8O3; 2) The growth of MGC803was significantly inhibited by ROCl-siRNA: increased apoptosis, providing a new experimental evidence for targeted therapy of gastric cancer.3) Inhibition of expression of ROC1by ROC1-siRNA can significantly increase expressions of tumor suppressor genes P21and P27in MGC803, suggesting a possible mechanism of inhibition of growth of tumor cells by ROC1silencing. |
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