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Background:Colorectal cancer is one of the commonly seen malignant tumors in digestive system. Most patients with colorectal cancer were diagnosed in advanced stage and the prognosis is poor. Early diagnosis is crucial to improve the overall survival of patient. Currently,molecular markers for diagnosis of colorectal cancer has attracted more and more attention of the investigators. Aims:To investigate the expression and significance of Rab5A in colorectal cancer. Methods:Colorectal cancerous tissue and the paired para-cancer noncancerous tissue of 135 patients who underwent resection for colorectal cancer were collected. Expression of Rab5A mRNA in cancerous tissue,para-cancer noncancerous tissue,colon cancer cell lines HCT116,HT-29,LoVo and SW480 and normal colonic epithelial cell line NCM460 was determined by real-time PCR. Protein expression of Rab5A in cancerous tissue and para-cancer noncancerous tissue of 4 patients was detected by Western blotting. Protein expression of Rab5A in cancerous tissue of 110 patients was detected by immunohistochemistry. Correlations between expression of Rab5A and clinicopathological characteristics and prognosis of colorectal cancer were analyzed. Results:Expression of Rab5A mRNA in cancerous tissue was significantly higher than that in para-cancer noncancerous tissue(P=0. 003). Expression of Rab5A mRNA in colon cancer cell lines HCT116, HT-29,LoVo and SW480 was significantly higher than that in normal colonic epithelial cell line NCM460(P﹤0. 05). Protein expression of Rab5A in cancerous tissue was significantly higher than that in para-cancer noncancerous tissue. In 110 cancerous tissues,51(46. 4%)had low expression of Rab5A and 59(53. 6%)had high expression of Rab5A. Protein expression of Rab5A was significantly correlated with tumor size,serum carcinoembryonic antigen(CEA)level and TNM stage(P =0. 008;P =0. 002;P =0. 010). 3-year survival rate of patients with high Rab5A expression was significantly lower than that of patients with low Rab5A expression(52. 1% vs. 77. 5%,P ﹤0. 05). Rab5A protein expression,serum CEA level and TNM stage were correlated with the prognosis of patients with colorectal cancer( P =0.009;P =0. 006;P =0. 017)and were independent factors for prognosis(P =0. 026;P =0. 032;P=0. 014). Conclusions:Expression of Rab5A correlates with the malignant behavior of colorectal cancer and its high expression indicates a poor prognosis. Rab5A might be used as a predictor for prognosis of colorectal cancer.
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Objective:Using the macrophage cell lines RAW264.7 stably expressing Rab5a and its dominant negative mutant Rab5aN133I as models to analyze the effect and the mechanism of Rab 5a,Rab5aN133I on CpG-induced production of pro-inflammatory cytokines and type Ⅰ IFN.Methods: The eukaryotic expression vectors of Rab5a and Rab5aN133I were transfected into RAW264.7 cells by liposome,and screened with G418.The G418-resistant colonies were obtained and amplified.The transformed cell lines were i-dentified by RT-PCR,Real time-PCR and Western blot.The production of cytokines were measured after transformed cell lines of Rab5a and Rab5aN133I was stimulation with CpG for 8 h.Results: Rab5a expression in transfected cells was significantly higher than the control cell group (P<0.05).Overexpression of Rab5a significantly promoted the production of TNF -α,IL1-β(P<0.01) and IFN-β( P<0.05) in CpG stimulated RAW264.7.The production of cytokines was restored in Rab 5aN133I transfected cell line.Conclusion:Rab 5a promotes CpG-induced pro-inflammatory cytokines and typeⅠIFN in macrophages,it may be act as a positive regulator in TLR9 signaling pathway.
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Objective:To establish cell lines stably expressing Rab5a and its the inactive mutant Rab5aN133I,analyze the effect of Rab5a on the expression of cytokines in LPS-stimulated RAW264.7 cells .Methods:RAW264.7 cells were transfected with Rab5a and its inactive mutant vector Rab5a N133I separately,and then screened by G418.Rab5a stable expressing cell lines were identified by Real time-PCR.The growth of the stable cell lines was analyzed by MTT assay.After the stable cell lines were stimulated by LPS for different time periods,the expression of iNOS,TNF-αand IL-6 was detected.Results:Rab5a and Rab5aN133I transfection resulted in elevated Rab5a mRNA expression compared with the control cells ( P<0.05 ).Rab5a overexpression enhanced the proliferation of RAW264.7 cells.However,the proliferation of Rab5aN133I cells was significantly slower than the control cells ( P<0.05).Overexpression of Rab5a promoted LPS-induced production of iNOS,TNF-αand IL-6 in RAW264.7 cells (P<0.01). Conversely,overexpression of Rab5aN133I abolished the stimulating effects of Rab5a.Conclusion: Rab5a promoted LPS-induced expression of iNOS,TNF-αand IL-6 in RAW264.7 macrophages in a GTP-binding ability-dependent manner.