AIM:To determine how the oncogene mi R-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.METHODS:RAS p21 GTPase activating protein 1(RASA1) protein expression in six colon cancer cell lin...AIM:To determine how the oncogene mi R-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.METHODS:RAS p21 GTPase activating protein 1(RASA1) protein expression in six colon cancer cell lines was assessed by Western blot.Colon cancer RKO cells were chosen for transfection because they are KRAS wild type colon cancer cells whose RASA1 expression is significantly decreased.RKO cells were transfected with vectors overexpressing or downregulating either mi R-21 or RASA1.Furthermore,a luciferase reporter assay was used to determine whether RASA1 is a gene target of mi R-21.Then,changes in m RNA and protein levels of RASA1,RASGTP,and other components of the RAS signaling pathways were assessed in transfected RKO cells by real-time quantitative reverse transcription-polymerase chain reaction,Western blot and immunoprecipitation.Finally,cell proliferation,apoptosis,invasion,and tumorformation ability w ere assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay,flow cytometry,transwell assay,and animal experiment,respectively.RESULTS:RASA1 protein levels were significantly decreased in RKO cells compared with the other 5 colon cancer cell lines,and RASA1 was confirmed as a target gene of mi R-21.Interestingly,RASA1 m RNA and protein levels in pre-mi R-21-LV(up-regulation of mi R-21) cells were lower than those in anti-mi R-21-LV(down-regulation of mi R-21) cells(P < 0.05).In addition,pre-mi R-21-LV or si RASA1(down-regulation of RASA1) cells showed higher cell proliferation,reduced apoptosis,increased expression of RAS-GTP,p-AKT,Raf-1,KRAS,and p-ERK1/2,and higher invasion and tumor formation ability,compared with control,antimi R-21-LV or pc DNA3.1-RASA1(up-regulation of RASA1) cells(P < 0.05).CONCLUSION:RASA1 is a target gene of mi R-21,which promotes malignant behaviors of RKO cells through regulation of RASA1 expression.展开更多
BACKGROUND: KRAS mutation plays an important role in the pathogenesis of pancreatic cancer. However, the role of wild-type KRAS in the progression of pancreatic cancer remains unknown. The present study was to investi...BACKGROUND: KRAS mutation plays an important role in the pathogenesis of pancreatic cancer. However, the role of wild-type KRAS in the progression of pancreatic cancer remains unknown. The present study was to investigate the expression of the Ras GTPase activating protein (DAB2IP) in pancreatic cancer and its clinical significance. METHODS: The expression of DAB2IP in pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was analyzed by Western blotting and realtime quantitative reverse transcription-PCR (qRT-PCR). The KRAS mutational types of pancreatic cancer tissues obtained from pancreatic cancer patients (n=20) were also analyzed. Subsequently, DAB2IP expression was detected in pancreatic cancer tissues, adjacent and normal pancreatic tissues (n=2) by immunohistochemistry, and the relationship between DAB2IP expression and the clinical characteristics of patients was evaluated. RESULTS: Western blotting and qRT-PCR results showed that DAB2IP expression in pancreatic cancer cells with wild-type KRAS was lower than that in those with mutation-type KRAS and normal human pancreatic ductal epithelial cells (P【0.05). Immunohistochemistry showed that DAB2IP expression was lower in pancreatic cancer tissues than that in adjacent and normal pancreatic tissues (Z=-4.000, P=0.000). DAB2IP expression was lower in pancreatic cancer patients with the wild-type KRAS gene than that in those with KRAS mutations (WilcoxonW=35.000, P=0.042). Furthermore,DAB2IP expression in patients with perineurial invasion was lower than that in those without invasion (WilcoxonW=71.500, P=0.028). DAB2IP expression was lower in patients with more advanced stage than that in those with early clinical stage (WilcoxonW=54.000, P=0.002). CONCLUSIONS: DAB2IP expression was reduced in patients with pancreatic cancer compared with those with no cancer. DAB2IP expression was correlated with the KRAS gene, perineurial invasion and clinical stage of the disease. Our data indicated that DAP2IP expression can be used as a potential prognostic indicator and a promising molecular target for therapeutic intervention in patients with pancreatic cancer.展开更多
基金Supported by National Natural Science Foundation of China,No.81272770Grants from Guangdong Natural Science Foundation,No.S2013020012746Foundation of Guangdong Provincial Department of Science and Technology,No.2012A030400018
文摘AIM:To determine how the oncogene mi R-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.METHODS:RAS p21 GTPase activating protein 1(RASA1) protein expression in six colon cancer cell lines was assessed by Western blot.Colon cancer RKO cells were chosen for transfection because they are KRAS wild type colon cancer cells whose RASA1 expression is significantly decreased.RKO cells were transfected with vectors overexpressing or downregulating either mi R-21 or RASA1.Furthermore,a luciferase reporter assay was used to determine whether RASA1 is a gene target of mi R-21.Then,changes in m RNA and protein levels of RASA1,RASGTP,and other components of the RAS signaling pathways were assessed in transfected RKO cells by real-time quantitative reverse transcription-polymerase chain reaction,Western blot and immunoprecipitation.Finally,cell proliferation,apoptosis,invasion,and tumorformation ability w ere assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay,flow cytometry,transwell assay,and animal experiment,respectively.RESULTS:RASA1 protein levels were significantly decreased in RKO cells compared with the other 5 colon cancer cell lines,and RASA1 was confirmed as a target gene of mi R-21.Interestingly,RASA1 m RNA and protein levels in pre-mi R-21-LV(up-regulation of mi R-21) cells were lower than those in anti-mi R-21-LV(down-regulation of mi R-21) cells(P < 0.05).In addition,pre-mi R-21-LV or si RASA1(down-regulation of RASA1) cells showed higher cell proliferation,reduced apoptosis,increased expression of RAS-GTP,p-AKT,Raf-1,KRAS,and p-ERK1/2,and higher invasion and tumor formation ability,compared with control,antimi R-21-LV or pc DNA3.1-RASA1(up-regulation of RASA1) cells(P < 0.05).CONCLUSION:RASA1 is a target gene of mi R-21,which promotes malignant behaviors of RKO cells through regulation of RASA1 expression.
基金supported by grants from the Project of International Cooperation in Guangzhou Province (2010B050700014)the Science and Technology Planning Project of Guangzhou(2011J4100006)
文摘BACKGROUND: KRAS mutation plays an important role in the pathogenesis of pancreatic cancer. However, the role of wild-type KRAS in the progression of pancreatic cancer remains unknown. The present study was to investigate the expression of the Ras GTPase activating protein (DAB2IP) in pancreatic cancer and its clinical significance. METHODS: The expression of DAB2IP in pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was analyzed by Western blotting and realtime quantitative reverse transcription-PCR (qRT-PCR). The KRAS mutational types of pancreatic cancer tissues obtained from pancreatic cancer patients (n=20) were also analyzed. Subsequently, DAB2IP expression was detected in pancreatic cancer tissues, adjacent and normal pancreatic tissues (n=2) by immunohistochemistry, and the relationship between DAB2IP expression and the clinical characteristics of patients was evaluated. RESULTS: Western blotting and qRT-PCR results showed that DAB2IP expression in pancreatic cancer cells with wild-type KRAS was lower than that in those with mutation-type KRAS and normal human pancreatic ductal epithelial cells (P【0.05). Immunohistochemistry showed that DAB2IP expression was lower in pancreatic cancer tissues than that in adjacent and normal pancreatic tissues (Z=-4.000, P=0.000). DAB2IP expression was lower in pancreatic cancer patients with the wild-type KRAS gene than that in those with KRAS mutations (WilcoxonW=35.000, P=0.042). Furthermore,DAB2IP expression in patients with perineurial invasion was lower than that in those without invasion (WilcoxonW=71.500, P=0.028). DAB2IP expression was lower in patients with more advanced stage than that in those with early clinical stage (WilcoxonW=54.000, P=0.002). CONCLUSIONS: DAB2IP expression was reduced in patients with pancreatic cancer compared with those with no cancer. DAB2IP expression was correlated with the KRAS gene, perineurial invasion and clinical stage of the disease. Our data indicated that DAP2IP expression can be used as a potential prognostic indicator and a promising molecular target for therapeutic intervention in patients with pancreatic cancer.