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
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.
