[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌...[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌组织石蜡切片进行杂交实验建立肺癌荧光原位杂交(fluorescence in situ hybridization,FISH)的检测体系。[结果]RBM5 15℃标记12h可获得合适的探针,探针与样本杂交后样本细胞内出现清晰明亮的绿色荧光信号,通过与呈橘红色荧光信号的CEP-3探针比较,可以判断肺癌细胞是否存在RBM5的缺失。[结论]RBM5探针制备的最佳条件是15℃标记12h,FISH实验的参数为10μg/ml蛋白酶K处理样本100 min、探针与样本37℃杂交16h、2×SSC/0.3%NP-40洗涤杂交样本5min。该实验体系适用于肺癌组织RBM5的FISH检测。展开更多
Background We were interested in determining how the tumor suppressor gene RBM5 is regulated in lung cancers. Previous studies suggested that the gene expression is related to histological subtype and smoking exposure...Background We were interested in determining how the tumor suppressor gene RBM5 is regulated in lung cancers. Previous studies suggested that the gene expression is related to histological subtype and smoking exposure, since in small cell lung cancers the RBM5 gene is deleted whereas in non-small cell lung carcinomas （NSCLC） RBM5 expression is reduced. Of particular interest was the recent finding that in lung adenocarcinomas, a histological subtype of NSCLC, smoking exposure correlated with mutational activity in the transforming growth factor alpha （TGF-c~） signaling pathway. Lung adenocarcinomas from smokers were associated with activating KRAS mutations, whereas lung adenocarcinomas from never-smokers were associated with activating epidermal growth factor receptor （EGFR） mutations. We hypothesized that inhibition of RBM5 in lung adenocarcinomas is achieved indirectly via these activating mutations. The objective of the research described herein was to determine if EGFR activation and RBM5 expression are negatively correlated. Methods EGFR expression in the lung adenocarcinoma cell line NCI-H1975 was inhibited using small interfering RNA. RBM5 expression was examined by real-time quantitative polymerase chain reaction and Western blotting. Results Reduced EGFR expression did not correlate with any change in RBM5 expression at either the RNA or protein level. Conclusion These results suggest that RBM5 expression is not directly regulated by EGFR in non-smoker related lung adenocarinomas, and that some other mechanism operates to inhibit either the expression or function of this potential tumour suppressor in lung cancers that retain the RBM5 gene.展开更多
文摘[目的]研究RBM5(RNA-binding motif protein 5)荧光探针的制备方法,确立RBM5荧光探针用于肺癌组织检测的原位杂交技术体系。[方法]以RP11-493K19菌株为材料,提取含有RBM5的质粒进行PCR验证,采用缺口平移法制备RBM5荧光探针,并与人肺癌组织石蜡切片进行杂交实验建立肺癌荧光原位杂交(fluorescence in situ hybridization,FISH)的检测体系。[结果]RBM5 15℃标记12h可获得合适的探针,探针与样本杂交后样本细胞内出现清晰明亮的绿色荧光信号,通过与呈橘红色荧光信号的CEP-3探针比较,可以判断肺癌细胞是否存在RBM5的缺失。[结论]RBM5探针制备的最佳条件是15℃标记12h,FISH实验的参数为10μg/ml蛋白酶K处理样本100 min、探针与样本37℃杂交16h、2×SSC/0.3%NP-40洗涤杂交样本5min。该实验体系适用于肺癌组织RBM5的FISH检测。
文摘Background We were interested in determining how the tumor suppressor gene RBM5 is regulated in lung cancers. Previous studies suggested that the gene expression is related to histological subtype and smoking exposure, since in small cell lung cancers the RBM5 gene is deleted whereas in non-small cell lung carcinomas （NSCLC） RBM5 expression is reduced. Of particular interest was the recent finding that in lung adenocarcinomas, a histological subtype of NSCLC, smoking exposure correlated with mutational activity in the transforming growth factor alpha （TGF-c~） signaling pathway. Lung adenocarcinomas from smokers were associated with activating KRAS mutations, whereas lung adenocarcinomas from never-smokers were associated with activating epidermal growth factor receptor （EGFR） mutations. We hypothesized that inhibition of RBM5 in lung adenocarcinomas is achieved indirectly via these activating mutations. The objective of the research described herein was to determine if EGFR activation and RBM5 expression are negatively correlated. Methods EGFR expression in the lung adenocarcinoma cell line NCI-H1975 was inhibited using small interfering RNA. RBM5 expression was examined by real-time quantitative polymerase chain reaction and Western blotting. Results Reduced EGFR expression did not correlate with any change in RBM5 expression at either the RNA or protein level. Conclusion These results suggest that RBM5 expression is not directly regulated by EGFR in non-smoker related lung adenocarinomas, and that some other mechanism operates to inhibit either the expression or function of this potential tumour suppressor in lung cancers that retain the RBM5 gene.
