Liu Chang-zheng, Liu Wei, Zheng Yi, Su Jin-mei, Li Jing-jing, Yu Lan, He Xiao-dong, Chen Song-sen
Department of Biochemistry, National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, and Department of General Surgery, Peking Union Medical College Hospital, Beijing 100005, China.
Chin Med Sci J. 2012 Jun;27(2):65-72.
To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tissues and to validate its bona fide targets in human cholangiocarcinoma cells.
The expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholangiocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respectively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarcinoma tissues by using real-time PCR, locked nucleic acid in situ hybridization (LNA-ISH), and immunohistochemistry analysis.
Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bile duct tissues (P<0.05). The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholangiocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN (P<0.01) and PDCD4 (P<0.05) and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue.
microRNA-21 expression is up-regulated in human cholangiocarcinoma and PTEN, PDCD4 are direct effectors of microRNA-21.
研究微小RNA-21在人胆管癌组织中的表达谱，并验证其在人胆管癌细胞中的真正靶标。
采用实时荧光定量PCR分析评估微小RNA-21在人胆管癌组织及胆管癌细胞系QBC939中的表达谱。分别通过双荧光素酶报告基因检测和蛋白质免疫印迹法分析并确认微小RNA-21的真正靶标。采用实时荧光定量PCR、锁核酸原位杂交（LNA-ISH）和免疫组织化学分析检测人胆管癌组织中微小RNA-21与其靶标的表达相关性。
实时荧光定量PCR分析显示，与配对的正常胆管组织相比，微小RNA-21在人胆管癌组织中的表达显著上调约5.6倍（P<0.05）。双荧光素酶报告基因检测显示，胆管癌细胞系QBC939中的内源性微小RNA-21抑制野生型PTEN（P<0.01）和PDCD4（P<0.05）的荧光素酶报告活性，而对突变型PTEN和PDCD4无此作用。此外，微小RNA-21功能缺失导致QBC939细胞中PTEN和PDCD4蛋白水平显著升高。在同一胆管癌组织中，微小RNA-21水平升高伴随着PTEN和PDCD4表达的显著降低。
微小RNA-21在人胆管癌中表达上调，PTEN、PDCD4是微小RNA-21的直接效应分子。
