实时荧光定量RT-PCR检测原发性肝细胞癌中Dicer mRNA的表达

Real time fluorescent quantitative RT-PCR in determination of Dicer mRNA in primary hepatocellular carcinoma

目的:建立Dicer mRNA表达量检测的实时荧光定量逆转录聚合酶链反应(reverse transcriptase polymerase chainreaction,RT-PCR)系统,检测肝癌细胞系与20例原发性肝细胞癌(primary hepatocellular carcinoma,PHC)组织中Dicer mR-NA的表达水平。方法:根据Dicer mRNA的全序列设计特异性引物,结合荧光染料SYBR GreenⅠ,RT-PCR扩增目的片段并实时检测产物的荧光强度,进行熔解曲线分析排除非特异性扩增,根据标准品绘制标准曲线,由软件自动计算出待测样品Dicer mRNA的准确含量,并以Dicer mRNA和18S rRNA含量的比值作为Dicer表达水平的指标。结果:该方法检测的线性范围为5×102~5×109copies/μl,样本的批内变异系数与批间变异系数为4.13%~19.72%和6.25%~18.76%。Dicer mR-NA在HBV阳性肝癌细胞系HepG2.2.15和HBV阴性肝癌细胞系HepG2的表达水平明显低于正常肝细胞系WRL-68(P<0.05);在PHC组织的表达水平明显低于癌旁配对组织(P<0.05)。结论:该方法具有较好的灵敏度、特异性和重复性。Dic-er mRNA表达量在肝癌细胞系和PHC组织中的检测为以后更好的研究PHC的发病机制奠定了理论基础。

Objective：To develop a real time fluorescent quantitative reverse transcriptase polymerase chain reaction （RTPCR） system for determining the expression of Dicer mRNA in human hepatoma cell lines and 20 samples of primary hepatocellular carcinoma（PHC）tissues. Methods： The specific primers, designed according to the complete sequence of Dicer mRNA, and the fluorescence dye SYBR Green I were used for RT-PCR amplification. The fluorescence was monitored in a real time manner. The expression levels of Dicer mRNA in samples were calculated according to the standard curve and the nonspecific amplifications were excluded by melting curve analysis. The mRNA levels of Dicer were presented as the ratios of Dicer mRNA to 18S rRNA. Results： The linear detection range of this system was from 5 × 10^2to 5 × 10^9 copies/btl and the coefficient of variation values for intra-experimental and inter-experimental reproducibility ranged from 4. 13% to 19. 72% and from 6.25% to 18.76%, respectively. The expression levels of Dicer mRNA in HBV positive hepatoma cell line HepG2.2.15 or in HBV negative hepatoma cell line HepG2 were significantly lower （P^0.05） compared to those of the normal liver cell line WRL-68; and those in PHC tissues were lower compared to those in the corresponding adjacent tissues of PHC （P〈0. 05）. Conclusion： Real time fluorescent quantitative RT-PCR has good sensitivity, specificity and reproducibility in determining Dicer mRNA levels. Determination of Dicer mRNA levels in hepatoma cell lines and PHC tissues lays a good theoretical foundation for further studying the mechanism of PHC.