松针提取物α-蒎烯对肝癌HepG2细胞miR-221及其下游靶基因表达的影响

Effects of α-pinene extracted from pine needle on expression of miR-221 and its potential target genes in human hepatocellular carcinoma cells

为探讨松针提取物α-蒎烯的抗肝癌作用及机制,该实验首先使用流式细胞技术检测α-蒎烯对HepG2细胞周期的影响,然后选取与G2/M期调控相关的miR-221,采用荧光定量PCR技术检测α-蒎烯干预对其表达的影响,同时通过Target Scan等在线生物信息学软件分析miR-221的靶基因,最后对相关靶基因的表达用定量PCR进行检测。结果显示,α-蒎烯呈剂量依赖性抑制HepG2细胞增殖,可阻滞细胞于G2/M期(P<0.05),显著下调HepG2细胞内miR-221的表达(P<0.05),生物信息学分析显示CDKN1B/P27和CDKN1C/P57可能是miR-221的下游靶基因,荧光定量PCR显示α-蒎烯处理后二者的表达显著上调(P<0.001,P<0.05)。上述结果表明,α-蒎烯可能通过阻滞细胞周期于G2/M期从而发挥抗肝癌作用,其对G2/M期的调控可能与下调miR-221表达和上调其靶基因CDKN1B/P27和CDKN1C/P57的表达有关。

To investigate the anti-hepatoma mechanism of α-pinene,HepG2 cell was treated with α-pinene and the change of cell cycle was examined by flow cytometry. The expression of miR-221,which was related the regulation of G2/M phase,was detected by quantitative Real-time PCR. Meanwhile,Target Scan and other online bioinformatics methods were used to analyze and predict the target genes of miR-221,then the expression level of related target genes were detected by quantitative Real-time PCR. The results showed that α-pinene inhibited the proliferation of HepG2 cells in dose-dependent manner. It was also proved that HepG2 cells were arrested at G2/M phase by α-pinene（ P〈0. 05）. The expression of miR-221 was down-regulated in α-pinene treated HepG2 cell. The bioinformatics analysis showed that CDKN1 B/P27 and CDKN1 C/P57 may be the protential targets of miR-221 and both of them were significantly up-regulated（ P〈0. 001,P〈0. 05） by α-pinene treatment. According to these results,it was believed that α-pinene may inhibit the proliferation of hepatocellular carcinoma cells through arrest the cell at G2/M phase,which may be associated with the down-regulate of the miR-221 expression and up-regulate of the CDKN1 B/P27 and CDKN1 C/P57 expression.