杨杰波+李明+谢菁菁+杨梦蝶+卢昕烁+王芳+陈伟强

[摘要]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TargetScan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，可阻滞細胞于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。

[关键词]α-蒎烯； G₂/M期； miR-221

[Abstract]To investigate the anti-hepatoma mechanism ofα-pinene， HepG2 cell was treated withα-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， TargetScan 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 thatα-pinene inhibited the proliferation of HepG2 cells in dose-dependent manner. It was also proved that HepG2 cells were arrested at G₂/M phase byα-pinene （P<0.05）. The expression of miR-221 was down-regulated inα-pinene treated HepG2 cell. The bioinformatics analysis showed that CDKN1B/P27 and CDKN1C/P57 may be the protential targets of miR-221 and both of them were significantly up-regulated（P<0.001，P<0.05）byα-pinene treatment. According to these results， it was believed thatα-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 CDKN1B/P27 and CDKN1C/P57 expression.

[Key words]α-pinene； G₂/M phase； miR-221

doi：10.4268/cjcmm20162118

肝癌是影响人类健康的重大疾病，其治疗一直是研究关注的焦点和难点。中医认为肿瘤本质是恶毒，常用具有活血、解毒功效的药物防治肿瘤^[1]。松针（pine needle）为松科松属植物的叶，在《本草纲目》中记载其“无毒，入肝、肾诸经，治各脏肿毒，强健肝、肾”，笔者据此推测其可能对肝癌具有治疗作用，并在前期实验中证实该假说^[2]。本课题组进一步从松针中分离得到了松针主要成分α-蒎烯，并证实α-蒎烯在体内具有一定的抗肝癌作用，可能是松针抗肝癌的主要活性成分^[3]。α-蒎烯属于双环单萜类化合物，具有碳碳双键和碳环结构，丰富的化学结构使α-蒎烯能发生异构化、氧化、氢化、加成、酯化、聚合等一系列化学反应，深入研究α-蒎烯抗肝癌的作用机制是其开发利用的关键，因此本实验采用体外细胞实验来观察α-蒎烯对人肝癌HepG2细胞细胞周期的影响，并根据结果利用定量PCR和生物信息学等方法，从分子水平进一步研究了α-蒎烯对G₂/M期相关miR-221及其下游靶基因表达的影响，为进一步阐明α-蒎烯抗肝癌机制提供研究基础。……