miR-374b-5p在黄芩素抑制人肝癌细胞增殖中的作用及分子机制

Baicalein has been proved to possess significant antitumor activity. It has been demonstrated that the microRNA (miRNA) plays important roles in the antitumor effect of traditional Chinese herbs. However, the role of miRNA on the antitumor effect of baicalein has not been reported. Our previous study found that the expression of miR-374b-5p in hepatoma cells was negatively correlated with concentration of baicalein by miRNA microarray analysis. The bioinformatics analysis implies PETN, a tumor suppressor gene, is the potential target gene of miR-374b-5p, it is indicated that miR-374b-5p might be correlated with AKT/mTOR signaling pathway. To prove the hypothesis of “baicalein → down-regulation of miR-374b-5p → up-regulation of PTEN → suppression of AKT/mTOR pathway → proliferation inhibition of hepatoma cells”, we will investigate the cell proliferation, cell cycle, apoptosis of HCC through over-expressed miR-374b-5p and knockdown miR-374b-5p, validate the pathway that miR-374b-5p suppress AKT/mTOR signaling via PTEN, and analysis the effect of change of PTEN, AKT expression on the proliferation inhibition induced by baicalein. This Program will provide new clues and new ideas for revealing the molecular mechanism of anti-hepatoma of baicalein from miRNA perspective, and will provide a basis of development and application of baicalein.

黄芩素具有广泛的抗肿瘤活性，研究发现miRNA可参与中药的抗肿瘤效应，但其是否参与黄芩素的抗肿瘤作用还未见报道。本课题组前期采用miRNA芯片分析发现肝癌细胞中miR-374b-5p的表达与黄芩素处理浓度呈负相关，生物信息学分析显示其与靶基因PTEN及AKT/mTOR通路相关，为此我们提出“黄芩素→下调miR-374b-5p表达→上调抑癌基因PTEN表达→抑制AKT/mTOR通路→抑制肝癌细胞增殖”的假设。本研究拟通过对比miR-374b-5p过表达和沉默细胞株经黄芩素处理后的增殖、凋亡、周期、裸鼠成瘤情况的变化；明确miR-374b-5p对PTEN和AKT/mTOR通路的调节作用；并检测PTEN、AKT的变化对黄芩素抑制肝癌细胞增殖的影响，从而证实提出的假设，旨在从miRNA角度研究黄芩素抗肝癌作用，为揭示黄芩素抗肝癌效应的分子机制提供新线索和新思路，为黄芩素药物开发和应用提供理论依据。

研究表明黄芩素可通过抑制细胞增殖而具有显著的抗肝癌活性。黄芩素的抗增殖作用是否与其对肝细胞癌中miRNA表达的调节有关仍未知。我们通过检测细胞增殖、周期及周期相关分子的表达变化来分析黄芩素对肝癌细胞Bel-7402的抗增殖作用。进行微阵列分析检测黄芩素处理后Bel-7402细胞中的miRNA表达谱变化，并通过qRT-PCR进行验证。通过qRT-PCR测定miR-3127-5p在不同HCC细胞系中的表达水平，并在细胞中转染miRNA mimics检测miR-3127-5p的抗增殖作用。结果发现黄芩素通过上调p21和p27的表达、抑制PI3K / AKT通路诱导细胞周期阻滞在S和G2/M期从而抑制肝癌细胞系Bel-7402的增殖活性。黄芩素可改变肝癌细胞株Bel7402的表达谱。其中表达上调的miR-3127-5p在不同肝癌细胞系中表达下降，过表达miR-3127-5p通过上调p21和p27的表达、抑制PI3K / AKT通路诱导细胞周期阻滞在S和G2/M期从而抑制肝癌细胞系Bel-7402的增殖活性，与黄芩素的抗肝癌活性类似。因此调节miRNA表达可能是黄芩素抗肝癌作用的重要机制。