复合纳米微粒靶向IL-8/miR-370-3p/Twist1和Snail通路抑制肝癌转移的机制及意义

Metastasis is one of the most fatal causes of therapeutic failure of hepatocellular carcinoma (HCC). Elucidation of the metastasis mechanism and exploration of new targets for HCC therapy are urgently needed. Our previous studies had demonstrated that, miR-370-3p down-regulated Twist1 and Snail, and inhibited Huh7 cells metastasis. Further results showed that interleukin 8 (IL8) up-regulated Twist1 and Snail, and simultaneously down-regulated miR-370-3p in Huh7 cells, indicating a possible pathway that IL8/miR-370-3p/Twist1 and Snail regulates HCC metastasis. In this study, we plan to perform a series experiments to investigate the mechanism and clinical relevance of IL8/miR-370-3p /Twist1 and Snail pathway in HCC by following steps. ① Multi HCC cell lines will be utilized to further confirm the pro-metastasis function of IL8/miR-370-3p/Twist1 and Snail pathway in vitro and in vivo. ② In order to test whether miR-370-3p serve as a therapeutic target to inhibit HCC metastasis, the experiment of in vivo anti-tumor effect using nanoparticles containing miR-370-3p mimic and doxorubicin will be performed in xenografts mice. ③ Data of serum IL8, tumor tissue miR-370-3p, Twist1, and Snail levels in HCC patients will be collected, and correlations between these factors and tumor metastasis will be evaluated. Then, the predictive value of these multi markers and relevance with clinical features will be investigated. The present study will elucidate the mechanism of IL8/miR-370-3p/Twist1 and Snail pathway in promoting HCC metastasis, and the results may provide new prognostic predictors and/or therapeutic targets for HCC.

肝癌转移常导致治疗失败，探究其转移机制及新治疗靶点是临床的重大需求。我们预实验结果显示：miR-370-3p可同时负调控Twist1和Snail、抑制肝癌Huh7细胞侵袭和转移，并拮抗IL8促癌作用。另外，IL8下调miR-370-3p表达，提示肝癌可能存在IL8/miR-370-3p/Twist1和Snail通路。本项目拟：①用多个肝癌细胞系，在细胞水平和动物体内实验进一步明确该通路对转移的作用；②在移植肝癌模型中，用纳米载体携带miR-370-3p类似物和多柔吡星，探讨同时抑制转移和增殖是否可改善疗效；③统计分析患者血清IL8、癌组织miR-370-3p、Twist1和Snail表达水平与肝癌转移的临床相关性，建立肝癌转移预测模型并评估其价值。本项目完成将阐明IL8/miR-370-3p/Twist1和Snail通路在肝癌转移中的机制和意义，为肝癌转移的预测或/和治疗提供新思路和靶标。

肝癌转移是治疗失败的主要原因之一，探究其转移机制及新治疗靶点是临床的重大需求。我们在TCGA数据和肝癌病人术后样本中发现促炎因子IL-8与肝细胞癌(HCC)患者预后不良有关，高水平IL-8的HCC患者表现出更多的转移和更短的生存期。在分子机制上，我们发现IL-8通过上调Snail和Twist1表达，诱导肝癌细胞发生上皮间充质转化(EMT)，促进肝癌细胞在体内外的迁移、侵袭和转移，然而这种调控是否依赖于microRNAs尚不清楚。通过Western blot实验，我们发现IL-8/ERK1/2/Snai1和IL-8/STAT3/Twist1两种不同的信号通路参与了肝癌细胞中IL-8诱导的EMT。有趣的是，我们通过in silico方法筛选出Hsa-miR-370-3p同时靶向Snail和Twist1。荧光素酶测定和RNA电泳迁移率变化测定(EMSA)用于评估miR-370-3p与靶向mRNA之间的相互作用，我们的结果显示，miR-370-3p与Twist1和Snail mRNA的3'-UTR区域直接特异性结合。继而我们在探索IL-8与miR-370-3p的关系时发现miR-370-3p能阻断IL-8的促EMT作用。通过Co-IP、荧光素酶和ChIP实验来研究IL-8介导的miR-370-3p修饰的机制，我们发现 IL-8通过激活STAT3募集HDAC1到miR-370-3p的启动子，从而抑制miR-370-3p转录活性。STAT3和HDAC拮抗剂可以抑制肝癌细胞迁移和侵袭。运用细胞株过表达和敲低等研究方法、transwell试验和异种移植裸鼠模型等来验证促肿瘤和抗肿瘤活性，同时分析IL-8和miR-370-3p水平在HCC患者中的临床相关性。在临床样本中，我们验证发现高miR-370-3p和低IL-8水平的患者总生存期较长。因此我们的研究阐明了IL-8/STAT3/miR-370-3p/Twist1和Snail轴调控肝癌转移的作用机制，miR-370-3p和IL-8是非常有价值的预后预测因子，我们的研究为肝癌转移的预测或/和治疗提供了新思路和靶标。