慢病毒介导人工microRNA沉默HIF-1α基因联合TACE治疗肝癌的实验研究

Transcatheter arterial chemoembolization (TACE) is the preferred treatment for unresectable liver cancer. However, the rates of tumor metastasis and recurrence after TACE therapy are extremely high, and thus seriously affect the therapeutic effect of TACE. It has been shown that high rates of metastasis and recurrence are closely associated with high levels of HIF-1α induced by local embolization. Meanwhile, our previous studies have shown that inhibiting the expression of HIF-1α effectively enhances the chemosensitiivity of tumor cells and inhibits the growth of liver cancer. Given the results and findings above, we firstly establish the lentiviral-mediated plasmid with artificial microRNA (amiRNA) targeting HIF-1α gene for silencing (amiRNA-HIF-1α). Rabbit models of VX2 live cancer would also be established and separated into four groups with the following treatments: normal saline solution, TACE, TACE + amiRNA-HIF-1α, and TACE + sorafenib. Through detecting the tumor sizes, microvessel density, rates of tumor metastasis and recurrence, and tests on rabbit including routine blood test, liver function and kidney funcion, we aim to assess the therapeutical efficacy and toxicity of lentivirus-mecondiated amiRNA targeting HIF-1α combined with TACE treatment for liver cancer. Meanwhile, through detecting the expression of protein associated with tumor invasion，metastasis and multidrug resistance, we intend to further reveal the underlying mechanisms of each group. The present project is expected to discover new means to improve the efficacy of liver cancer with TACE treatment, and also provide theoretical foundation for further clinical trials.

经肝动脉化疗栓塞术(TACE)是不能手术切除肝癌的首选疗法，然而TACE术后转移复发率甚高，严重影响TACE的疗效。研究表明，TACE术后转移复发与栓塞导致HIF-1α表达上调密切相关。我们前期研究发现降低HIF-1α能有效增强化疗敏感性和抑制肝癌的生长。有鉴于此，我们拟构建慢病毒介导人工microRNA靶向沉默HIF-1α基因载体（amiRNA-HIF-1α），以兔VX2肝癌模型为研究对象，分四组实施治疗：生理盐水、TACE、TACE+amiRNA-HIF-1α、TACE+索拉菲尼。通过检测各组肿瘤体积、微血管密度、转移复发率、荷瘤兔血常规和肝肾功能，评估amiRNA-HIF-1α联合TACE治疗肝癌的疗效及毒副作用；通过检测侵袭、转移和耐药蛋白的表达，进一步阐明各治疗组的分子机制。本项目为优化肝癌TACE治疗方案提供新思路，为临床应用人工microRNA基因靶向治疗提供理论基础。

经肝动脉化疗栓塞术（TACE）是治疗晚期、特别是不可切除肝癌的首选方法。然而，长期来看病人行TACE术后，总的生存期并没有明显的延长，导致这一结果的主要原因是TACE诱导 HIF-1α表达增加，进而引起肿瘤血管形成和复发。因此，我们推测CRISPR/Cas9介导的 HIF-1α基因敲除可以增强TACE术治疗肝癌的疗效。在本研究中，我们构建基于慢病毒为载体的 HIF-1α敲除的肝癌细胞（SMCC-7721）及其裸鼠原位肝癌模型。裸鼠被分为四组，分别为空白对照组（Ctrl）、肝动脉结扎组（HAL）、肝动脉结扎+注射对照慢病毒组（HAL+LV-Ctrl）、肝动脉结扎+实验慢病毒组（HAL+LV-H721），每组8只裸鼠。活体成像技术监测肿瘤大小，记录裸鼠存活时间。结果显示： HIF-1α基因被敲除后，缺氧环境下的SMMC-7721细胞侵袭、迁移能力显著下降，凋亡率增加，细胞周期阻滞。动物实验中，HAL + LV-H721组的肿瘤大小较对照组显著降低，裸鼠总生存时间明显延长。同时，在HAL + LV-H721组裸鼠移植瘤中，微血管形成显著减少，肿瘤细胞的凋亡率明显增加，裸鼠外周血血清中VEGF、MMP2 和 MMP9的表达显著降低。我们的研究结果表明：基于CRISPR/Cas9技术的 HIF-1α基因敲除，能显著增强TACE治疗晚期特别是不可切除肝癌的疗效。