基于microRNA调控高效靶向肝癌干细胞的基因-溶瘤腺病毒治疗系统研究

Cancer stem cell (CSCs) play important roles in metastasis, radiation & drug resistance, cancer relapse, and are associated with poor prognosis of cancer patients.To date, various therapeutic strategies have been developed for targeting CSCs. Among them, oncolytic adenovirus after fiber modification(such as Ad5/35 chimeric fiber adenovirus) has many advantages, making it an ideal vector system to target and eradicate CSCs. However, CSCs share many same features with normal stem cells or progenitor cells, raising the possibility to harm normal stem cells or progenitor cells during the treatment targeting to CSCs. In our former exploration of gene-virology therapy, a series of regulation strategies have been established to limit oncolytic adenovirus conditionally replicate and kill cancer cells, and the efficiency to transfect cancer cell, especially CSCs has been greatly improved through modifications on adenoviral coat and driving transgene expression using the endogenous adenoviral promoter. Furthermore, we have established a universal system to obtain CSCs from both cell lines and primary tissues of hepatocellular carcinoma. In this project, we will carry out to screen microRNAs which are specific down-regulated in CSCs of hepatocellular carcinoma and utilize their corresponding target sequences to regulate the replication of oncolyic adeovirus, specificly killing CSCs while leaving normal stem cell or progenitor cells unharmed.Further more, we will use the endogenous adenoviral gene expression machinery, such as promoter, to drive therapeutic transgene expression in order to limit oncolytic adenovirus conditionally replicate and kill cancer cells, and the efficiency to transfect cancer cell, especially CSCs.

肿瘤干细胞（CSCs）是肿瘤复发、转移、耐药等的潜在根源，是肿瘤靶向治疗的重点之一。溶瘤腺病毒经改造后能高效感染CSCs，是CSCs靶向治疗的理想系统。然而，CSCs具有许多与正常干细胞相似的特性，在靶向CSCs的同时，可能造成对正常细胞的损害，具有潜在的应用风险。我们是国际上最早提出并开展肿瘤基因-病毒治疗的实验室之一，建立了多种溶瘤腺病毒靶向性调控策略，如改造腺病毒外壳蛋白及通过腺病毒内源性启动子启动和调控外源基因的表达，显著提高腺病毒对肿瘤细胞尤其是CSCs的感染效率，并减少对正常细胞的毒副作用；并且建立了肝癌干细胞的通用性培养体系。在本项目中，拟以肝癌干细胞为模型，筛选获得肝癌干细胞特异性下调的microRNA，研究将其作为溶瘤腺病毒的肿瘤靶向性的调控靶点，以腺病毒内源性启动子启动和调控表达外源基因，建立高效靶向肝癌干细胞、对正常干细胞或祖细胞基本无毒性的基因-溶瘤腺病毒治疗系统.

目的：基因-病毒治疗是继手术、放疗、化疗之后，肝癌靶向治疗的一种非常有前景的生物治疗方案。其中，溶瘤腺病毒具有宿主范围较广与外源基因容量大等优势，被认为是肿瘤靶向治疗的理想载体系统之一。我们根据肝癌中特定基因（miR-133a）的缺失而改造后的溶瘤腺病毒克服了野生型腺病毒对肝脏具有较强的嗜性，靶向肿瘤细胞能力差及转染肿瘤细胞效率低等问题。.方法：通过qPCR, WB, 划痕实验，免疫荧光等分子体外评价含有miR-133a靶序列的溶瘤腺病毒用于治疗肝细胞癌的效果及毒副作用。结果：体外qPCR实验和荧光素酶实验结果表明，miR-133a在肝癌细胞系中相较于普遍低表达且有效的干扰上游基因，验证miR-133a作为候选分子进行溶瘤腺病毒靶向性研究。紧接着，我们成功构建含有miR-133a靶向序列和突变序列的溶瘤腺病毒（ miR-133aT ）。细胞活力检测（CCK8法）表明，在保留有优于对照病毒WAd5肿瘤杀伤力的同时，miR-133aT 对正常细胞杀伤能力较弱。同样地，划痕实验和Transwell实验证明miR-133aT有效抑制肝癌细胞的迁移能力。我们分别选择miR-133a高表达和低表达细胞系进行体内皮下荷瘤的抑癌选择实验。 miR-133aT和miR-133aT在靶基因低表达（ Hep3B ）的体内均可以显著抑制肿瘤且毒性较低，在miR-133a高表达（HepG2）的体内却丧失了相应的杀伤活性。.结论：综上所述，我们成功构建含有miR-133a靶序列的溶瘤腺病毒，体内外实验均表明其可以特应性杀伤肝癌细胞对正常组织毒性较低。