高效基因打靶介导的多基因联合干预鼻咽癌转移的研究

Incidence of metastasis and relapse of nasopharyngeal carcinoma (NPC) were unusually high, but there are not effective therapy methods against NPC metastasis and relapse. NPC metastasis involves multi-target, multi-gene. The multi-locus gene targeting mediated by zinc-finger nucleases (ZFN) established by our group can be used for multi-gene combined intervention. Based on general mechanisms of tumor metastasis and specific molecular mechanisms for NPC metastasis, four target genes (E-cad, MMP-9, endostatin and Bmi-1) whose roles are well-characterized, are selected to establish the multi-locus gene targeting method mediated by ZFN. The technology platform of multi-gene combined intervention for NPC metastasis will be set up by different transfection strategies and multiple-level identification strategies. The metastatic characteristics and malignant phenotypes will be detected in vitro and in vivo. The effects of different combinations of multi-gene intervention for NPC metastasis will be compared. The most promising strategies will be further optimized. In this study, we will first apply multi-locus gene targeting technology to cancer cells to solve some important problems found in conventional gene therapy technologies such as low efficiency of targeted integration, unstable expression and low safety. Multi-gene intervention strategy holds the promise of overcoming the limitations of single-gene intervention and revealing more subtle interactions of multiple target genes in tumor metastasis. The technique can been applied to other tumor types, or combined with other biological treatments such as cell therapy and oncolytic virus therapy, thereby contribute to the development of comprehensive cancer therapies with broad application prospect.

鼻咽癌转移与复发率高，但对其复发与转移却无有效治疗方法。其转移涉及多靶点、多基因，课题组前期建立的锌指核酸酶介导的多位点基因打靶技术可实现多基因联合干预。本研究针对肿瘤转移共性和鼻咽癌转移特定分子机制，选择作用较明确的4种靶基因(E-cad、MMP-9、Endostatin和Bmi-1），在鼻咽癌建立锌指核酸酶介导的多位点基因打靶方法；通过不同转染策略和多层次鉴定策略建立多基因联合干预鼻咽癌转移的技术平台；通过对肿瘤转移特性和恶性表型的体内外检测，比较不同配伍组合的多基因联合抑制鼻咽癌转移的效果，并优化方案。本研究率先将多位点基因打靶专利技术应用于肿瘤，将解决外源基因定点整合效率低、表达不稳定、安全性差等基因治疗部分问题；多基因联合干预将克服单基因治疗的局限性，并可揭示肿瘤转移中多靶点相互作用的机制；不仅可向其它肿瘤推广，更可结合细胞和溶瘤病毒生物治疗，促进肿瘤综合治疗模式的发展。

鼻咽癌转移与复发率高，但对其复发与转移却无有效治疗方法，其转移涉及多靶点、多基因，该项目旨在建立结合TALEN介导的多位点基因打靶和TALEN的多基因干预鼻咽癌细胞技术。. 首先结合课题组建立的多位点基因打靶技术，构建分别由E-cadherin基因、Endostatin基因、Bmi-1 基因shRNA真核表达元件与同源重组引导序列组成的多位点基因打靶载体。然后设计、筛选和鉴定特异性识别且切割人rDNA的类转录激活因子效应核酸酶核酸酶(TALEN)，获得切割效率高达78.5%的TALEN。接着将单个携带靶基因的多位点基因打靶载体与TALEN表达载体共转染鼻咽癌细胞CNE2，利用PCR检测外源基因整合，Western blot检测基因打靶载体转染后Bmi-1及E-cad蛋白表达情况，建立鼻咽癌细胞TALEN介导的多位点基因打靶方法。各靶基因的多位点基因打靶载体以不同配伍组合与TALEN真核表达载体共转染鼻咽癌细胞CNE2，结果发现多位点基因打靶介导的E-cad或Bmi-1 基因干预均可显著抑制鼻咽癌细胞体外迁移能力、侵袭能力及上皮间质转化（EMT）进程，。且双基因干预比单基因干预更能显著降低鼻咽癌细胞体外迁移能力，更能显著降低EMT标志物vimentin蛋白表达水平。，通过体外细胞研究建立鼻咽癌转移的多基因联合干预技术平台。制备各靶基因的多位点基因打靶载体腺病毒，构建鼻咽癌细胞裸鼠皮下移植瘤的实验模型，瘤内注射各种多位点基因打靶载体腺病毒，单独注射Endostatin载体腺病毒组、同时注射Endostatin载体 和Bmi-1 shRNA载体腺病毒组均可抑制瘤体生长，但两组间在抑瘤率上没有统计学上的差异，说明多位点基因打靶介导的endostatin干预可显著抑制裸鼠移植瘤的生长。. 该研究率先将多位点基因打靶技术结合TALEN应用于肿瘤多基因干预，不仅可克服单基因治疗的局限性，且可结合细胞治疗和其它生物治疗，促进肿瘤综合治疗模式的发展；也可用于探讨肿瘤发病过程中多基因相互作用的机制。