靶向沉默MiR-221/222调控PUMA基因及PTEN基因增强口腔鳞癌放射敏感性

Improving the sensitivity of tumor cells to radiation is the key to the success of radiotherapy. How to improve the radiosensitivity of OSCC has become a hot research by experts and scholars. PUMA, with a significant effect on apoptosis, is a main protein in apoptosis process induced by radiotherapy. PTEN is the earliest discovered tumor suppressor gene, which has phosphatase activity, and the deletion of PTEN could lead tumor cells to be radiation-resistant. Both of PUMA and PTEN are the target genes of miR-221/222. The expression of PUMA and PTEN can be raised simultaneously by silencing miR-221/222, hence enhancing cell's radiosensitivity. In our previous work, we have successfully synthesized radiation-inducible promoter and confirmed its role in radiation-induction. This study intends to construct the plasmid with a promoter to regulate the expression of antisense miR-221/222 sponge, and transfect the plasmid to OSCC cells with nanocarriers to silence miR-221/222, raising the expression of PUMA and PTEN simultaneously. PUMA could promote cell apoptosis by activating casepase enzyme, while PTEN could block the signaling pathway of PI3K/Akt to inhibit the growth of tumor and the repairation of radiation damage. With joint action enhance the sensitivity of tumor cells to radiation. In this study, we combine gene therapy with radiotherapy organically, attaining radiotherapy and sensitization effect efficiently, and achieving the target property of radiotherapy at the same time, in order to provide a new strategy for the treatment of OSCC.

肿瘤对放射线的敏感性是决定放疗成败的关键，如何提高OSCC放射敏感性是目前研究热点。PUMA具有强大促凋亡作用，是放疗引起细胞凋亡过程的关键蛋白。PTEN是最早发现具有磷酸酶活性的抑癌基因，其缺失可导致肿瘤细胞辐射抗性。PUMA和PTEN是miR-221/222共同靶基因，沉默miR-221/222可同步上调PUMA和PTEN表达，增强细胞放射敏感性。本课题组已成功合成放射诱导启动子并证实其辐射诱导性，本研究拟构建含有该启动子调控表达反义miR-221/222海绵体的质粒，纳米载体介导转染，沉默miR-221/222，上调PUMA表达，活化casepase，诱导凋亡，同时上调PTEN表达，阻断PI3K/Akt信号通路，抑制肿瘤生长与放射损伤修复。通过双通路共同作用增强放射敏感性。本研究将基因治疗与放疗有机结合，放疗同步增敏，同时实现基因治疗靶向性，为口腔鳞癌治疗提供一种新思路。

口腔鳞状细胞癌( OSCC) 具有恶性程度高、淋巴结易转移及预后差等特点，是口腔颌面部最常见的恶性肿瘤，严重威胁人类健康。根据美国NCCN头颈癌治疗指南，放疗是OSCC综合序列治疗的主要手段之一，对中晚期患者尤为重要。如何增强放射敏感性和靶向性对优化OSCC的放疗模式具有非常重要的指导价值。近年来利用基因治疗联合放射增敏成为研究热点，其目的是期望通过调节肿瘤相关基因表达增加肿瘤细胞放射敏感性，以降低放疗剂量、提高疗效、减少并发症，为OSCC的放射治疗提供新思路。.本课题组前期研究发现，OSCC中miR-221/222呈阳性表达，其沉默可靶向上调PUMA和PTEN基因表达，促进OSCC细胞凋亡。.本课题通过细胞、分子及动物水平的研究，探讨miR-221/222沉默对OSCC细胞的放射增敏作用及其机制。实验首先检测miR-221/222在OSCC组织与口腔正常组织中的表达；通过miR-221/222 mimics和miR-221/222 inhibitor转染上调或沉默HSC3和HSC6细胞中miR-221/222表达，检测miR-221/222表达变化对OSCC细胞放射敏感性的影响；分析并验证miR-221/222的直接靶基因，进而探讨miR-221/222沉默发挥放射增敏作用的机制；最后通过体内实验进一步验证miR-221/222沉默对OSCC细胞裸鼠移植瘤的放射增敏作用及其机制。研究发现，OSCC组织中miR-221/222表达明显升高；miR-221/222沉默可减少细胞克隆形成率，抑制细胞增殖，促进凋亡，增加OSCC细胞的放射敏感性；PUMA与PTEN均为miR-221/222的直接靶基因，miR-221/222沉默可靶向上调PUMA与PTEN基因表达，上调的PUMA可活化Caspase酶，促进细胞凋亡，PTEN表达增加则可阻断PI3K/Akt信号通路，抑制细胞生长与放射损伤修复，协同发挥放射增敏作用；miR-221/222沉默可显著抑制裸鼠移植瘤的生长，增加裸鼠移植瘤的放射敏感性。本课题利用放射诱导启动子充当调控As-miR-221/222海绵体表达的分子开关，将基因治疗与传统放疗有机结合，增加细胞放射敏感性的同时实现放疗靶向性，为口腔鳞癌的基因放射治疗提供新策略。