miR-17-5p通过靶向XRRA1基因调节干细胞引起鼻咽癌放疗抵抗的实验研究

Radiotherapy is the most successful nonsurgical treatment for nasopharyngeal carcinoma (NPC). Although NPCs initially respond well to a full course of radiation, recurrence is frequent cause by radioresistance in cancer stem cells (CSCs). miR-17-5p has a crucial role in DNA damage repair and apoptosis, also can regulate cell cycle and play an important role of the cancer stem cell maintain and proliferation. In our previous study, stem cell-like subpopulation (PKH26+) was identified from NPC cell lines using a label-retention technique, PKH26+ cells were slow cell cycle cells that have stemness and resistance to radiotherapy. We found that, miR-17-5p was up-expression in PKH26+ cells and CNE2-R cells (a radioresistance cell line) , this results show that miR-17-5p may play an important role in cancer stem cells and radioresistance. Our research was carry out for study the influence of miR-17-5p in the stemness in NPC; and further understand the role of miR-17-5p to drives radioresistance in CSCs through targets X-ray radiation resistance associated 1(XRRA1). Furthermore, this research will help to reveal a potential therapeutic application in reversal of radioresistance, and provide a support to study the new treatment strategies for the NPC therapy.

放疗是鼻咽癌的主要治疗方式，但是鼻咽癌中存在着少数的肿瘤干细胞（CSCs）是放疗耐受，导致复发的根源。miR-17-5p可以调控细胞周期，与肿瘤干细胞的增殖和生长密切相关，是决定细胞受损伤后修复还是凋亡的重要分子。在前期研究中，我们发现鼻咽癌中存在着一种慢周期的PKH26+细胞亚群，具有干细胞特性并对放疗抵抗。miR-17-5p在PKH26+及耐放疗细胞株（CNE2-R）中表达明显上调，提示miR-17-5p与鼻咽癌干细胞的放疗耐受相关。本研究主要探讨miR-17-5p对鼻咽癌CSCs数量及功能的影响，及其对细胞DNA损伤修复的调控是否为CSCs放疗耐受的主要机制；在此基础上研究 miR-17-5p通过调控其下游靶基因X射线放射辅助因子1（XRRA1）而改变鼻咽癌干细胞的放疗耐受。通过本项目的实施，将帮助我们深度认识鼻咽癌放疗抵抗的机制，为开发新的治疗策略提供依据。

放疗是肿瘤治疗的主要方式之一，有研究表明肿瘤干细胞（CSCs）是放化疗耐受，导致复发的根源。miR-17-5p可以调控细胞周期，与肿瘤干细胞的增殖和生长密切相关，是决定细胞受损伤后修复还是凋亡的重要分子。在前期研究中，我们发现鼻咽癌中存在着一种慢周期的PKH26+细胞亚群，具有干细胞特性并对放疗抵抗。在此项研究中，我们为了明确miR-17-5p通过靶向调节CDKN1A影响鼻咽癌细胞的增殖，将miR-17-5p mimics转染人鼻咽癌细胞系CNE2，使用qRT-PCR和Western blot检测miR-17-5p对CDKN1A的影响；生物信息学预测miR-17-5p有CDKN1A的结合位点；荧光素酶实验检测miR-17-5p 可靶向调节CDKN1A；通过Western blot和克隆形成实验验证miR17-5p通过调节CDKN1A影响鼻咽癌细胞株CNE2的增殖。接着我们在CD133+、PKH26+及耐放疗细胞株（A549-R和H1299-R）中检测miR-17-5p发现表达下调，过表达miR-17-5p对肺癌CSCs数量及对细胞增殖有影响，给予耐放疗细胞株4Gy的照射，过表达miR-17-5p可逆转放疗耐受。接着我们研究了XRRA1的功能，发现过表达XRRA1促进直肠癌细胞的增殖和细胞周期进程，为了研究XRRA1 与放射及药物敏感性的关系 我们在 HT29 及 H116 上分别过表达及敲除 XRRA1，然后使用CBP， CAPE，IR进行处理，观察对细胞生长的影响， 发现抑制 XRRA1 后，可提高细胞的存活率。过表达 XRRA1 后细胞的克隆形成率降低，凋亡率明显增加，通过本项目的实施，将帮助我们深度认识肿瘤放疗抵抗的机制，为开发新的治疗策略提供依据。