长链非编码RNA RP.11-461A8.5：调控乳腺癌耐药的新机制

Paclitaxel is the cornerstone of breast cancer chemotherapy, but drug resistance is a difficult problem in the process of chemotherapy. Using lncRNA array technology, we found that RP.11-461A8.5 significantly down-expressed in the MCF-7/PTX cells. We further found that down-regulate of RP.11-461A8.5 in MCF-7 cells can induced MCF-7 cell PTX resistance; we predicted TRAP1 as a potential target gene of RP.11-461A8.5 by employing lncRNA target gene prediction softwares, and we also found that down-regulate RP.11-461A8.5 could up-regulate the expression of TRAP1, which indicated that RP.11-461A8.5might be involved in the drug resistance of breast cancer through TRAP1. In this study, we will further uncover the biology characteristics of RP.11-461A8.5; we want to detect the effect of RP.11-461A8.5 on breast cancer drug resistance through over-expression and down-expression strategy, we use TRAP1 as clue, we want to explore the undergoing molecular mechanism of RP.11-461A8.5 induced breast cancer drug resistance. There was few information about the relationship between lncRNA and breast cancer drug resistance. This study will provide a potential intervention target for reversing the drug resistant for breast cancer therapy.

紫杉醇是乳腺癌化疗的基石，但其诱发的化疗耐药是临床治疗瓶颈。RP.11-461A8.5是我们采用芯片技术筛选获得、差异低表达于乳腺癌耐药细胞中、功能未知的lncRNA。前期研究发现，乳腺癌细胞中抑制RP.11-461A8.5可诱导细胞耐药；生物信息学及实验结果初步确定TRAP1为其下游靶基因；RP.11-461A8.5表达沉默可上调TRAP1表达；而文献报道TRAP1与乳腺癌耐药密切相关，提示RP.11-461A8.5可能通过TRAP1影响乳腺癌耐药性。本课题拟阐明RP.11-461A8.5在乳腺癌中的表达规律；采用过表达和沉默策略，明确RP.11-461A8.5对乳腺癌耐药的影响；以TRAP1为机制线索，揭示RP.11-461A8.5调控乳腺癌耐药的分子机制。RP.11-461A8.5在乳腺癌耐药方面尚无研究报道，研究可能为逆转乳腺癌耐药提供新靶标。

化疗药物耐药是乳腺癌治疗的瓶颈，目前研究证明LncRNA可能参与到肿瘤发生发展、治疗耐药等过程。本研究采用LncRNA基因芯片检测筛选建立的阿霉素耐药株MCF-7/ADM及其亲本细胞系LncRNA差异表达，发现1597条lncRNA上调，1514条lncRNA下调，经过在细胞株和人源肿瘤组织及其癌旁组织中的验证，发现在耐药的细胞株和肿瘤组织中，RP.11-461A8.5均表现为低表达，生物信息学分析，提示TRAP1 基因可能是RP.11-461A8.5的直接调控对象，且与TRAP1基因为Natural antisense关系。我们观察MCF-7/ADM较其亲本细胞株，临床样本肿瘤组织较其对应的癌旁组织中TRAP1在mRNA和蛋白表达水平均增高。通过构建RP.11-461A8.5 过表达的慢病毒载体，使MCF-7/ADM细胞过表达RP.11-461A8.5，结果发现其对阿霉素的敏感性显著增高， 阿霉素诱导其凋亡率升高，且TRAP1在RNA水平和蛋白水平表达均降低，阿霉素5μg/ml孵育6h，细胞内ROS生成增多，线粒体膜电位降低。阿霉素5μg/ml孵育48h，过表达RP.11-461A8.5的MCF-7/ADM细胞Caspase3，Caspase9活性显著增高，为验证RP.11-461A8.5对乳腺癌细胞耐药性的影响是通过调节TRAP1，影响线粒体功能而实现的，我们在过表达RP.11-461A8.5 MCF-7/ADM细胞中过表达TRAP1，发现在MCF-7/ADM细胞上过表达RP.11-461A8.5的所产生的上述效应可被取消。构建RP.11-461A8.5 表达沉默慢病毒载体，使MCF-7细胞RP.11-461A8.5表达沉默，结果发现其对阿霉素的耐药性显著增高，TRAP1表达增高，阿霉素5μg/ml孵育6h，RP.11-461A8.5表达沉默的MCF-7细胞较干预前ROS生成显著降低，线粒体膜电位升高，caspase3，caspase9活性显著降低，而在RP.11-461A8.5表达沉默的MCF-7细胞中使TRAP1表达沉默，其对阿霉素敏感性增加，细胞内ROS水平升高，线粒体膜电位水平降低，caspase3，caspase9 活性显著增高。以上结果表明，RP.11-461A8.5可能通过调节TRAP1的表达，调控线粒体内稳态，从而影响乳腺癌细胞耐药。