Sp1诱导miR-205表达促进食管癌细胞放疗抵抗的研究

Esophageal cancer is the fourth death of cancer in china. Radiotherapy is one of the important treatment of esophageal carcinoma, radiotherapy resistance is thought to be an important reason for the failure of esophageal cancer recurrence or radiation. .In current study, we analyzed miR-205 expression in 150 esophageal carcinomas (ESC) samples and revealed that miR-205 expression was up-regulated in most primary tumor compared with the normal esophageal tissue, and miR-205 was significantly upregulated in response to Radiation. We try to investigate the effects of miR-205 on radioresistance in vitro and in vivo..We also analyzed potential miR-205 transcription factor, which can activated by ionizing radiation, through Transfac and ALGGEN, and found Sp1 as a predicted transcription factor,which can be activated by ATM protein. We try to investigated the molecular mechanism of Sp1 dependent up-regulation miR-205 by ionizing radiation..MicroRNAs inhibit translation or induce mRNA degradation in general by binding to the 3’ untranslational region (3’UTR) of target mRNAs,so we analyzed potential miR-205 target genes through targetscan，Microlnspector and targetscan, and found its target genes including apoptosis and cell cycle regulation of TP53BP2, PTEN. We found miR-205 was matched with TP53BP2 and PTEN 3’UTR seperately. Further, we try to investigate the underlying molecular mechanisms of TP53BP2 and PTEN down-regulated by miR-205. .Our findings will present functional and mechanistic insight into the critical role of miR-205 on radiosensitivity in the esophageal carcinomas.

放疗抵抗是食管鳞癌复发和治疗失败的重要原因。miR-205在多种鳞癌组织中表达上调，我们前期研究也表明miR-205在食管鳞癌中高表达，且辐照能够诱导miR-205表达上调，提示miR-205可能与参与了食管癌放疗敏感性的调控。首先，我们拟通过体外细胞实验及体内裸鼠食管癌模型明确miR-205诱导放疗抵抗的作用。进一步，我们通过Transfac和ALGGEN软件分析miR-205的启动子区，找到多个转录因子Sp1的结合位点，通过ChIP和转录活性检测等实验阐明Sp1对miR-205的转录调控机制，揭示辐照后miR-205表达上调的机制。最后，我们通过MiRanda、Pictar和Targetscan靶基因预测软件对miR-205靶基因预测并挑选出与凋亡及增殖相关基因TP53BP2和PTEN作为候选靶基因，拟进一步探讨miR-205调控放疗敏感性的分子机制，为促进食管癌放疗敏感性提供新策略。

食管癌是损害人类健康的主要恶性肿瘤之一，侵袭性高。我国是食管癌高发病区，每年的新发病人数占全世界的一半以上。食管癌患者预后差，5年生存率为5%-45%。放疗是治疗食管癌的重要手段之一，而放射抵抗是食管癌治疗失败的重要原因。最新研究发现，射线可以引起组织或细胞的microRNAs表达谱发生变化，诱发肿瘤放疗抵抗性的发生，但目前关于miRNA作用于肿瘤放疗抵抗的具体机制尚不透彻。前期研究发现miR-205在食管鳞状细胞癌中高表达，而它与食管癌放疗的关系国内外目前尚未有报道。因此，我们通过构建食管癌细胞的放疗抵抗株，对比miR-205在放疗抵抗株与亲本株中的表达水平，发现miR-205在三株放疗抵抗株中的表达水平均显著上调，提示miR-205参与了食管癌放疗抵抗性的调控。进一步研究发现，转录因子SP1诱导miR-205表达参与调控食管癌放疗抵抗；通过靶基因预测，我们锁定miR-205下游靶基因PTEN，发现miR-205通过抑制PTEN表达，激活Akt通路，进而诱导放疗抵抗的发生。为了进一步验证体外实验结果，我们通过裸鼠成瘤的体内实验进一步证实miR-205可促进食管癌放疗抵抗。本研究从体内、体外两个层面阐释了miR-205在食管癌放疗抵抗中的调控及作用机制，为预防食管癌放疗抵抗提供了新的作用靶点。