miR-221通过靶向PTEN调节胃癌放射敏感性的研究

Gastric adenocarcinoma is one of the most common cancer in China.Radiation therapy is one important method for both adjuvant treatment and palliative treatment, however because of the diversity of gastric cancer, not all of the gastric cancer patients can benefit from radiotherapy. Therefore, further understanding molecular mechanisms of radiation sensitivity and identifying radiation sensitivity involved markers are of great clinical value. Recently, many miRNAs were found to be differentially expressed in different type of gastric cancers, and could represent the alteration in biological characteristics,miRNAs can regulate radiosensitivity involved gene, then influence the radiation sensitivity. Therefore, exploring the miRNA expression profiling of radiation sensitive and radiation resisitance gastric cancer and the role of differentially expressed miRNAs in gastric cancer would provide new insight into the molecular mechanisms of individulised radiation therapy of gastric cancer.In our study, we profiled gastric cancer tissue samples of different radiation sensitivity by miRNA microarray , then screened out the miRNAs aberrantly expressed in radiation sensitive and radiation ressistance gastric cancer miR-221. We have also confirmed that the target gene of miR-221 is PTEN through biological information analysis. Interestingly, PTEN can regulate the pathway of PI3K/mTOR which is involed of radiosensitivity. In our study, the role and its mechanism of miR-221 in the radiation sensitivity of gastic cancer was examined to obtain more evidence whether miR-221 could serve as an new biomarker for radiation therapy choice of gastric cancer.

胃癌是我国最常见的恶性肿瘤之一。放疗不论做为姑息治疗或辅助治疗都是一种重要的手段，然而由于胃癌的多样性，不是所有的胃癌患者都能从放射治疗中获益。因此探索胃癌放射敏感分子机制仍是胃癌研究的重要内容。近年来研究发现，不同的胃癌组织类型中存在miRNA差异表达，可能代表胃癌生物特性的不同，而这些差异表达的miRNA又调控者放射敏感相关的基因和信号通路，从而进一步影响胃癌放射敏感性的差异。因此分析不同放射敏感性胃癌的miRNA表达谱，探讨差异表达miRNA影响胃癌放射敏感性的机制，将为胃癌放射治疗的选择提供新的思路和理论依据。本研究通过前期miRNA芯片分析不同放射敏感胃癌miRNA表达谱，筛选差异表达miRNA为miR-221，且通过生物信息学方法获得其靶基因为PTEN，而PTEN对于放射敏感相关的信号通路PI3K/mTOR有一定的调控作用，本研究拟探讨miR-221对胃癌放射敏感性的影响机制。

II期临床实验被2014年ASTRO收录为POSTER。患者分为2组，每组4例治疗前标本，MicroRNA芯片分析和基因信息学分析。.首先，miR-221，显示miR221在放射抵抗的胃癌组织样本和细胞中高表达。假设miR221过表达能引起胃癌细胞的放射抵抗。分析其机制及启动的相关信号通路。在MGC-803细胞中成功建立miR221过表达稳定细胞株，miR221过表达后，PTEN的表达受到抑制，进一步证明了PTEN是miR221的靶基因。miR221具有促进MGC-803生长的功能，但对细胞周期影响甚小； miR221过表达后，细胞迁移能力明显下降，明显抑制细胞凋亡，MGC-803/ miR221形成的克隆数要多于对照组细胞。确定miR221过表达后能引起MGC-803的放射抵抗，PTEN作为其靶基因被下调后发挥了部分作用，但不参与到miR221对细胞行为改变的全过程，而我们的结果显示miR221对细胞周期改变很小，MGC803/ miR221的凋亡明显受到抑制。因此miR221通过作用不同的靶基因而共同影响肿瘤细胞的生物学行为。miR221过表达后细胞周期在G2/M期受到明显阻滞，而DNA损伤标记H2AX在MGC-803/ miR221中出现的高峰期也要早于对照细胞，随后由于细胞修复开始而开始降低，修复要早于对照细胞，但两者修复速度接近。.其次 miR-197，miR-197在放射抵抗的患者肿瘤组织中高表达，与放射抵抗相关。LQ存活曲线表明细胞修复增多，计算的α值减小，也表明细胞放射敏感性降低。miR-197过表达组，S期细胞增多，由于DNA合成复制，为细胞同源重组修复提供模板，增加了细胞对放射性的抵抗。ATR通路明显活化，P-CHK1增强，parp表达增强，细胞DNA修复增强。细胞增殖实验显示miR-197能够促进细胞生长。过表达miR-197后细胞成瘤能力明显增强。.通过miRNA数据库targetscan和miRDB比对分析，发现PTPN9是miR-197的潜在靶基因。构建PmiGLO-PTPN9-3‘-UTR luciferase报告载体，分析luciferase活性变化，证实了PTPN9是miR-197的靶基因，转入miR-197mimics和抑制剂分别下调和增强luciferase活性。.miR-197能够促进肿瘤的生长，抑制细胞凋亡，增强对射线的抵抗