血清miRNA预测食管癌放化疗疗效及其作用机制的探索性研究

Concurrent radiochemotherapy is the main treatment for locally advanced esophageal squamous cell cancer (ESCC), but at expense of severe toxicities. The overall treatment outcome is still poor since some tumors are resistant to radiochemotherapy. Early prediction of the efficacy of radiochemotherapy can identify patients with resistant tumors to receive other optimal therapy, thus avoid the toxicity and damage from concurrent radiochemotherapy. Serum miRNAs, steadily expressed and easily to test, have recently been proved to be closely related with tumor prognosis. So theoretically, serum miRNAs can be the ideal markers in predicting the efficacy of radiochemotherapy. Our previous study divided patients with locally advanced ESCC into radiochemotherapy sensitive and resistant groups according to the treatment outcomes. Many differentially expressed serum miRNAs between the two groups have been detected by ABI TaqMan miRNA chip method, among which some were significantly associated with treatment efficacy. In the following study in the expanding sample, we plan to use qRT-PCR method to further choose the serum miRNAs closely relating to radiochemo-sensitivity. By using Cox proportional hazard model, a comprehensive model including the serum miRNAs and the prognosis related clinical factors will be established to predict the sensitivity of radiochemotherapy. And then the prediciton model will be validated in testing cohort. In the study of realted mechanisms, target genes of the selected miRNAs will be determined by bioinformatics software, luciferase reporter gene system, and chromatin method. By the further research in the DNA damage, cell cycle and apoptosis, the molecular mechanism of how the miRNAs regulate radio-sensitivity though their target genes will be explored. This study, focusing on the clinical problems of radiochemotherapy for ESCC, can improve individualized therapy, and provide theoretical basis to explore the new treatment method, which has prominent scientific and potential clinical values.

放（化）疗是局部晚期食管癌的主要治疗手段，但总体疗效欠佳且毒副作用大。因此早期预测其疗效能促进食管癌个体化治疗。血清miRNA稳定性好且易于检测，与肿瘤预后密切相关，有望成为预测放化疗疗效的理想指标。我们前期的miRNA芯片研究显示食管癌放（化）疗敏感和抗拒组患者血清miRNA的表达差异显著，其中部分miRNA与放化疗疗效密切相关。本课题拟扩大样本量，利用qRT-PCR确定与疗效相关的血清miRNA，然后联合临床因素根据Cox比例风险模型建立食管癌放（化）疗的疗效预测模型，并进行独立验证；其次将利用生物信息软件、荧光素酶报告基因系统及染色质等方法确定miRNA的靶基因，并深入研究在DNA损伤反应中,miRNA通过其靶基因影响放疗敏感性的分子机制。本课题能够为探索新的治疗方法提供理论基础，有较好的科学性和潜在的临床价值。

背景：放化疗是局部晚期食管癌的主要治疗手段，但总体疗效欠佳且毒副作用大。因此早期预测其疗效能促进食管癌个体化治疗。血清miRNAs稳定性好且易于检测，与肿瘤预后密切相关，有望成为预测放化疗疗效的理想指标。.材料与方法：建立放射抵抗细胞系进差异表达miRNAs的筛选。利用原位杂交及qRT-PCR测定miRNAs在食管鳞癌组织、血清及细胞中的表达情况。通过MTS法研究miRNAs对食管鳞癌细胞增殖能力的影响，通过Transwell法研究miRNAs对食管鳞癌细胞迁移和侵袭能力的影响。从血清及体外培养基分离外泌体测定miR-339的表达并进行靶基因的相关研究。.结果：在食管鳞癌中，miR-323a-3p高表达与预后好相关(P=0.004)。在大多数食管鳞癌细胞中，相对食管正常上皮细胞，miR-323a-3p呈现低表达状态。与对照组细胞相比，miR-323a-3p高表达能够抑制食管鳞癌细胞的增殖、迁移和侵袭能力。MiR-339及miR-181低表达与放射抵抗相关。体内及体外实验结果表明，miR-181高表达抑制食管鳞癌的侵袭，促进DNA损伤所诱导的细胞凋亡。在T分期较晚的食管鳞癌患者中，miR-339表达下调(P=0.04)。肿瘤组织或患者血清中miR-339低表达与总生存差显著相关(P=0.036，P=0.012)，与DFS差亦相关(P=0.037)。此外，在TCGA数据库中，组织内miR-339下调与生存差相关（P=0.037）。MiR-339以外泌体形式分泌至血清中，可以预测局部晚期食管鳞癌术前放疗的病理反应。MiR-339通过下调CDC25A从而增强DNA损伤所诱导的细胞凋亡。.意义：外泌体miR-339与放射敏感性相关，高表达miR-339促进放射敏感性。MiR-339可以预测术前放疗后的病理反应情况。MiR-339有望成为指导个体化治疗的非侵入性检查指标。