miR-381调控食管鳞癌放疗敏感性的分子机制研究

Radiotherapy failure or local recurrence is associated with radioresistance.Enhanced survival ability of tumor cell (enhanced proliferation, anti-apoptosis, abnormal cell cycle) plays a key role in radioresistance.As a critical factor for gene-regulation, the role of miRNAs in radioresistance-regulation of esophageal squamous cell carcinoma(ESCC)is becoming a research focus. Our prophase study found that miR-381 was the most significantly downregulated gene in radioresistant ESCC cells in prophase.CTNNB1/LEF1、CDK1、XIAP and CXCR4, as potential targets of miR-381, are key regulators of cell survival ability. So, miR-381 may play an important role in the regulation of ESCC radiosensitivity.This project intends to do as follows:①Using a variety of ESCC cells and nude mouse-human tumor model to identify the role of miR-381 in radioresistance;②By Reporter gene, regulation of miRNA expression, siRNA, RT-PCR and Western blot, we will investigate the association among miR-381, potential targets and survival pathway, and reveal a new mechanism of ESCC radioresistance;③To uncover the predictive significance of miR-381 in radiation response from clinical specimens. Implementation of the program will help to elucidate the molecular mechanism of ESCC radioresistance and support a new way for target therapy in ESCC.

肿瘤细胞生存能力（增殖、周期及抗凋亡）的增强赋予肿瘤放疗抵抗从而导致放疗失败或局部复发。miRNAs作为调控基因表达的重要分子，在食管鳞癌放疗抵抗调控方面是新的研究热点。本项目组前期发现放疗抵抗食管鳞癌细胞中miR-381表达下调最显著,生物信息学技术筛选示miR-381有5个潜在靶点（CTNNB1、LEF1、CDK1、XIAP和CXCR4）是细胞生存途径的关键基因，提示其可能在调控食管鳞癌放疗抵抗中发挥关键作用。基于上述设想，本项目拟：①利用体内外实验明确miR-381表达水平对食管鳞癌放疗敏感性的影响；②采用分子生物学相关技术验证miR-381与5个靶点的调控关系、明确放疗抵抗的调控机制；③收集临床标本，了解miR-381及5个靶点表达水平对放疗反应的临床预测意义。本项目的实施有助于明确食管鳞癌放疗抵抗的形成机制，为食管鳞癌提供新的靶向治疗策略。

背景：前期研究发现miR-381表达量随放疗抵抗性增加而下降，但miR-381对食管鳞癌放疗的调控作用及机制未明。肿瘤细胞的增殖及抗凋亡能力增强会促进放疗抵抗，而生物信息学显示miR-381的潜在靶基因与增殖、凋亡通路相关。本研究探讨了miR-381对食管鳞癌放疗敏感性的调控作用及机制。.方法：应用RT-PCR检测不同放疗敏感性食管鳞癌细胞株及食管鳞癌组织中miR-381的表达情况。调控miR-381表达情况，利用MTT、Transwell小室、克隆形成实验检测miR-381表达改变对食管鳞癌细胞增殖、凋亡及放疗敏感性的调控作用，并在动物模型中验证其对放疗敏感性的影响。双荧光报告基因分析miR-381对靶基因XIAP的表达调控，分析食管鳞癌组织标本中miR-381与XIAP的蛋白表达关系。应用shRNA技术沉默XIAP表达，比较其与miR-381对食管鳞癌细胞增殖、凋亡及放疗敏感性的调控作用。.结果：miR-381在食管癌组织及细胞中表达下调，且在不同食管鳞癌细胞株中随放疗敏感性下降，miR-381表达下调。上调miR-381表达降低KYSE450 /150的增殖活性，增加凋亡及放疗敏感性；下调miR-381表达增加TE1/ECA109的增殖活性，减少凋亡及放疗敏感性。体内实验也证实上调miR-381表达增加肿瘤的放疗敏感性。miR-381与XIAP在食管鳞癌组织中表达负相关，报告基因证实miR-381可结合XIAP的3’-非编码区域。沉默XIAP表达与上调miR-381表达均可抑制细胞增殖、活化凋亡途径，从而增加放疗敏感性。.结论：miR-381通过靶向XIAP活化凋亡通路在食管鳞癌中发挥放疗敏感性的调控作用。