5-Aza-dC去甲基化调控microRNA-320a/E2F-1轴逆转非小细胞肺癌顺铂耐药的机制研究

Cisplatin is a common chemotherapy drug for non-small cell lung cancer（NSCLC）, which has drawn public attention to improve its curative effect. It is widely proved that microRNA is closely related to drug resistance in tumor and that DNA methylation is one of the important resistance mechanisms. E2F-1 could lead to cisplatin resistance through NF-κB and MRN/TAM signal pathway. We previously reported that nontoxic dose of demethylation agent 5-Aza-dC (5-aza-2'-deoxycytidine) could reverse the cisplatin-resistance in NSCLC A549/DDP cells. However, the mechanisms are still not identified. In our preliminary experiments, microRNA profiling analyses were performed to determine miRNAs expression levels. MiR-320a hypermethylation were detected in cisplatin-resistant A549/DDP cells and tumor tissues. Besides, miR-320a expression significantly increased in 5-Aza-dC processed A549/DDP cells. Further bioinformatic prediction suggested E2F-1 as a possible downstream target gene. We therefore hypothesize that 5-Aza-dC could reverse cisplatin resistance in NSCLC by modulating the methylation of microRNA-320a and affecting the downstream target gene E2F-1, which consequently impact the NF-κB and MRN/TAM signal pathway. In order to clarify our hypothesis, molecular, cellular, animal models and tumor tissues of patients would be utilized to explore the function and mechanism of miR-320a in the 5-Aza-dC mediated reversal of cisplatin-resistance in NSCLC. Accordingly, we aim to provide theoretical and experimental evidences for improving the chemotherapy effect in NSCLC.

顺铂是非小细胞肺癌（NSCLC）化疗常用药物，提高其疗效是临床亟待解决的难题。研究表明microRNA表达与肿瘤耐药密切相关，DNA甲基化是重要调控机制之一，E2F-1可作用于参与顺铂耐药的NF-κB及MRN/TAM通路。我们已报道，小剂量去甲基化药物5-Aza-dC可逆转人肺癌耐药细胞株A549/DDP顺铂耐药；预实验发现，miR-320a在A549/DDP细胞及顺铂耐药肿瘤组织中表现为高甲基化；5-Aza-dC处理后的A549/DDP细胞中其表达上调；预测E2F-1是其下游靶基因。基于此，我们认为：5-Aza-dC去甲基化调控miR-320a/E2F-1轴，作用于NF-κB及MRN/TAM通路，逆转NSCLC对顺铂的耐药性。本项目拟通过细胞和动物模型，运用分子生物学方法，探究miR-320a在5-Aza-dC逆转NSCLC顺铂耐药中的调控及作用机制，为提高NSCLC化疗疗效提供新思路。

顺铂是治疗非小细胞肺癌(NSCLC)的常用化疗药物，其耐药性制约了临床疗效，是临床亟待解决的问题。microRNA与化疗耐药密切相关，DNA甲基化是化疗耐药的重要机制之一。我们已报道，小剂量去甲基化药物5-Aza-dC（去甲基化）可以逆转顺铂耐药的人肺癌细胞株A549/DDP对顺铂耐药性。因此，我们提出：在NSCLC中，microRNA的DNA位点甲基化可导致microRNA下调，并介导了顺铂耐药。在已有的研究基础上，我们用5-Aza-dC处理A549/DDP得到逆转顺铂耐药的A549/DDP。然后，通过Microarry分析进一步筛选差异表达的microRNA。我们的结果显示：5-Aza-dC处理后的A549/DDP细胞之miR-320a较非处理组表达上调，Real-Time-PCR结果也显示5-Aza-dC处理后后A549/DDP细胞miR-320a表达上调。我们进一步利用GEO和TCGA数据库数据验证miR-320a在肺腺癌中的表达，以及miR-320a与肺腺癌预后的关系。本研究为NSCLC顺铂耐药机制提供了新的见解。