长链非编码RNA AC006050.3-003调控肺鳞癌顺铂化疗敏感性及其机制研究

Cisplatin is currently used as the backbone of combination chemotherapy strategies in the management of lung squamous cell carcinoma (SCC) patients. However, the effectiveness of chemotherapy is often hindered by the emerging drug resistance. As demonstrated in recent studies, lncRNAs actively participate in the generation of chemoresistance. A new lncRNA AC006050.3-003, identified in our previous study, was overexpression in the resistant tumor tissues of lung SCC patients with cisplatin-based chemotherapy compared with sensitive tumor tissues. RNA interference against the lncRNA AC006050.3-003 was able to promote cisplatin induced apoptosis and enhance the cisplatin sensitivity in lung cancer SCC cells. Several recent studies report that lncRNA may function as competing endogenous RNA in modulating the concentration and biological functions of miRNAs. Bioinformatics analysis and preliminary experiments indicated that lncRNA AC006050.3-003 could bind miR-338-3P and miR-138, and that HIF-1α was the target gene of miR-338-3P and miR-138. In addition, previous studies reported that HIF-1α was involved in cisplatin resistance by anti-apoptotic mechanism. The current project will explore whether lncRNA AC006050.3-003 could modulate the sensitivity of lung SCC cells to cisplatin via miR-338-3P/miR-138-HIF-1α pathway by in vitro and in vivo experiments. Moreover, the value of AC006050.3-003 as a biomarker to predict treatment response of lung SCC patients with cisplatin-based chemotherapy will be assessed by detecting the expression of AC006050.3-003 in tumor tissues and plasma of lung SCC patients. This project will provide more information and details of cisplatin resistance of lung SCC.

顺铂是肺鳞癌化疗的基石，但耐药导致化疗失败。长链非编码RNA（lncRNA）参与肿瘤化疗耐药。我们前期研究发现lncRNA AC006050.3-003在肺鳞癌顺铂耐药组织表达增高，下调其表达可促进顺铂诱导的细胞凋亡，提高顺铂敏感性。lncRNA可通过竞争性内源RNA机制与miRNA结合，上调miRNA靶基因表达。生物信息学分析及预实验证实AC006050.3-003可与miR-338-3P、miR-138结合，HIF-1α是miR-338-3P和miR-138的共同靶基因。文献报道HIF-1α可通过抗凋亡机制参与顺铂耐药。本项目将从细胞和动物水平研究AC006050.3-003是否通过miR-338-3P/miR-138-HIF-1α通路介导抗凋亡导致肺鳞癌顺铂耐药。通过检测肺鳞癌肿瘤组织和血浆AC006050.3-003的表达，评价其预测顺铂耐药的价值。本研究将完善肺鳞癌顺铂耐药机制。

我们前期研究发现KRT17P3可能参与调控肺癌顺铂敏感性，但其分子机制尚不清楚。根据AC006050.3-003的初始基因号（Gene Symbol ENSG00000231870），HGNC（HUGO Gene Nomenclature Committee）已将ENSG00000231870命名为KRT17P3，其转录子为KRT17P3-001（ENST00000420566），故本报告将AC006050.3-003统一称为KRT17P3。lncRNA可通过竞争性内源RNA机制与miRNA结合，上调miRNA靶基因表达。本研究旨在进一步证实KRT17P3对非小细胞肺癌顺铂耐药的调节作用，并探索其可能的分子机制。结果显示在非小细胞肺癌A549和 SK-MES-1细胞系中高表达KRT17P3导致细胞对顺铂耐药，低表达KRT17P3导致细胞对顺铂敏感性升高。体外实验证实高表达KRT17P3的A549细胞移植瘤对顺铂耐药。生物信息学分析提示KRT17P3可能与miR-497-5P结合，mTOR是miR-497-5P的靶基因。回复实验证实miR-497-5P可逆转KRT17P3导致的细胞对顺铂敏感性改变。荧光素酶报告基因实验、MS2-RIP、anti-Ago2 RIP实验证实KRT17P3与miR-497-5P结合，Western blot实验证实KRT17P3通过miRNA-497-5p调控mTOR蛋白的表达。本课题通过体外实验和体内实验证实KRT17P3可以调控NSCLC细胞顺铂敏感性，KRT17P3通过miR-497-5p/mTOR通路调控NSCLC顺铂敏感性。本课题还初步证实了KRT17P3在顺铂耐药患者外周血中表达升高，且其高表达与耐药相关。本课题丰富了NSCLC顺铂耐药机制，为逆转NSCLC顺铂耐药提供分子靶标。同时对寻找新的NSCLC顺铂耐药相关标记物，指导临床用药具有重要意义。