血浆miR-362-5p促进非小细胞肺癌复发转移的机制研究及临床意义

Non-small cell lung cancer (NSCLC) is one of common malignant tumor in China. Recurrence and metastasis is an important reason to poor prognosis. Find a sensitive and specific biomarkers and elucidate the molecular mechanism for the early detection of NSCLC recurrence and metastasis are urgently needed to reduce the high morbidity and mortality of the disease. Our previously study showed that some plasma miRNAs differential expressed in NSCLC with recurrence compared with patients without recurrence by Illumnia RNA sequencing. MiR-362-5p, one of deregulated miRNA, is correlated with recurrence in NSCLC patients, who received sequential adjuvant chemotherapy after surgical resection, and may play an important role in promoting NSCLC recurrence and metastasis, but the molecular mechanism is not clear. To determine whether the expression profile of plasma miRNAs can serve as a NSCLC fingerprint and the function and molecular mechanism of interesting differential miRNA (miR-362-5p) in NSCLC metastasis, we performed quantitative RT-PCR assay to selected differentially expressed plasma miRNAs from a sample set including 200 NSCLC cases with or without recurrence, and risk score analysis to evaluate the diagnostic value of the plasma miRNA profiling system. We utilize the knockouts for miR-362 in NSCLC cells to explore the role of plasma miRNAs in cross talk of tumor cells and environment, elaborate the function and molecular mechanism of miR-362-5p in promoting NSCLC metastasis and reveal the mechanisms of epigenetic regulating the expression of miR-362-5p in NSCLC by multi-methods in cell biology, molecular biology and bio information.

复发转移是非小细胞肺癌临床治疗效果不佳的原因之一。本课题组前期利用二代测序筛选出与NSCLC术后复发转移相关的血浆miRNAs；现有结果提示血浆miR-362-5p表达与非小细胞肺癌术后复发转移密切相关，并可促进肺癌细胞的迁移运动。以上结果提示：血浆miRNAs分子可作为非小细胞肺癌术后复发转移的评价指标，miR-362-5p可能在肿瘤复发转移中发挥重要作用，但其分子机制并不清楚。本项目拟以此为基础，以miR-362基因敲除细胞为工具，综合运用细胞生物学、分子生物学、生物信息学等研究手段，探讨以下问题：①建立NSCLC复发转移相关的术前血浆miRNAs评估体系；②探讨NSCLC患者血浆中miRNAs的产生来源和作用靶细胞问题；③miR-362-5p在NSCLC复发转移过程中发挥的功能及分子作用机制；④miR-362-5p上调的分子机制，为临床复发转移风险评估及抗复发转移治疗提供理论依据。

非小细胞肺癌是中国常见的恶性肿瘤之一，复发转移是非小细胞肺癌临床治疗效果不佳的主要原因之一。本课题组利用二代测序筛选出与NSCLC术后复发转移相关的血浆miRNAs，其中miR-362-5p高表达预示非小细胞肺癌术后复发转移的可能性高，且对多西他塞和顺铂药物敏感性不高。为明确该分子的作用机制，课题组首先利用CRISPr技术敲除A549和95-D细胞中的miR-362-5p。研究结果表明miR-362-5p可通过下调细胞中SEMA-3a的表达水平，促进了细胞骨架重排、侵袭转移、药物耐药等功能，而过表达SEMA-3a可抑制上述改变，表明SEMA-3a是miR-362-5p的作用靶点。另外，课题组还通过分子生物学等方法，探讨miR-362-5p在非小细胞肺癌细胞中高表达的机制。以上结果为临床复发转移风险评估及抗复发转移治疗提供理论依据。