血小板外泌体来源的长非编码RNA Linc01021调控肺腺癌转移的机制研究

Patients with distant metastases of lung adenocarcinoma usually have poor prognosis. The number of platelet counts in peripheral blood of those patients with metastasis is significantly higher than that in those without metastasis, suggesting that abnormal platelet count and platelets dysfunction are closely related to lung cancer metastasis. In this study, we found that long non-coding RNA linc01021 of platelet was significantly increased in patients of lung adenocarcinoma with metastasis by single cell RNA-sequencing，and linc01021 in platelets exosomes（P-EXOs）of those patients was also significantly increased. Fluorescence tracking technique showed that P-EXOs could be transported from platelets to the lung cancer cell microenvironment, and then uptaked by lung cancer. Interference or overexpression of linc01021 in cancer cells, the invasion and metastasis of lung cancer were significantly affected. Meanwhile bioinformatics analysis suggested that linc01021 could bind EZH2 to control the expression of FATs. So the hypothesis is that platelets, as vectors, transport linc01021 to lung cancer microenviroment via P-EXOs, increasing the invasion of lung cancer cells and promoting the metastasis of lung adenocarcinoma. This project is intended to use the next generation of sequencing, RIP, ChIP and other technologies to reveal the above hypothesis, through in vitro and in vivo experiments as well as clinical analysis to verify the our hypothesis,in order to uncover new mechanism of lung adenocarcinoma metastasis.

肺腺癌发生远处转移的患者预后不良,且常见其外周血中血小板计数显著高于未转移者，提示血小板数目异常及其功能失调与肺癌转移密切相关。本课题组通过单细胞测序技术发现肺腺癌转移患者血小板长非编码RNAlinc01021明显升高，而血小板外泌体（P-EXOs）linc01021也显著升高，荧光跟踪技术显示P-EXOs可被血小板运输至肺癌细胞微环境中，并被肺癌细胞吞入细胞内。干扰或过表达linc01021后，肺癌的侵袭和转移能力受到明显影响。同时生物信息学分析提示linc01021可绑定EZH2调控FATs的表达。据此推测血小板作为载体，通过P-EXOs将高表达的linc01021运输到肺癌细胞微环境中，增加了肺癌细胞的侵袭能力，从而促进肺腺癌的转移。本项目拟运用二代测序、RIP、ChIP等技术揭示上述假设，通过体内外实验和临床样本分析加以验证，以期获得肺腺癌患者发生转移的新机制。

肺腺癌发生远处转移的患者预后不良,且常见其外周血中血小板计数显著高于未转移者，提示血小板数目异常及其功能失调与肺癌转移密切相关。本课题组通过单细胞测序技术发现肺腺癌转移患者血小板长非编码RNAlinc01021明显升高，而血小板外泌体（P-EXOs）linc01021也显著升高，荧光跟踪技术显示P-EXOs可被血小板运输至肺癌细胞微环境中，并被肺癌细胞吞入细胞内。干扰或过表达linc01021后，肺癌的侵袭和转移能力受到明显影响。同时生物信息学分析提示linc01021可绑定EZH2调控FATs的表达。以上结果提示血小板作为载体，通过P-EXOs将高表达的linc01021运输到肺癌细胞微环境中，增加了肺癌细胞的侵袭能力，从而促进肺腺癌的转移。此外我们对7名健康个体和15名非小细胞肺癌(NSCLC)患者的血小板进行了RNA测序并完成分析。我们的数据显示，与健康个体相比，转移性非小细胞肺癌的血小板中存在一个近乎普遍的差异表达基因(DEG)谱，包括626个上调的RNA (mRNA和ncRNAs)和1497个下调的基因。显著的过表达基因在局灶性黏附、血小板活化、缝隙连接和粘附连接通路中富集。DEGs中还包括了既往报道的肿瘤相关基因如PDGFR、VEGF、EGF等，验证了血小板RNA-Seq在肿瘤学研究中的一致性和意义。我们还验证了一些上调的DEGs参与了肿瘤细胞诱导的血小板聚集(TCIPA)和肿瘤的形成。对肺腺癌和肺鳞状细胞癌的亚组分析结果表明，血小板RNA-Seq在鉴别肿瘤组织学类型方面具有潜在的诊断价值。..另外我们还探究了WNT5B在肺腺癌进展中的致癌作用及其受miR-5587-3p的调控作用。从临床标本和细胞实验阐明WNT5B在肺腺癌（LAD）组织存在高表达，体内、体外外实验揭示WNT5B基因致癌作用；通过基因转录组测序筛选出WNT5B可能影响的下游通路，通过数据库预测WNT5B潜在上游miRNA并通过临床LAD标本筛选出与WNT5B表达显著相关的miR-5587-3p，通过体外实验证实miR-5587-3p-WNT5B-LAT1信号轴存在，靶向该信号轴具有潜在抗肿瘤效应。