长链非编码RNA MALAT1作为食管鳞癌诊断标志物及其促进食管癌恶性增殖的机制研究

Previous studies showed that lncRNA MALAT1 acts as an oncogene in esophageal squamous cell carcinoma (ESCC) by promoting tumor proliferation and metastasis. Knockdown of MALAT1 activated the ATM-CHK2 pathway which responds to DNA injury signals, indicating the involvement of MALAT1 in maintenance of chromosome integrality, or in metabolism of DNA-toxic substances. Following screening of target mRNAs by microarray chip combined with bioinformatics analysis suggested that MALAT1 mainly participates in cell metabolism pathway, and GLO1 is the most possible target gene of MALAT1. Product of GLO1 participates in degradation of methylglyoxal (MG), which is a by-product during glycolysis of tumor cell, and methylglyoxal may bind with chromosome to form advanced glycation endproducts (AGEs) that are highly toxic to DNA and may inhibit tumor growth. Degradation of methylglyoxal by GLO1 promises rapid proliferation of tumor cells. Further miRNA microarray screening combined with ceRNA network analysis found common miR-382 binding site shared by MALAT1 and 3'-UTR of GLO1, indicating that MALAT1 may play as ceRNA to regulate GLO1 expression. Moreover, we found that MALAT1 may be secreted by ESCC cells and transferred by exosomes. Expression level of serum MALAT1 in pre-operative ESCC patients is obviously higher than in post-operative patients and normal controls, suggesting that the circulating MALAT1 level may be used as a potential non-invasive diagnostic biomarker for ESCC.

前期研究表明，MALAT1在食管鳞癌中具有促进肿瘤生长及迁移侵袭的作用，其表达干扰可激活DNA损伤应答通路，表明MALAT1可能参与染色质稳定性维持或核酸毒性物质的代谢。进一步的mRNA芯片筛查联合生物信息学分析结果表明，MALAT1主要参与细胞代谢信号通路，甲基乙二醛代谢基因GLO1的表达受其调控最为明显。甲基乙二醛是肿瘤细胞糖酵解代谢的产物，可与染色质结合形成晚期糖基化终末产物，产生强大的细胞毒性，抑制肿瘤生长。GLO1对甲基乙二醛的降解作用保证了肿瘤细胞的快速生长。miRNA芯片筛查联合ceRNA网络分析结果表明，MALAT1与GLO1共享miRNA-382结合位点，可能作为ceRNA调控GLO1的表达。此外，我们还发现MALAT1可经食管癌细胞分泌并经外泌体跨细胞转运及进入血液循环，食管癌患者术前血清中MALAT1表达水平明显高于术后及健康对照,可能作为食管癌无创诊断标志物。

长链非编码RNA在肿瘤中的作用与机制日益受到关注，在前期的研究中，我们发现，lncRNA MALAT1可以促进食管鳞癌的生长增殖及迁移侵袭，并发现MALAT1促癌作用可能与其调控食管鳞癌的DNA损伤修复通路ATM-CHK2相关。在本研究中，我们进而采用表达谱芯片筛查技术对MALAT1所调控的靶基因进行了筛查，结果发现乙二醇酶GLO1受MALAT1的调控最为明显。甲基乙二醇是肿瘤酵解代谢的中间产物，可以产生强大的细胞毒性导致细胞的生长抑制，在肿瘤细胞中，GLO1的高表达可以促进甲基乙二醛的降解，保证肿瘤细胞的快速生长。我们的研究表明，GLO1在食管鳞癌中起到促癌生长的作用，且MALAT1通过GLO1发挥其促癌生长的作用。GLO1的表达抑制可以导致细胞终末糖基化产物AGEs的蓄积，进而导致DNA损伤，激活DNA损伤修复通路，抑制食管鳞癌细胞生长。对GLO1的基因区进行生物信息学分析发现其启动子区存在大量的组蛋白修饰信号，表明其可能受到组蛋白修饰的调控，在对MALAT1的靶基因进行深入分析时我们发现，组蛋白乙酰转移酶KAT2B受到MALAT1的调控亦十分明显，对KAT2B的表达干扰可以导致GLO1基因区的组蛋白乙酰化修饰信号减弱，说明KAT2B可能对GLO1的组蛋白修饰进行调控。进而我们通过miRNA芯片筛查联合生物信息学分析，预测了MALAT1与KAT2B的共结合miRNA mi-206，荧光素酶报告基因实验证实了miR-206对MALAT1与GLO1的降解作用，进而说明MALAT1可能通过ceRNA机制调控KAT2B的表达，而KAT2B对GLO1的组蛋白乙酰化修饰则进一步对GLO1的表达水平进行调控。此外，我们的研究还表明MALAT1可经食管癌细胞分泌的外泌体进行转运，从而导致食管鳞癌恶性特征的扩散。食管鳞癌分泌的外泌体可以进入血液循环，使得血液中的MALAT1表达水平有望成为食管鳞癌的无创诊断标志物。本研究的开展为解释食管鳞癌的发病机制及寻找食管鳞癌的治疗方案提供了新的策略，并为食管鳞癌的无创筛查提供了潜在靶点。