长链非编码RNA-MEG3作为ceRNA的调控网络在多发性骨髓瘤发生发展中的分子机制研究

Competing endogenous RNA (ceRNA) as a novel regulatory model provides us new insights on cross-talk between RNAs, both coding and non-coding, through miRNA recognition elements (MREs). Recent studies highlight the roles of long non-coding RNA (lncRNA), one kind of non-coding RNAs, function as ceRNA in human tumorigenesis. Maternally expressed gene3 (MEG3) is the first discovered lncRNA with anti-tumor effect. Previous studies have proved that the expression of MEG3 is down-regulated or absent in many varieties of tumor tissues, and it plays a role in anti-tumor effect mainly by the regulation of p53 signaling pathways. Our previous work showed that the expression level of MEG3 gradually decreased and the levels of miR-181a/b increased with the MM disease progression. We further found that the over-expression of MEG3 in ARP-1 MM cell line with absence of p53 mRNA could inhibit the MM cell proliferation and promoted the MM cell apoptosis, as well as inhibited the migration and the tube formation of human umbilical vein endothelial cells. Using the bioinformatics tool, we predict the 3'UTR of MEG3 mRNA has miR-181a/b binding sites. Therefore, we put forward the hypothesis that lncRNA-MEG3 function as ceRNA plays roles in MM pathogenesis, development, prognosis and angiogenesis by competitively combining with miR-181a/b to regulate the expression of targeted mRNAs. To verify the hypothesis, combined with lentivirus transfection, microarray assays, luciferase reporter gene assays and RNA immunoprecipitation, we will investigate it from several levels of molecular, cell line and clinical MM patients and expound ceRNA regulatory networks of MEG3-miR-181a/b-mRNA in MM. This project aims to elucidate the molecular mechanisms of MEG3 in the development and progression of multiple myeloma from the new viewpoint, which will provide theoretical and experimental foundation for the choice of treatment regimens and target therapy.

竞争性内源RNA（ceRNA）作为一种全新的调控机制，为我们揭示RNA之间的相互作用提供新的思路。lncRNA作为ceRNA在肿瘤发病中的作用已成为研究热点。我们前期工作表明随着MM疾病进展lncRNA-MEG3表达水平逐渐降低，miR-181a/b表达水平增高；且发现在p53mRNA缺失的ARP-1细胞株中过表达MEG3，可抑制MM细胞增殖，促进MM细胞凋亡。生物信息学软件预测MEG3的3’UTR区含有miR-181a/b结合位点，由此提出MEG3可作为ceRNA通过竞争性结合miR-181a/b调控靶向mRNA的表达在MM发生发展中发挥作用的假说。本项目拟采用荧光素酶报告系统、基因芯片及RNA免疫沉淀等方法从分子、细胞，体内和体外等多个层面对此假说进行论证，阐明MEG3作为ceRNA在MM中发挥作用的MEG3-miR-181a/b-mRNA调控网络，为寻找新的治疗靶点奠定理论基础。

lncRNA作为竞争性内源RNA（ceRNA）以一种全新的调控机制在肿瘤发病中发挥作用。本研究揭示了MEG3作为第一个被发现的具有抑癌作用的lncRNA在多发性骨髓瘤中的作用。我们通过cck-8，流式凋亡和周期检测技术证明了MEG3能够抑制骨髓瘤细胞增殖，促进凋亡。同时，5-Aza-CdR可以通过去甲基化作用上调MEG3的表达，抑制骨髓瘤细胞生长，阻滞细胞周期在G0/G1期。生物信息学软件预测MEG3的3’UTR区含有miR-181a/b结合位点，由此提出并证明MEG3可通过竞争性结合miR-181a/b调控靶向mRNA HOXA11的表达在MM发生发展中发挥抑癌功能。此外，我们通过CCK-8和流式凋亡技术证明MEG3能够通过抑制自噬途径促进硼替佐米的杀伤作用。综上，本项目从分子、细胞，体内和体外等多个层面进行论证，阐明MEG3作为抑癌分子在MM中发挥作用，为寻找新的治疗靶点奠定理论基础。