MMSA-1基因调控多发性骨髓瘤细胞增殖、凋亡及迁移的机制研究

Wnt/β-catenin/TCF4 signaling pathway and RAS participate in multiple myeloma (MM) oncogenesis. As a key member in regulating RAS activation, whether ZDHHC9 overexpressed in MM and the underlying pathogenic mechanisms in MM remains unclear. Previously we identified a novel splice variant of ZDHHC9 named MMSA-1, and have proved its over-expression both in myeloma cells lines and primary myeloma cells. Furthermore, we found a close correlation between MMSA-1 expression level and myeloma cells’proliferation, apoptosis and migration, as well as disease progression. Moreover, bioinformatic technology showed that there may be TCF4 binding sites in MMSA-1’s promotor region. Therefore we hypothesized that hyperactived Wnt signaling pathway in MM made TCF4 consistently binding to MMSA-1 promotor and induced MMSA-1 over expression, followed by RAS and the downstream signaling pathways hyperactivation and bone marrow microenvironment changes, which ultimately resulted in MM cells proliferation and migration. Herein, we aim to explore the regulation mechanism of Wnt/β-catenin/TCF4 on MMSA-1 expression; after that, RAS localization, palmitoylation level as well as activity of downstream signaling pathways will be evaluated, and bone marrow microenvironment related characteristics will also be examined. By doing so, we hope to elucidate the underlying mechanisms of MMSA-1 gene in the pathogenesis of MM and at the same time pave way for MMSA-1 targeted gene therapy of MM in the future.

Wnt/β-catenin/TCF4通路及RAS与多发性骨髓瘤（MM）关系密切，而ZDHHC9作为调节RAS活性的关键，其在MM中的表达情况及可能的致病机制仍不明确。我们近期发现MMSA-1作为ZDHHC9的新型剪接变体，在MM细胞中高表达，并与细胞增殖、凋亡、迁移及疾病进展密切相关，且生物信息学方法预测其启动子区可能存在TCF4的结合位点。因此我们假设：在MM中Wnt通路活化导致TCF4持续激活MMSA-1，进而活化RAS及下游通路，同时通过影响骨髓微环境促进MM细胞迁移。本项目拟研究Wnt/β-catenin/TCF4对MMSA-1的表达调控作用，随后通过检测RAS定位、棕榈酰化水平、下游通路活性及骨髓微环境相关指标，探索MMSA-1在调控MM细胞增殖、凋亡、迁移中的具体机制。本研究有望阐明MMSA-1基因激活及参与MM发病的机理，为未来将MMSA-1作为MM的基因治疗靶点提供依据。

本项目探索了多发性骨髓瘤相关新抗原基因MMSA-1调控多发性骨髓瘤细胞增殖、凋亡及迁移的具体分子机制。本项目内容分为以下两部分，第一部分，通过荧光素酶报告实验等探索Wnt/β-catenin/TCF4 信号通路对 MMSA-1基因表达的调控作用；随后构建MMSA-1基因干扰/过表达骨髓瘤U266细胞系，探索MMSA-1基因表达水平对RAS蛋白活性的调节及其下游信号通路的活化作用以及对MM细胞凋亡（线粒体凋亡）、迁移能力、克隆形成能力的影响；最后，通过观察 MMSA-1 表达水平变化后MM细胞的血管生成因子、低氧诱导因子及粘附分子的表达，明确其对MM细胞及骨髓微环境之间相互作用的影响。我们的研究结果表明，高表达于骨髓瘤细胞U266上的MMSA-1基因表达水平受Wnt/β-catenin/TCF4信号通路的调控。在U266细胞内，转录因子TCF4与MMSA-1基因启动子区发生异常结合，导致MMSA-1启动子异常活化而高表达。过表达的MMSA-1蛋白与骨髓瘤细胞表面RAS蛋白相互作用，上调RAS蛋白活性，进而活化下游RAS/RAF/MEK/ERK、RAF/PI3K/AKT等信号通路，促进U266细胞增殖、抑制凋亡，同时使骨髓瘤细胞的克隆形成能力大大增强、迁移能力增强。另外，过表达的MMSA-1还通过促进血管生成、下调低氧诱导因子及黏附分子的表达削弱骨髓瘤细胞与骨髓微环境之间的作用，进而促进了MM细胞的侵袭和迁移。本项目从基因及分子生物学层面揭示了多发性骨髓瘤新抗原基因MMSA-1的调控机制以及其在促进多发性骨髓瘤发生发展中的分子学机制，深入揭示了MMSA-1在骨髓瘤细胞中的生物学功能，为未来作为免疫治疗靶点应用于MM的治疗提供了理论依据。