USP22在非小细胞肺癌发生和发展中的作用及分子机制

As so far, the origin and development mechanism of non-small cell lung cancer (NSCLC) has not yet been fully elucidated. Our initial study found that there were close relations between USP22 and the multiplying of cancer cells. We found that USP22 is overexpressed in human NSCLC tissues and cell lines. USP22 silencing by shRNA inhibits proliferation, induces apoptosis and arrests cells at the G0/G1 phases in NSCLC cells and curbs human NSCLC tumor growth in a mouse xenograft model. Moreover, USP22 USP22 promotes NSCLC tumorigenesis, on one hand through MDMX upregulation and subsequent p53 inhibition, and on the other hand through COX-2 desubiquitination followlled by the enhancement of PGE2 expression. With the study of literatures, we considered that USP22, on the one hand, may be involved in the interactions between MDMX/MDM2 and p53, participate in the regulation of p53 pathway by deubiquitination MDMX/MDM2, and promote the cell proliferation, migration and invasion of NSCLC; on the other hand, may participate in the regulation of miRNAs and their downstream target genes to promote the development of NSCLC. This research plans to demonstrate the interactions between MDMX/MDM2 and p53 pathway, USP22-mediated miRNAs and the target genes in the NSCLC cell lines A549 and NCI-H460. We will also assess the express relevance among USP22, MDMX/MDM2 and p53, along with the relevance among USP22, miRNAs and target genes in NSCLC patients using clinical samples of NSCLC. We will confirm the effect of USP22 on NSCLC proliferation, migration and invasion in vivo and in vitro, and make attempt to elucidate the molecular mechanism in promoting development of NSCLC from two aspects: USP22 participates in the regulation effects of MDMX/MDM2 and p53, and also participates in the regulation of miRNAs and their downstream target genes. We hope this study will provide the theory and practical evidences in the diagnosis and treatment of NSCLC.

非小细胞肺癌的发生发展机制未完全阐明。我们前期研究发现：USP22在人NSCLC组织和细胞株中呈高表达；USP22基因沉默引起NSCLC细胞凋亡和细胞周期停滞在G0/G1期，并抑制裸鼠体内肿瘤生长；USP22通过与MDMX作用激活p53通路，还能通过去泛素化作用调节COX-2而增强PGE2的表达，发挥促癌作用。结合文献，我们认为“USP22可能通过去泛素化MDMX/MDM2而参与调控p53和NF-κB通路间的交互作用，促进NSCLC的发展；还可能通过调节miRNA激活下游靶基因，影响NSCLC的发生发展”。本课题拟在NSCLC 细胞系A549和NCI-H460中，从体内、外两方面证实USP22在NSCLC细胞生长增殖中的作用；从USP22参与miRNAs和靶基因作用、参与MDMX/MDM2和p53通路以及p53和NF-κB通路间交互作用的调控等方面，阐述其促进NSCLC发生发展的分子机制。

肺癌（Lung Cancer, LC）是全球范围内发病率及死亡率最高的恶性肿瘤，其中约80-85%为非小细胞肺癌（Non Small Cell Lung Cancer, NSCLC），并且发病率正逐年增高，严重威胁着人类的生命和健康。泛素特异肽酶22（Ubiquitin Specific Protease 22，USP22）是近年来发现的泛素水解酶家族的一个新成员，属于UBPs家族。USP22参与肿瘤细胞周期相关基因的调控，并与肿瘤细胞增殖密切相关。在前期研究中，我们对非小细胞肺癌肿瘤组织和不同非小细胞肺细胞株中USP22的表达情况进行了测定，发现USP22在非小细胞肺癌组织和细胞中表达明显上调。随后我们构建了USP22 RNAi稳定表达非小细胞肺癌细胞株，有效地沉默USP22基因表达，利用MTT法、流式细胞技术和体内成瘤实验在体外、体内证实了USP22基因沉默可以显著抑制非小细胞肺癌细胞的增殖。同时，通过免疫沉淀验证了USP22和MDMX两者之间的相互作用。通过过表达和沉默MDMX，实验表明USP22通过上调MDMX，抑制p53及相关通路蛋白发挥促进非小细胞肺癌增殖的作用。随后证明了miR-30e-5p通过与USP22中的特定序列结合而负向调节USP22的表达，结果表明miR-30e-5p可通过下调USP22，使Sirt1 / JAK / STAT3信号通路失活，从而抑制NSCLC的发生。在后续研究中，我们发现与USP22同家族的USP21在NSCLC的发生发展过程中也有一定的作用。在NSCLC细胞中，USP21通过去泛素化YY1,使其蛋白水平表达稳定。随后进一步验证了YY1可结合至SNHG16的启动子区域并上调SNHG16的表达水平，同时SNHG16通过调控miR-4500可增强USP21表达水平。这样USP21/YY1/SNHG16可形成一个反馈回路调控非小细胞肺癌的增殖、迁移和侵袭。本课题为NSCLC的诊断和治疗提供理论依据与实践基础。