中国科学院昆明动物研究所机构知识库

肺癌作为所有肿瘤中发病率和死亡率最高的癌症，导致全球大约27%的癌症患者死亡。肺癌的复发率很高，5年生存率仅为17.4%左右，肿瘤分期较高的肺癌5年生存率甚至只有2%。近年来，科研工作者对肺癌的发病机制及其治疗进行了大量的研究，但是对于提升肺癌患者生存期效果甚微，因此对于肺癌进行更深入的研究仍非常有必要。蛋白质翻译后修饰作为维持细胞自身稳态的一种重要调节方式，主要包括乙酰化、甲基化、糖基化、羟基化、磷酸化和泛素化等。其中泛素化作为一类作用方式复杂且作用结果多样的蛋白质翻译后修饰方式，在蛋白质的降解和功能调节中起着十分重要的作用，几乎参与了细胞增殖、凋亡、分化、基因表达、信号传递等一切生命活动的调控。泛素化通过调控不同底物的稳定性与活性，影响肿瘤的发生和发展。其中E3连接酶是泛素化过程中底物特异性的决定者，被视为癌症发生的重要调节因子，特别是含HECT结构域的连接酶，已然成为研究、开发新药物的新靶标。HUWE1（HECT，UBA and WWE domain containing 1，E3 ubiquitin protein ligase，又名MΜLE、Ib772、LASU1、UREB1、ARF-BP1等）是一种含HECT结构域的E3连接酶，其底物有N-MYC、C-MYC、MIZ-1、BCL-1、P53、H1.3等。HUWE1通过调节其底物的稳定性，控制着细胞内大量与肿瘤发生密切相关的生物学过程，例如DNA损伤修复、细胞增殖、凋亡、分化以及细胞内稳态等。但是HUWE1是原癌基因还是抑癌基因目前仍存在很大争议。本研究利用免疫组化以及Western blot 分析发现，癌组织的HUWE1蛋白表达水平显著高于癌旁组织。到目前为止，HUWE1过表达对肺癌的发生发展的具体作用尚不清楚，因此我们构建了四环素诱导敲降HUWE1的慢病毒载体并感染A549细胞，发现敲降HUWE1后A549细胞的增殖以及克隆形成能力降低。为了验证HUWE1对A549细胞体内成瘤能力的影响，我们将感染了敲降HUWE1病毒的A549细胞接种于免疫缺陷小鼠皮下，成瘤后利用四环素诱导敲降HUWE1，结果表明肿瘤生长速度显著变慢。取移植瘤石蜡切片染色观察，发现随着HUWE1的敲降，Ki67阳性率降低，Cleaved Caspase3表达升高。在机制研究中，通过对敲降HUWE1的A549细胞进行RNA-seq分析发现，变化的信号通路中与肿瘤相关的主要集中在P53信号通路，P53信号通路中变化的基因中与血管形成相关的主要是SERPINE1、CD82以及与细胞周期相关的是CDKN1A。我们通过Western blot发现在A549细胞中敲降HUWE1后P53和P21表达显著上调，同时对移植瘤的分析发现敲降HUWE1后，血管形成相关蛋白VEGF、CD31和Tie2的表达量下调。这些证据证明敲降HUWE1主要通过P53的累积来抑制细胞增殖，通过抑制血管形成来抑制肿瘤的增长，从而抑制肺癌的发生和发展。

Lung cancer is the highest of incidence and mortality in all tumors, which account for about 27% in cancer deaths. Lung cancer have extremely high rate in recurrence, and 5-year survival rate is only 17.4 %, 5-year survival rate of high grade lung cancer patient is only about 2%. Even in recent years, researchers carried out extensive research in the pathogeneses and treatment of lung cancer, but it did not show apparent effect on improving survival time for lung cancer patient. So it remains to be further studied for lung cancer.Post-translational modification of protein is one of the important regulating modes in maintaining cell steady state, which include acetylation, methylation, glycosylation, hydroxylation, phosphorylation and ubiquitination. Ubiquitination is a more complex and more diverse protein modification mode. It plays a very important role in the degradation and function of protein, which is involved in cell proliferation, apoptosis, differentiation, gene expression and signal transmission. Ubiquitination affects the occurrence and development of tumor by regulating the stability and activity of different substrates. As the decider of the substrate specificity in the process of ubiquitination, the ubiquitin E3 ligase is regarded as important regulatory factor of cancer, especially with HECT structure domain, which has become the new target for the research and development of new drug.HUWE1(HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase, alternate name MΜLE, Ib772, LASU1, UREB1, ARF-BP1) is E3 ligase containing HECT domain. Its substrates include N-MYC, C-MYC, MIZ-1, BCL-1, P53 and H1.3 etc. By regulating the stability of the substrates, HUWE1 regulates a large number of biological processes related to tumorigenesis, such as DNA damage repair, cell proliferation, apoptosis, differentiation and intracellular homeostasis. However, there is still a lot of controversy that HUWE1 is oncogene or tumor suppressor gene.In this article, we found HUWE1 expression level of carcinoma tissue was significantly higher than para-carcino ma tissue by immunofluorescence technique and western blot. So far, how HUWE1 overexpression effects the occurrence and development of lung cancer is unclear. So we knocked down HUWE1 in lung cancer cells A549 by building the tetracycline to induce knockdown HUWE1 lentivirus and infected A549 cells with the virus, we found the ability of A549 cell proliferation and clone formation were reduced after knockdown HUWE1. To verify HUWE1 influence on the capacity to initiate a new tumor of A549 cells in vivo, we infected A549 cells with the virus then inoculated into immunodeficiency mice subcutaneously. After the tumor had formed we used tetracycline to knockdown HUWE1, and found the growth of tumor was significantly slower. Staining the paraffin section of the transplanted tumor, we found Ki67 positive rate reduced and Cleaved Caspase3 positive rate increased with the knockdown HUWE1. These results proved knockdown HUWE1 significantly reduced the malignant degree of A549 cells. In the study of mechanism, the change of signal pathway mainly focused on P53 signal pathway which takes part in tumor. The change genes were mainly tumor angiogenesis SERPINE1, CD82 and cell cycle inhibiting CDKN1A. We found that the expression of P53 and P21 increased and the expression of VEGF, CD31, Tie2 decreased after knockdown HUWE1 by western blot. These evidences proved that knockdown HUWE1 could inhibit the cell proliferation and tumor angiogenesis through the accumulation of P53, thus inhibited the occurrence and development of lung cancer.