中国科学院昆明动物研究所机构知识库

蛋白质翻译后修饰是细胞维持自身稳态的重要调节方式，包括乙酰化、甲基化、糖基化、羟基化、磷酸化和泛素化等。泛素化作为一类作用方式更加复杂且作用结果更加多样的蛋白质修饰，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。肿瘤的发生是癌蛋白和抑癌蛋白调控紊乱的结果，而泛素化对于蛋白质的稳定和降解至关重要，从这一点上来说泛素化与肿瘤的发生密不可分。肿瘤的重要特征之一就是细胞分裂不受控制，而控制细胞分裂的细胞周期蛋白以及细胞周期蛋白激酶抑制剂都受泛素介导的蛋白酶体系调控。泛素介导的蛋白水解体系主要通过泛素活化酶（E1）、泛素结合酶（E2）以及泛素连接酶（E3）将需要降解的底物标记上泛素分子，随后被转移至蛋白酶体中被降解。目前发现的E1酶有2种，E2酶有40种，E3酶超过500种。由于E3泛素连接酶的底物特异性使其越来越成为肿瘤治疗的靶点。HUWE1（ HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase，又名MULE、Ib772、LASU1、UREB1、ARF-BP1等）是一种分子量为482 kDa的E3 泛素连接酶，其3’末端含 HECT结构域。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。自从其被发现开始，HUWE1在肿瘤中的作用就备受争议。不同的研究小组在U2OS细胞中，分别发现HUWE1既可以降解促凋亡蛋白P53导致细胞生存率增加又可以通过泛素化抗凋亡蛋白Mcl-1促进细胞凋亡。HUWE1在乳腺癌、肺癌、结肠癌和胰腺癌等多种肿瘤中高表达，这说明在这些肿瘤中HUWE1可能作为一种癌基因，但是在皮肤癌、甲状腺肿瘤、肝癌种作为抑癌基因起作用。这主要与其底物有关：HUWE1既可以降解癌蛋白，比如N-MYC、C-MYC、MCL1，又可以降解抑癌蛋白，比如P53、MYC、MIZ1。这些结果证明了HUWE1极可能在癌症发生中具有重要作用，但对于HUWE1是促进、还是抑制肿瘤发生发展与其作用底物有关。卵巢癌是造成女性死亡的第五大肿瘤。由于其发病位置的隐蔽性，以及缺乏有效的早期检测指标，导致卵巢癌致死率居于所有妇科肿瘤之首，对女性生命造成严重威胁。最新研究数据表明恶性程度高的卵巢癌病人五年生存率甚至低于30%。然而到目前为止，卵巢癌的发病机理尚未完全清楚，因此对卵巢癌发生和发展的分子机制进行更深入的研究显得非常有必要。TCGA数据库显示，在卵巢癌中HUWE1突变率高达13%，这其中既包括错义突变、基因片段缺失和RNA表达下调，也包括多拷贝和RNA表达上调。这两种病人的生存率，前者显著高于后者，说明HUWE1在卵巢癌中极有可能扮演着原癌基因的角色。为了寻找Huwe1在卵巢癌发生发展中的作用机制，我们首先从Huwe1条件性敲除小鼠中分离卵巢上皮细胞，并进行体外培养。根据TCGA人卵巢癌数据库分析，TP53、KRAS和MYC遗传学变异最为显著，因此我们构建了过表达MYC和KRASG12D，以及沉默p53基因的慢病毒载体，以求构建的卵巢癌模型最大程度上接近人卵巢癌特征。接着我们将包装的病毒感染小鼠卵巢上皮细胞（MOSE）使其恶性转化，建立Huwe1敲除的细胞株MOSE-Huwe1L/L-Cre和4羟基他莫昔芬诱导敲除Huwe1的细胞株MOSE-Huwe1 L/L-CreER。体外研究表明，Huwe1敲除后，细胞的增殖和克隆形成能力显著降低。我们将这两株细胞分别移植到BALB/c Nude小鼠皮下，发现MOSE- Huwe1 L/L -CreER细胞移植后小鼠完全成瘤(10/10)，而MOSE- Huwe1 L/L -Cre细胞移植后小鼠则完全不成瘤(0/10)，证明在卵巢癌起始阶段Huwe1是必需的。为了研究Huwe1对肿瘤发展的影响，我们将MOSE- Huwe1 L/L -CreER细胞皮下移植到BALB/c Nude小鼠皮下，将小鼠分为以下几组：玉米油对照组（A）、细胞注射后32天肿瘤直径5mm时他莫昔芬处理组（B）以及细胞注射后16天肿瘤尚未形成时他莫昔芬处理组（C）。我们发现：与A组相比，Huwe1敲除的B组与C组的肿瘤体积明显减少。取小鼠肿瘤组织进行免疫组化染色分析，发现随着Huwe1的敲除，肿瘤KI67阳性率降低，Caspase-3阳性率升高，这说明敲除Huwe1对肿瘤体积的抑制作用主要通过抑制细胞增殖并促进细胞凋亡来实现的。为了寻找Huwe1在维持卵巢癌发生和发展种的作用机制，我们将MOSE- Huwe1 L/L -CreER和MOSE- Huwe1 L/L -Cre两组细胞进行转录组分析，发现当Huwe1敲除后，非编码长链RNA H19的表达量显著降低。在MOSE- Huwe1 L/L -CreER细胞中敲降H19，发现细胞增殖能力和软琼脂克隆形成率均降低，能够重现Huwe1敲除后的细胞表型。根据文献报道，组蛋白H1.3能够抑制H19的表达，同时Huwe1是降解组蛋白的主要E3泛素化连接酶。因此我们在MOSE- Huwe1 L/L -CreER 细胞中敲除Huwe1后检测H1.3的表达，发现Huwe1敲除后，H1.3表达显著上调。为了更深入地研究Huwe1、H1.3和H19三者之间的关系，我们在MOSE- Huwe1 L/L -CreER和MOSE- Huwe1 L/L -Cre细胞中敲降H1.3，发现H1.3表达下调可以部分营救Huwe1敲除导致的细胞增殖抑制和H19 RNA表达抑制。软琼脂克隆形成实验进一步证明了在Huwe1缺失的细胞中敲降H1.3，细胞克隆形成能力增加。在分子机制上，我们利用CO-IP实验发现，Huwe1可与H1.3结合并促进其泛素化。此外，在人卵巢癌样本中我们发现HUWE1与H1.3的表达呈负相关，并且在人卵巢癌细胞系SKOV3中敲降HUWE1，能够重现小鼠卵巢癌模型中敲除Huwe1引起的表型，且机制与小鼠中的一致。这些证据证明Huwe1是维持卵巢癌发生和发展所必需的，且Huwe1通过降解H1.3维持H19的表达，进而维持肿瘤的发生和发展。 

Post-translational modifications (PTMs) of protein including acetylation, methylation, glycosylation, hydroxylation, phosphorylation and ubiquitination are important in maintaining cell homeostasis. Ubiquitination, as one of protein modifications which is more complex and more diverse plays critical role in protein degradation and functional regulation and involves in cell proliferation, apoptosis, differentiation, gene expression, signal transduction, DNA repair, protein quality control and antigen presentation. Tumorigenesis is the result in dysregulation of oncoprotein and tumor suppressor protein and ubiquitination is essential for the stability and degradation of these proteins. From this point, ubiquitination is closely related to cancer. One of the most important characteristics of cancer is uncontrolled cell division. Cyclins and cyclin-dependent kinase inhibitors which can regulate cell division are both rigorously regulated by ubiquitin-proteasome system (UPS). UPS is a major pathway for intracellular proteins degradation and more than 80% of intracellular proteins are degraded through UPS. Degradation of protein via UPS involve two distinct and successive pathways. Protein destined for proteolysis are initially tagged by a covalently linked polyubiquitin chain of three enzymes: E1(ubiquitin-activating enzyme), E2(ubiquitin-conjugating enzyme) and E3(ubiquitin ligase). Finally, the tagged proteins are degraded by 26S proteasome. Up to date, there are 2 E1, more than 40 E2 and over 500 E3 enzymes. Because of the substrates specificity and diversity, E3 ligases are regarded as anti-tumor therapy targets.HUWE1 (HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase, also known as MULE, Ib772, LASU1, UREB1, ARF-BP1, etc.) is a E3 ligase with the molecular weight of 482 kDa. As a HECT E3 ligase, HUWE1 regulates many cellular biological processes involved in tumorigenesis, such as DNA repair, cell proliferation, apoptosis, differentiation through stabilizing or degrading its substrates. Since it was cloned, the role of HUWE1 in tumors is controversial. Different research groups have shown that HUWE1 targets P53 and MCL-1 for degradation, which promotes cell apoptosis or survival respectively in U2OS cells. Furthermore, many of its substrates have been shown to play critical roles in tumorigenesis. These include a variety of tumor-promoting and tumor-suppressing substrates such as MYC, histones, CDC6, N-MYC, POLB, TopBP1, PA2G4 (isoform 2), Miz-1, HDAC2, Pol λ, MyoD, BRCA1, TIAM1 and H2AX. All the results above imply that HUWE1 is critical for the development of cancer. However, whether it is promoting or inhibiting the tumorigenesis is still in debate.Ovarian cancer is the fifth leading cause of death by cancer in women. Due to the hidden location and the lack of effective early detection methods, ovarian cancer is the first leading cause of death by cancer in female genital organs which seriously threats women’s life. The recent data demonstrate that the five-year survival rate of high malignant ovarian cancer is less than 30%. However, up to now, the pathogenesis of ovarian cancer has not been fully understood. So, it is very necessary to study the molecular mechanism of the occurrence and development of ovarian cancer. The TCGA data shows that the mutation rate of HUWE1 in ovarian cancer is 13% which not include missense mutation, deletion and down-regulation of RNA expression but include multiple copies and up-regulation of RNA expression. The survival rate of former patients is higher than the latter indicating that HUWE1 maybe act as an oncogene in ovarian cancer. In order to discover the role and mechanism of Huwe1 in ovarian cancer, we isolated the mouse ovarian epithelial cells(MOSE) from Huwe1 conditional knockout mice. According to TCGA database, the most significantly genetic alterations in ovarian cancer are TP53, KRAS and MYC. So, in order to establish the ovarian cancer model that is mostly close to the characteristics of humans, we constructed the lentiviral vector overexpressing KRASG12D, MYC and silencing the TP53. IRES-Cre and IRES-CreER elements are also adopted in the lentiviral vector to achieve the knockout of Huwe1. The MOSEs are infected with the above two lentiviruses for malignant transformation to establish the Huwe1 knockout cell line (MOSE- Huwe1 L/L -Cre) and inducible Huwe1 knockout cell line (MOSE- Huwe1 L/L -CreER) by 4-Hydroxytamoxifen. The capacity of cell proliferation and colony formation is reduced significantly after Huwe1 was knockout in vitro. So, we transplanted the MOSE- Huwe1 L/L -Cre and MOSE- Huwe1 L/L -CreER cells into BALB/c Nude mice subcutaneously and found that the former is tumor-free (0/10) while the latter are all tumorigenic (10/10). So, we conclude that Huwe1 is essential for the initial stage of ovarian cancer. In order to study the role of Huwe1 in the development of ovarian cancer, we injected the MOSE- Huwe1 L/L -CreER cell subcutaneously into BALB/c Nude mice. The mice were divided into three groups: the corn oil group (A), the treatment of TAM when the diameter of tumor reaches 5mm at the 32 days after cell injection (B) and the treatment of TAM when no tumor forms at the 16 days after cell injection (C). We found that compared with A group, the tumor volume was significantly reduced after Huwe1 knockout induced by 4-OHT. The immunochemistry results demonstrate that after Huwe1 knockout, the number of Ki67 positive cell was decreased while the number of caspase3 positive cell was increased. We analyzed the gene expression differences between MOSE-Huwe1L/L -Cre and MOSE-Huwe1L/L-CreER cells to discover the mechanism of Huwe1 in the origin and development of ovarian cancer. Long non-coding RNA H19 decreased significantly after Huwe1 knockout. Knockdown of H19, the capacity of cell proliferation and colony formation were reduced significantly in MOSE-Huwe1 L/L -CreER cells and the phenotype is very similar to the Huwe1 knockout. It was reported that Huwe1 can ubiquities and degrades Histone and Histone H1.3 which can inhibit H19 expression. So, we suspect there are some relationship among these three molecules. We detected the expression of H1.3 in MOSE-Huwe1 L/L-CreER cells after Huwe1 was knockout and found that H1.3 was upregulated after Huwe1 was knockout. In order to study the relationship between Huwe1, H19 and H1.3, we induce H1.3 knockdown in MOSE-Huwe1 L/L -Cre and MOSE- Huwe1 L/L -CreER cells and found that silencing H1.3 can partially rescue the inhibition of cell proliferation and the downregulation of H19 caused by knockout of Huwe1. And the capacity of colony formation is increased after H1.3 was knockdown in Huwe1-deficient cells. The CO-IP results demonstrated that Huwe1 can binds and ubiquities H1.3. Furthermore, there is a negative relationship between HUWE1 and H1.3 in human ovarian cancer samples. In human ovarian cancer cell line SKOV3, knockdown of Huwe1 can restore the phenotype of Huwe1 knockout in MOSE. Based on these data above, we can conclude that Huwe1 can degrade H1.3 and maintain H19 expression to sustains normal ovarian epithelial cell transformation and tumor growth.