【作者】 林杨；

【导师】 崔满华；

【作者基本信息】 吉林大学 ， 妇产科学， 2010， 博士

【摘要】 卵巢癌是女性生殖系统最常见的恶性肿瘤之一,在美国卵巢癌已居妇科恶性肿瘤死亡原因的首位,我国卵巢癌的发病率也呈逐年上升的趋势。即使实施理想的肿瘤细胞减灭术及铂类为主的化疗,但卵巢癌5年生存率徘徊在25%～30%。近几年来,寻找新的卵巢癌的治疗方法已经成为迫在眉睫的问题。RNA干扰又称为转录后基因沉默,通过将与mRNA序列相对应的正义和反义RNA组成的21-23个双链RNA导入靶细胞,从而使细胞内特异性基因的mRNA降解和基因沉默。目前国内外研究学者已常规利用RNAi抑制或下调在卵巢癌发生、发展过程中起关键作用的癌基因、抑癌基因、凋亡相关基因等卵巢癌相关基因的表达,从而达到基因治疗的目的。人NOB1基因是近来发现的基因,在白血病等多种肿瘤组织和卵巢癌的多药耐药性细胞中发现有过表达。进一步研究发现,NOB1能够抑制细胞增殖,使细胞周期停留在G1期,调节细胞生长和增殖。提示NOB1在癌症发生和转移过程中可能发挥着重要作用。目前国内外尚无卵巢癌组织中NOB1的表达及其二者关系的研究,故而本文研究的目的是验证NOB1基因的过表达可以促进卵巢癌的发生,并通过RNAi抑制NOB1的表达。采用半定量RT- PCR方法检测NOB1在卵巢癌组织、良性肿瘤组织及正常组织中的表达情况。设计并化学合成针对NOB1的siRNA,用慢病毒载体转染法将其转SKOV3、HEY细胞内,通过MTT、细胞克隆形成实验,观察NOB1-siRNA对SKOV3、HEY生长活性和增殖能力的影响;流式细胞术观察NOB1-siRNA对SKOV3、HEY细胞分裂能力的影响。建立SKOV3裸鼠皮下移植瘤模型,体内观察发现NOB1-siRNA对卵巢癌生长的影响。半定量RT- PCR实验结果发现NOB1在卵巢癌组织中的表达明显高于良性肿瘤组织及正常组织,且其表达与肿瘤的大小、细胞分化程度相关,与年龄、组织学分型、肿瘤分期无相关性。MTT、细胞克隆形成实验结果发现转染NOB1-siRNA的SKOV3、HEY细胞生长活性和克隆形成率明显低于对照,流式细胞术实验结果发现转染NOB1-siRNA的SKOV3、HEY细胞阻滞于G0/G1期,细胞分裂减缓;成功建立SKOV3裸鼠皮下移植瘤模型,体内观察实验结果发现注射NOB1-siRNA的小鼠的转移瘤体积及重量均低于对照组。由此得出结论:NOB1在卵巢癌组织中的表达明显上调,同时NOB1-siRNA可有效抑抑制SKOV3、HEY的细胞生长活性、增殖能力并阻滞细胞分裂,同时显著抑制裸鼠的成瘤能力,提示NOB1可能与卵巢癌发生、发展相关,NOB1有望成为卵巢恶性肿瘤基因靶向治疗的新靶点。更多还原

【Abstract】 Ovarian cancer is one of the most common malignanc tumors in the female genital organs, which was about 4% of the total malignant tumors and 5% of the cancers. The incidence of ovarian cancer is lower than the cervical cancer and endometrial cancer and the mortality mortality rate was rank at the top of gynecologic cancer. With a high degree of malignancy of ovarian cancer and poor prognosis, the ovarian cancer was a serious threat to women’s health.Since the embryonic development, anatomy organization and endocrine function is more complicated, the clinical early diagnosis of ovarian cancer on the type and nature is very difficult. Most patients diagnosed were found in the advanced phase of ovarian cancer. Moreover, the majority of ovarian cancer had spread to the bilateral uterine attachmentuterus, omentum and pelvic organs by laparotomy. Therefor, both in the diagnosis and treatment effects for ovarian cancer were not good enough. For patients with advanced ovarian cancer, even after surgery and chemotherapy, the majority of them recurred two or three years later. So far, the five-year survival rate of ovarian cancer was only 20% to 30% from the clinical data on the domestic and international statistics, significantly lower than that of cervical cancer and endometrial cancer. Every year, the death number of ovarian cancers was even more than the sum of all other gynecological tumors. In recent years, the clinical treatment of ovarian cancer has made some progress as the surgery and adjuvant therapy improved, but the late survival rate of ovarian patients did not significantly increased.Ovarian was exposed to the abdominal cavity, which instantly after Penetrating, ovarian cancer cells can extensively distributed, landing onto the surface of main organs in abdominal cavity, namely diaphragm, liver, intestine, mesentery, omentum and other abdominal viscera which consequently lead to difficulties to lesions complete resection by Surgery. The residual lesions and a large number of sub-clinical lesions mainly rely on chemotherapy after Surgery, but chemotherapy drugs can not be completely eliminated the ovarian cancer which was moderate sensitive to the chemotherapy drugs. The remaining tumor cells exposed to chemotherapy drugs got multi-drug resistance for long period of time, which made the treatment for ovarian cancer more difficult. Therefore, to search for a more effective treatment method and alleviatethe side effects was a intractable problem for researchers in gynecologic oncology.Tumor-specific molecular targeted therapy provides a new way for the treatment of ovarian cancer. It greatly enhanced the efficiency and patient survival for its special role in tumor cells and less toxicity to normal tissue. Those drugs targeted to the tumors were applied to the clinic therapy, including epidermal growth factor receptor (EGFR) inhibitors, monoclonal antibody for specific cell marker, drugs with genetics signs and against certain cancer genes and cells, anti-angiogenesis drugs. These drugs have brought a good therapeutic effect in cancer therap, however, there are still some limitations for their widely use. It was mainly due to that different tumors have different genetic backgrounds and biological properties, which affected the scope of application of molecular targeted drugs. Thus, in the treatment of ovarian cancer, it shouldn’t simply rely on migration from other tumors. It is of important practical significance to independent research cancer-causing genes and find suitable molecular target of the ovarian cancer.Oncogenes of ovarian cancer involve in the cell proliferation, cell cycle regulation, apoptosis signal, invasion, metastasis and other aspects. A new gene NOB1 (NIN1/RPN12 binding protein 1 homolog) was cloned in 2005, which is a RNA-binding protein. This type of protein is often associated with the occurrence of malignant tumors. Structurally, NOB1 is a component of 26S proteasome which is a protelytic complex of eukaryotes. It comprises two major complexes: the catalytic 20S proteasome and the 19S regulatory particle, which degrades proteins that have been targeted for destruction by the ubiquitination (Ub) pathway. It has been found that an increasing number of proteins were included in the subunits of 26S protease. Among them, yeast Nob1p (Nin one binding protein) was first separated by the yeast two-hybrid screening method using Rpn12, a subunit of the 19S regulatory particle. Nob1p played an important role in the function of protease and RNA metabolism process.In the past studies, Nob1p was found to be associated with the proteasome in the cells with exponential growth period, and is degraded during the transition to the stationary phase. Nob1p can also form a complex with nucleoprotein Pno1, which is required for nuclear transfer of the 20S proteasome. Nob1p is a molecular chaperone combined with 20S proteasome and 19S regulatory particle, as well as facilitated to the maturation of 26S proteasome.Yeast Nob1p equally involved in the process of ribosome RNA (rRNA) development and assembling and rRNA is one of the most important structural and functional components in the ribosome assembly process. Nob1p can be co-precipitated with 20S pre-rRNA and pre-40S. The precipitated 20S pre-rRNA is dimethylated at the 3 ’region. Depletion of Nob1p will damage the synthesis of 18S rRNA, accompanied by an accumulation of 20S pre-rRNA at a high level.In humans, Nob1p homologous gene NOB1 and its encoding product were first cloned and analyzed from human kidney cDNA library in 2005. The human NOB1 gene consisted of nine exons and eight introns and located at 16q22.3. The assembled cDNA of NOB1 was 1749 bp in length, containing an ORF of 1239 bp. The ORF encoded a protein with a predicted molecular weight of 50 kDa through the western experiments. While the results of RT-PCR showed that the expression of NOB1 was mainly detected in adult human lung, liver, and spleen. In vitro cultured mammalian cells, the expression of NOB1 protein mainly distributed in nucleus, and some can be detected in cytoplasm. This information provides valuable reference for further study of NOB1 gene function. However, up to date, there was no tumor-related report about NOB1 gene. The role of the NOB1 gene in ovarian cancer occurrence and development process is still unknown. The function of NOB1 and the correlation with the prognosis of ovarian cancer need further validation, which will undoubtedly contribute to the clinic application of NOB1 and the treatment of ovarian cancer.In the present study, the expression of NOB1 in ovarian cancer and its relevance to the clinical pathological feature was explored, which was not reported previously. The inhibition effect of NOB1 on the cell proliferation and cell cycle in vitro and the tumor-forming ability in nude mice was investigated. NOB1 is expected to become the new molecule targeted to ovarian tumor in gene therapy. However, the function and application of NOB1 gene in human tumor therapy need further investigation. Together with our results, it provides new ideas and references for the functional mechanism of NOB1 and its therapeutic applications for malignant tumors, especially ovarian cancer.Objective: To explore the role of NOB1 gene in the ovarian cancer and its relationship with clinical pathological characteristics, the expression of NOB1 gene in the ovarian cancer was investigated. To elucidate the effect of NOB1 gene on the cell proliferation and cell cycle, RNAi technology was applied to silence NOB1 gene in ovarian cancer cell lines SKOV3 and HEY. Moreover, to further confirm the effect of decreased NOB1 levels on tumorigenesis in vivo, tumorigenicity experiments were performed in the ovarian cancer cells following lentivirus infection.Methods:In the present study, we tested the expression of NOB1 in 50 ovarian tumor samples and 50 non-ovarian tumor samples by quantitative PCR, and explored the correlation between the expression of NOB1 and the ovarian clinic-pathologic feature.We designed several sequences of siRNA which targeted NOB1 gene and screened the most effective silence site for RNAi, and pack it with lentiviral particles.Using the packed lentiviral vector to infect human ovarian tumor SKOV3 and HEY cell line, MTT assay was performed to evaluate the cell proliferation. DNA synthesis in proliferating cells was determined by BrdU incorporation assay. Colony formation ability and cell cycle change was also investigated after the NOB1 gene inhibition.Nude mice transplanted human ovarian cancer model was established. The weight of the tumor weight and size were measuredafter the targeted shRNA lentiviral particles and control lentiviral particles were injected into the mice. The inhibition rate was used as an index to calculate the effect NOB1 gene silence on the ovarian cancer tumorigenesis capacity in vivo.Results:The high expression of NOB1 gene in ovarian cancer was significant correlated with the tumor size and the cell differentiation, while no correlated with the age of patients, tumor histological types and staging from the quantitative PCR and statistical analysis results.The recombinant lentiviral vector of shRNA targeted NOB1 was successfully constructed and was infected into HEK293 cells together with an over-expression vector. Western blot results proved that the 3rd siRNA target sequence was the most effective on NOB1 silencing. The NOB1 mRNA and protein expression were significantly inhibited in two human ovarian cancer cell lines, SKOV3 and HEY cells, when infected by shRNA lentiviral vector.It was showed that the cell proliferation, DNA replication and colony formation ability were inhibited. Both cells were significantly blocked at G1 phase following the flow cytometric analysis. In the xenografts assays in nude mice, the tumor size and weight was measured. It was revealed that the tumorigenic capacity was significantly inhibited following the knockdown of NOB1 in ovarian cancer cells.Conclusions: The high expression of NOB1 in ovarian cancer was relevant to the clinical pathological feature, which may be a molecular incentive of malignant tumor formation. The NOB1 gene expression in ovarian cancer cells was effectively down-regulated by lentivirus-mediated RNA interference. Inhibition the NOB1 expression can inhibit the cell proliferation in vitro and the tumor-forming ability in nude mice, and affect the cell cycle. NOB1 is expected to become the new molecule targeted to tumor in gene therapy.更多还原