MicroRNA在宫颈癌发病机制的分子基础研究

发布时间：2018-03-19 08:49

本文选题：宫颈癌　切入点：miR-183　出处：《武汉大学》2014年博士论文　论文类型：学位论文

【摘要】：宫颈癌是最常见的妇科恶性肿瘤。据世界范围的资料统计,每年全球大约有50万新发宫颈癌病例,其中85%新发病例在发展中国家。宫颈原位癌高发年龄为30～35岁,浸润癌为45～55岁,近年来其发病有年轻化的趋势。无论是宫颈癌患者的临床表现还是宫颈癌细胞的分子生物学表现,均具有恶性肿瘤侵袭、转移的特性,给患者带去无尽的烦恼及痛苦,严重威胁妇女健康安全。至今宫颈癌的发病机制尚不明确,研究发现除了高危型人乳头瘤状病毒的持续感染与宫颈癌发病密切相关外,某些癌基因与宫颈癌的发病也是有关联的,如C-myc基因过度表达,宫颈癌远处转移的危险性估计增加了6倍,RAS基因表达增加与淋巴结转移有关,说明某些癌基因与宫颈癌的发病过程和肿瘤的扩散有着十分密切的关系。宫颈癌常呈局部扩散和淋巴结转移,肿瘤蔓延至阴道上段、宫旁、膀胱和直肠是最常见的部位。因此有必要寻求新的理论指导,并发现新的干预线索降低宫颈癌的发病率。微小核糖核酸(microRNA, miRNA)是真核生物体内一类长约22nt的内源性非编码单链小RNA,通过转录后调控作用,参与调节几乎所有的生命活动和疾病过程。近十年来,在多项宫颈癌的研究中发现miRNAs在宫颈癌组织与配对的正常宫颈组织之问存在差异表达,并被预测与宫颈癌的发病和侵袭有重要的关系。最近还有学者发现并认为血浆miRNAs的检测可能成为宫颈癌的生物学指标,用于宫颈癌的早期诊断与预后监测。这些研究结果极大地鼓励了我们进行宫颈癌中miRNAS的研究的信心。本研究利用基因芯片技术筛选宫颈癌组织、宫颈癌转移组织和正常宫颈组织中的差异miRNAs,结果发现：miR-183在宫颈癌组织、宫颈癌转移组织和正常宫颈组织中同时出现下调,在转移性宫颈癌组织中下降更明显,而miR-215则在宫颈癌组织中表达明显上调。目前有关miR-183和miR-215在宫颈癌发生发展中作用机制的研究并无相关报道。经实时荧光定量PCR (Quantitative Real-time PCR, qPCR)验证后发现结果与初筛结果基本一致,提示miR-183和miR-215在宫颈癌发生发展中可能具有重要作用。为了验证miR-183在宫颈癌发生发展中的作用,本研究分别通过过表达及沉默表达策略,从正反两方面探讨miR-183对宫颈癌细胞功能的影响： 1、细胞增殖与细胞周期：应用MTT法检测细胞增殖,PI-FACS法检测细胞周期,结果发现miR-183过表达或表达抑制对宫颈癌CASKI细胞增殖及细胞周期均无影响。 2、细胞凋亡：Annexin V-APC单染,流式细胞仪检测显示miR-183过表达增强CASKI细胞凋亡,而miR-183表达抑制则减少CASKI凋亡。 3、CASKI体外侵袭能力研究：Transwell实验结果显示miR-183表达抑制导致CASKI侵袭能力显著提高。结合宫颈癌临床上具有高侵袭转移能力的特点,推测miR-183在宫颈癌的发病和转移过程中发挥作用。综上所述,本研究通过筛查目的miRNAs,并结合实验验证的方法,系统分析了miR-183在宫颈癌细胞中的表达特征、对间质细胞功能的改变作用,研究发现miR-183低表达状态与宫颈癌发展、发生密切有关。同时发现miR-215的的高表达与宫颈癌的发生、发展等密切相关,为进一步寻找宫颈癌诊断及治疗的靶标提供了一定的研究基础。
[Abstract]:Cervical cancer is the most common gynecological malignant tumors worldwide. According to statistics, every year about 50 million new cases of cervical cancer cases, of which 85% new cases in developing countries. The high age of cervical carcinoma in situ was 30~35 years old, invasive carcinoma was 45~55 years old, in recent years the incidence of a younger age trend. Both molecules biology of cervical cancer patients with clinical manifestations or cervical cancer cells, with malignant tumor invasion and metastasis characteristics, to bring endless troubles and suffering, a serious threat to women's health and safety. Now the pathogenesis of cervical cancer is not clear, the research found that in addition to closely related to persistent infection with high-risk cervical cancer human papilloma virus, the incidence of certain cancer gene and cervical cancer is also related, such as overexpression of C-myc, increased by 6 times the risk of cervical cancer metastasis RAS based estimation. Because of increased expression and lymph node metastasis, and tumor pathogenesis that diffusion of oncogenes and cervical cancer has a very close relationship. Cervical cancer often showed local spread and lymph node metastasis, tumor spread to the upper vagina, uterine, bladder and rectum is the most common site. So it is necessary to seek the theory the guidance of the new, and the discovery of new clues intervention to reduce the incidence of cervical cancer.
MicroRNAs (microRNA, miRNA) is a kind of endogenous eukaryotic organisms 22nt long encoding non single stranded small RNA, through post transcriptional regulation, involved in the regulation process of almost all life activities and disease. Over the past ten years, in the study of a number of cervical cancer have been found in the differential expression of miRNAs in normal cervical tissue cervical cancer tissue and paired the question, and is predicted to have important relationship between pathogenesis and invasion and cervical cancer. Recent scholars have found and that the detection of plasma miRNAs may be a biological indicator of cervical cancer, for early diagnosis and prognosis of cervical cancer. The monitoring results of these studies greatly encouraged us to miRNAS cervical cancer in confidence.
Gene chip technology to screen cervical cancer using this study, differences in metastasis in cervical carcinoma tissue and normal cervical tissue, miRNAs, miR-183 were found in cervical cancer, cervical cancer metastasis tissue and normal cervical tissue and decreased in metastatic cervical carcinoma tissues decreased significantly, while miR-215 in cervical cancer tissue expression was up-regulated. The miR-183 and miR-215 in the study reported no mechanism in the occurrence and development of cervical cancer. By real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR) after verification and found the results of screening results showed that the miR-183 and miR-215 in cervical cancer may play an important role in the development.
In order to verify the role of miR-183 in the development of cervical cancer, we investigated the effect of miR-183 on the function of cervical cancer cells from two aspects: overexpression and silencing strategy.
1, cell proliferation and cell cycle: cell proliferation was detected by MTT assay. Cell cycle was detected by PI-FACS assay. It was found that miR-183 overexpression or expression inhibition had no effect on proliferation and cell cycle of cervical cancer CASKI cells.
2, apoptosis: single staining of Annexin V-APC, flow cytometry showed that miR-183 overexpression enhanced apoptosis of CASKI cells, while the inhibition of miR-183 expression reduced apoptosis of CASKI.
3, study of CASKI invasiveness in vitro: the results of Transwell experiment showed that the inhibition of miR-183 expression led to a significant increase in the invasiveness of CASKI.
Combined with the characteristics of high invasion and metastasis in cervical cancer, it is speculated that miR-183 plays a role in the pathogenesis and metastasis of cervical cancer.
In summary, this study through the purpose of screening miRNAs, and experiment method, systematic analysis of the expression of miR-183 in cervical cancer cells. The effect on the function of Leydig cells, the study found that low expression of miR-183 and cervical cancer development, closely related. At the same time that the high expression of miR-215 and cervical cancer the occurrence and development are closely related, provide the research basis for further diagnosis and treatment of cervical cancer targets.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

【共引文献】