miR-93靶向RAB11FIP1在宫颈癌发生中的作用及机制

发布时间：2018-02-13 16:43

  本文关键词： microRNA miR-93 宫颈癌 宫颈上皮内瘤变 microRNA miR-93 宫颈癌 增殖 凋亡 miR-93 RAB11FIP1蛋白 靶基因 慢病毒　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：官颈癌(Cervical Cancer, CC)是全球女性最常见的妇科恶性肿瘤之一。随着官颈癌筛查的普及以及对于官颈癌前病变的及时治疗,使得宫颈癌的发生率在发达地区显著下降,但在欠发达地区由于筛查未开展或不规范,官颈癌的发生率仍然保持较高的水平。同时宫颈癌也是死亡率较高的女性恶性肿瘤,严重威胁广大妇女的健康。 业已公认,高危人乳头瘤病毒(High Risk Human papillomavirus,HR-HPV)感染是官颈癌发病的必需因素,但不是惟一因素,除病毒感染以外,宿主因素也制约了疾病的发生和发展。研究这些因素在在官颈癌发生与进展过程中的作用与机制,对制定宫颈癌防控新策略具有十分重要的意义。 miRNA是一类非编码小分子单链RNA,可以与靶基因mRNA3'非翻译区结合,引起靶基因mRNA降解或翻译抑制,在转录后水平调节基因的表达。最近的研究发现,miRNAs的表达异常与肿瘤的发生发展关系密切,但niRNA在不同的组织和细胞中发挥的生物学功能和机制并不相同,具有组织与细胞特异性和时效性的特点。另外,同一个niRNA可以有多个不同的靶基因,多个不同的miRNA也可以同时作用于一个靶基因,从而造成不尽相同的生物学功能。因此,miRNA及其靶分子构成了一个巨大的错综复杂的生物调控网络。 已有研究发现,miRNAs在肿瘤的发生发展过程中的发挥了重要作用。迄今已发现多种miRNA在宫颈癌中表达下降,并通过各自不同的靶基因调控官颈细胞的生物学行为,参与宫颈癌的发生、侵袭、转移等过程。阐明miRNA在官颈癌发生发展过程中的作用及机制对探索宫颈癌防治新策略有重要意义。 本课题组前期通过官颈癌相关miRNA芯片筛查发现,在官颈癌组织表达改变的众多miRNA中,miR-93表达升高。在此基础上,本研究首先验证miR-93在宫颈癌、高级别CIN和正常组织中的表达差异,并比较分析miR-93的表达水平与宫颈癌临床不良预后因素的相关性。再通过基因功能实验,探寻miR-93的异常表达对宫颈癌细胞生物学行为的可能作用并确定其发挥作用的靶基因。旨在为探寻官颈癌发生与进展新机制及宫颈癌防控新策略提供科学依据。 第一部分官颈癌及高级别CIN组织中miR-93表达及意义 目的： 验证miR-93在宫颈癌及高级别CIN组织中表达上调,并探讨miR-93表达与宫颈癌不良预后因素之间的相关性。 方法： 收集宫颈鳞癌168例、CIN2-360例和正常上皮48例组织标本,采用Stem-loop RT-PCR和Real time PCR法检测miR-93表达水平,并收集官颈癌患者的临床病理资料,分析miR-93的表达水平与官颈癌临床不良预后因素的相关性。 结果： 1.miR-93在宫颈鳞癌和CIN2-3组织中的表达水平与宫颈正常上皮组织相比显著上调,但两者间无显著差异。 2.miR-93表达与宫颈癌FIGO分期、盆腔淋巴结转移、深间质浸润、阴道壁累及以及脉管浸润无关。 结论： 1.宫颈癌组织中miR-93表达水平显著高于宫颈正常组织,但与宫颈癌不良预后因素无显著相关性。 2.高级别CIN组织中miR-93表达水平显著高于宫颈正常组织,而高级别CIN组织与癌组织相比miR-93表达水平无差异,提示miR-93表达改变在宫颈癌发生发展进程中可能是一个早期事件。 第二部分miR-93对宫颈癌细胞生物学行为的影响 目的： 明确miR-93对宫颈癌细胞增殖、凋亡、侵袭等生物学行为的调控作用。 方法： 在宫颈癌细胞株Siha和CaSki中,利用细胞转染技术抑制miR-93的表达,利用MTT法检测细胞增殖能力,流式细胞技术检测细胞凋亡及细胞周期分布,Transwell侵袭试验检测细胞的侵袭能力。 结果： 1.在宫颈癌细胞中,下调miR-93表达抑制Siha和CaSki细胞的增殖,促进细胞的凋亡。 2.在宫颈癌细胞中,下调miR-93表达不改变细胞周期和侵袭力。结论： miR-93表达下调抑制宫颈癌细胞增殖并促进凋亡,但对G1期细胞比例和细胞侵袭力没有影响,与miR-93主要在官颈癌发生阶段发挥癌基因功能相符。 第三部分miR-93在宫颈癌细胞中靶向调控RAB11FIP1蛋白表达 目的： 确定miR-93的靶基因,以揭示miR-93促宫颈癌发生过程中的分子机制。 方法： 通过软件预测miR-93靶基因。用miR-93inhibitor转染官颈癌细胞株Siha和CaSki,用Real time RT-PCR检测RAB11FIP1mRNA表达,用Westen Blot检测RAB11FIP1蛋白表达。利用双荧光素酶报告基因的方法确认miR-93与RAB11FIP1的靶向关系。同时在宫颈癌细胞株Siha和CaSki中,利用慢病毒转染技术过表达RAB11FIP1蛋白,利用MTT法检测细胞增殖能力,流式细胞技术检测细胞的凋亡。用免疫组化的方法在宫颈组织中检测RAB11FIP1蛋白表达。 结果： 1.抑制miR-93的表达上调官颈癌细胞RAB11FIP1蛋白水平。 2.miR-93与含RAB11FIP13'UTR区的双荧光素酶报告质粒共转染,下调荧光素酶的活性,而不影响3'UTR区突变报告质粒的荧光素酶活性。 3.在宫颈癌细胞中,上调RAB11FIP1蛋白的表达抑制Siha和CaSki细胞增殖,促进细胞的凋亡。 4.在宫颈癌及高级别CIN组织中, RAB11FIP1蛋白低表达,且miR-93表达与RAB11FIP1蛋白水平呈负相关,但与宫颈癌不良预后相关因素无关。 结论： 1.RAB11FIP1是miR-93的直接靶基因,miR-93通过靶向RAB11FIP1调控宫颈癌细胞的生物学功能。 2.上调RAB11FIP1表达抑制宫颈癌细胞增殖并促进凋亡；在高级别CIN和宫颈癌组织中RAB11FIP1蛋白表达降低,但与宫颈癌不良预后因素无关,提示RAB11FIP1也在官颈癌发生的早期阶段发挥抑癌作用。
[Abstract]:Cervical cancer (Cervical Cancer CC) is one of the most common female gynecologic malignant tumors. With the popularity of cervical cancer screening and timely treatment for cervical precancerous lesions, the incidence of cervical cancer decreased significantly in developed areas, but in less developed regions due to screening did not carry out or not, the occurrence of cervical cancer rates still remain high level. At the same time, cervical cancer is a high mortality of malignant tumors of the female, a serious threat to women's health.
It has been well known that human papillomavirus (High Risk Human papillomavirus, HR-HPV) infection is a necessary factor of cervical cancer, but not the only factor, in addition to virus infection, the host factors also restrict the occurrence and development of disease. The effect and mechanism of these factors in the occurrence and progression of cervical cancer in the that is very important to develop new strategies of cervical cancer prevention and control.
MiRNA is a kind of non encoding single stranded RNA molecules, and target gene mRNA3'untranslated region of target genes by mRNA degradation or translation inhibition, regulation of gene expression at the post transcriptional level. Recent studies have found that the occurrence and development is closely related to abnormal expression of miRNAs and tumor, but the biological function and mechanism of niRNA in the play different tissues and cells are not the same, has the characteristics of tissue and cell specificity and timeliness. In addition, the same niRNA can have a number of different target genes, multiple different miRNA can also be applied simultaneously to a target gene, resulting in biological function is not the same. Therefore, miRNA and the target molecules constitute a huge biological network perplexing.
It has been found that miRNAs in the process of tumor progression has played an important role. So far it has been found that many decreased expression of miRNA in cervical cancer, and the biological behavior of different target gene regulation of cervical cells, participate in the occurrence of cervical cancer, invasion, metastasis. To clarify the effect and mechanism of miRNA development in the process of cervical cancer has important significance to explore the prevention and treatment of cervical cancer new strategies.
Ourprevious by cervical cancer related miRNA microarray screening in cervical cancer tissues the expression of numerous miRNA, increase the expression of miR-93. On this basis, this study firstly verified miR-93 in cervical cancer, differentially expressed high level of CIN and normal tissues, and the correlation analysis of the expression of miR-93 and cervical cancer clinical adverse prognostic factors. Through the experiment of gene function, to explore the abnormal expression of miR-93 may effect on the biological behavior of cervical cancer cell line and to determine its role. To play the target gene to explore cervical cancer to provide a scientific basis for the occurrence and progress of the new mechanism and new strategies for cervical cancer prevention and control.
The expression and significance of miR-93 in the first part of cervical cancer and high grade CIN tissue
Objective:
The expression of miR-93 was up-regulated in cervical cancer and high grade CIN tissues, and the correlation between the expression of miR-93 and the adverse prognostic factors of cervical cancer was investigated.
Method锛，

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