MiR-634通过调控mTOR信号途径抑制宫颈癌细胞增殖及诱导凋亡的实验研究

发布时间：2018-07-04 14:41

  本文选题：细胞增殖 + 宫颈癌　； 参考：《山东大学》2015年博士论文

【摘要】：研究目的与意义宫颈癌(cervical cancer)是全球女性发病率第二位的恶性肿瘤,仅次于乳腺癌,在某些局部地区和国家其发病率位居首位,每年大约有50万的新发病例,差不多其中45%的病例最终死亡。我国每年新增病例约15万,严重威胁女性的健康。近年来,我国加大了对宫颈癌的普查力度,使之早期发现、早期诊断、早期治疗的水平有了很大的提升,加之卫生条件的改善和保健意识的增强使宫颈癌发病率及死亡率不断下降。近20年来尽管对宫颈癌的研究越来越深入,可是中晚期宫颈癌的疗效未见明显提高,治疗方法的种类和特异性明显落后。为了提高宫颈癌的疗效,寻找治疗宫颈癌的新途径显得尤为迫切。雷帕霉素靶蛋白(target of rapamyein, TOR)基因是最早在酵母中发现的一类高度保守的蛋白激酶家族,随后在自然界其它物种中广泛发现。在哺乳动物细胞中也存在结构和功能相似的TOR蛋白,命名为哺乳动物雷帕霉素靶蛋白(mammalian target of rapamyein, mTOR)。mTOR基因位于人类第1号染色体短臂的3区6带2亚带中,是丝／苏氨酸蛋白激酶家族成员,mTOR基因的羧基端具有与磷脂酰肌醇3-激酶(P13K)高度同源的催化结构域,是经典信号传导通路：磷脂酰肌醇3-激酶、蛋白激酶B和哺乳动物雷帕霉素靶蛋白(P13K-Akt-mTOR)的下游基因,是该信号通路接受上游刺激并向下传递信号的节点；多种肿瘤的发生与该信号通路活化及mTOR高表达密切相关,这在结肠癌、淋巴瘤、乳腺癌及鼻咽癌等多种恶性肿瘤中早已有报道。MicroRNAs (miRNAs)是一类非编码调节性小RNA分子,由21到25个核苷酸组成,miRNA前体成熟后与辅酶因子结合成RNA诱导沉默复合体,与靶信使RNA (message RNA, mRNA)的3’UTR以碱基互补配对结合,导致mRNA降解或转录后水平的抑制,从而抑制靶基因mRNA的翻译并参与调控细胞进程,如炎症反应、应激反应、分化、凋亡及迁移等。目前已经发现miRNAs可以调控细胞周期、增殖、分化、凋亡及衰老；多数肿瘤中都存在miRNAs的异常表达,它与肿瘤的发生、进展、诊断、治疗及预后密切相关,是当前肿瘤领域研究热点。有报道称miR-634过表达能直接通过调控线粒体内稳态相关基因而激活线粒体凋亡通路、抗氧化能力及细胞自噬能力。同时在异种移植小鼠模型中,有研究发现miR-634抑制肿瘤的生长和增强紫杉醇的敏感性。有研究表明在恶性胶质瘤中miR-634过表达对mTOR途径起负调节作用；这为我们提供了研究其它miRNAs家族成员在宫颈癌致病机理中发挥作用指明了方向。本研究拟探讨miR-634是否可以通过调控mTOR基因的表达影响宫颈癌细胞的生物学行为,能否以miR-634为媒介逆转细胞的自我保护流程,影响宫颈癌细胞的增殖、侵袭及凋亡,从而提高宫颈癌化学治疗及靶向治疗的疗效,为调控宫颈癌细胞的信号传导通路及抑制肿瘤细胞的生长和促进肿瘤细胞的凋亡提供新的思路。研究方法与结果第一部分MiR-634在宫颈癌组织中的表达及其作用研究研究方法：1.自2013年8月至2013年12月收集烟台毓璜顶医院证实为宫颈鳞状细胞癌标本15例,所有患者术前均未进行放、化疗及其它治疗。应用Real-time PCR技术检测miR-634在宫颈癌组织和癌旁正常组织中的差异表达。2.培养HeLa及Siha细胞,通过构建miR-634过表达质粒,并检测所构建质粒的有效性及转染效率；然后将过表达质粒分别转染宫颈癌HeLa及Siha细胞中,通过WST-8实验、克隆形成实验、划痕实验和Transwell实验,研究过表达miR-634及封闭后对HeLa及Siha细胞的增殖、迁移和侵袭的影响。3.流式细胞仪检测miR-634过表达和封闭后对HeLa及Siha细胞凋亡的影响。研究结果：1.应用Real-time PCR技术检测发现与癌旁正常组织相比mi R-634在宫颈癌组织表达明显降低。2.通过构建好的miR-634过表达质粒转染HeLa及Siha细胞后发现,过表达miR-634的HeLa及Siha细胞其增殖、迁移及侵袭能力减弱,封闭后结果相反。3.流式细胞分析仪结果显示miR-634过表达诱导了HeLa及Siha细胞凋亡率的增加,而封闭miR-634时降低了HeLa及Siha细胞的凋亡率。第二部分MiR-634在宫颈癌细胞中与mTOR基因的相互关系研究方法：1.培养HeLa及Siha细胞,应用Real-time PCR方法及Western blot技术检测mTOR基因mRNA和蛋白在HeLa及Siha细胞中的水平。2.构建含mTOR基因3’-UTR段和含mTOR基因3'-UTR-mut段双荧光素酶报告基因载体,并验证其活性；使用lipofectamine 2000转染试剂将不同的质粒或重组质粒转染至HeLa及Siha细胞中,测定荧光素酶活性,分析miR-634对mTOR基因有无调控作用。3.通过Real-time PCR方法及Western blot技术检测miR-634过表达和封闭后HeLa及Siha细胞中mTOR mRNA的表达及蛋白水平的变化。研究结果：1.通过双荧光素酶报告载体实验证明mTOR是miR-634的靶基因之一,其3’非翻译区包含miR-634的结合位点；miR-634能够通过作用于mTOR基因的3’非翻译区(3’UTR)的特定位点,对其进行负性调节。2.通过Real-time PCR和Western blot技术检测发现过表达miR-634后HeLa及Siha细胞中mTOR基因的mRNA和蛋白水平均明显降低,封闭后结果相反。第三部分mTOR基因在宫颈癌组织中的表达及其作用研究研究方法：1.应用Real-time PCR技术检测mTOR基因在宫颈癌组织和癌旁正常组织中的差异表达。2.培养HeLa及Siha细胞,构建pSilencer/si-mTOR质粒,利用RNA干扰技术封闭HeLa及Siha细胞中靶基因mTOR。3.采用Western blot方法验证pSilencer/si-mTOR质粒的有效性。4.pSilencer/si-mTOR质粒转染HeLa及Siha细胞后通过WST-8实验、克隆形成实验、划痕实验和Transwell实验检测细胞生长特性的变化。研究结果：1.应用Real-time PCR技术检测发现与癌旁正常组织相比mTOR基因mRNA的表达水平在宫颈癌组织中明显升高。2.WST-8实验、克隆形成实验、划痕实验和Transwell实验结果显示,将mTOR基因封闭后HeLa及Siha细胞的增殖、迁移及侵袭能力均下降。研究结论1.miR-634能够抑制HeLa及Siha细胞的增殖侵袭和迁移,起到抑癌基因的作用。2.将mTOR基因封闭后,HeLa及Siha细胞增殖能力减弱,细胞集落形成和侵袭能力也明显减弱。3.在HeLa及Siha细胞中,高表达的miR-634对mTOR基因表达水平的抑制能力增强,使mTOR基因的促进增殖活性减弱,造成细胞生长速度减慢。4.通过阐明miR-634在宫颈癌中的具体作用机制及其靶基因的研究有利于我们对宫颈癌的发生发展进行深入理解,也为临床上宫颈癌的诊断和治疗提供一条新的途径。
[Abstract]:The purpose and significance of cervical cancer (cervical cancer) is a second place malignant tumor in the world. Second only to breast cancer, the incidence of the disease is the first in some local areas and countries, with about 500 thousand new cases each year, about 45% of them die out. The number of new cases in China is about 150 thousand a year, and it is a serious threat to women. In recent years, China has increased the survey of cervical cancer, making early detection, early diagnosis, the level of early treatment has been greatly improved, combined with the improvement of health conditions and health awareness, the incidence and mortality of cervical cancer have declined continuously. In recent 20 years, although the research of cervical cancer has become more and more in-depth, but In order to improve the curative effect of cervical cancer, it is very urgent to find a new way to treat cervical cancer. The target of rapamyein (TOR) gene is the first kind of highly conserved protein kinase family found in yeast. It is found widely in other species in nature. In mammalian cells, there are also TOR proteins with similar structure and function. The mammalian target of rapamyein, mTOR.MTOR gene is located in the 6 band 2 subband of the short arm of the human chromosome first, and is a member of the family of silk / threonine protein kinase. The carboxyl terminal of the mTOR gene has a catalytic domain that is highly homologous to the phosphatidylinositol 3- kinase (P13K). It is the classic signal transduction pathway: the downstream genes of phosphatidyl inositol 3- kinase, protein kinase B and mammalian rapamycin target protein (P13K-Akt-mTOR), which are the nodes of the signal through the upstream stimulation and the downward transmission of signals. The occurrence of the tumor is closely related to the activation of the signal pathway and the high expression of mTOR. It has been reported that.MicroRNAs (miRNAs) is a class of non coding and regulatory small RNA molecules in colon cancer, lymphoma, breast cancer and nasopharyngeal carcinoma, which is composed of 21 to 25 nucleotides, and miRNA precursor is combined with coenzyme factors into RNA induction. The silencing complex, combined with the 3 'UTR of the target messenger RNA (message RNA, mRNA), is combined with base pairs to lead to the degradation of mRNA or inhibition of post transcriptional levels, which inhibits the translation of the target gene mRNA and participates in the regulation of cell processes, such as inflammation, stress response, differentiation, withering and migration and so on. The cell cycle has been regulated by miRNAs. Proliferation, differentiation, apoptosis and senescence; most of the tumors have abnormal expression of miRNAs. It is closely related to the occurrence, progression, diagnosis, treatment and prognosis of tumors. It is a hot spot in the field of cancer. It is reported that miR-634 overexpression can activate mitochondrial apoptosis pathway directly by regulating the homeostasis related genes in mitochondria and the antioxidant energy can be activated. It is also found that miR-634 inhibits tumor growth and increases the sensitivity of taxol in xenotransplantation mice. Studies have shown that miR-634 overexpression in malignant gliomas can negatively regulate the mTOR pathway; this provides us with the study of the pathogenesis of its miRNAs family in the pathogenesis of cervical cancer. The purpose of this study is to indicate whether miR-634 can affect the biological behavior of cervical cancer cells by regulating the expression of mTOR gene, whether it can reverse the self protection process of cells with miR-634 as a medium, influence the proliferation, invasion and apoptosis of cervical cancer cells, so as to improve the treatment of cervical cancer and the treatment of targeted therapy. A new idea to regulate the signal transduction pathway of cervical cancer cells and to inhibit the growth of tumor cells and promote the apoptosis of tumor cells. Research methods and results of the first part of MiR-634 in cervical cancer tissue expression and its role: 1. collected from Yantai Yuhuangding Hospital from August 2013 to December 2013 proved to be 15 specimens of squamous cell carcinoma of the cervix were not performed, chemotherapy and other treatments were not performed before operation. The differential expression of miR-634 in the tissues of cervical cancer and normal tissues by Real-time PCR was used to detect the.2. culture of HeLa and Siha cells by constructing miR-634 overexpressed plasmids, and the effectiveness and transfection efficiency of the constructed plasmids were detected. The expression plasmids were then transfected into cervical cancer HeLa and Siha cells respectively. Through WST-8 experiment, clone formation, scratch test and Transwell experiment, the effects of miR-634 and miR-634 on the proliferation, migration and invasion of HeLa and Siha cells were studied..3. flow cytometry was used to detect miR-634 over expression and closed to HeLa and Siha cells. The effect of apoptosis. Results: 1. the Real-time PCR technique detected that the expression of MI R-634 in cervical cancer tissues was significantly lower than that of normal tissues adjacent to the carcinoma..2. was found to transfect HeLa and Siha cells through the constructed miR-634 overexpressed plasmid, and the proliferation of HeLa and Siha cells overexpressing miR-634, the ability to migrate and invasiveness were weakened. The result of reverse.3. flow cytometry showed that miR-634 overexpression induced the increase of apoptosis rate of HeLa and Siha cells, while blocking miR-634 decreased the apoptosis rate of HeLa and Siha cells. Second part MiR-634 in cervical cancer cells and mTOR gene interaction methods: 1. to cultivate HeLa and Siha cells, and to apply Real-time Methods and Western blot technique, the mTOR gene mRNA and protein were detected in the level.2. in HeLa and Siha cells to construct the 3 '-UTR segment containing mTOR gene and the 3'-UTR-mut segment double luciferase reporter gene carrier of mTOR gene, and to verify its activity. In the cell, the activity of luciferase was measured, and the effect of miR-634 on the mTOR gene was analyzed..3. was detected by Real-time PCR method and Western blot technique to detect the expression of mTOR mRNA in HeLa and Siha cells and the changes in protein level after miR-634's overexpression and closure. The results of the study: 1. through the double luciferase reporter carrier experiment proved that One of the target genes, its 3 'non translation region contains the binding site of miR-634, and miR-634 can negatively regulate it through a specific site that acts on the 3' non translation region (3 'UTR) of the mTOR gene, and.2. is detected by Real-time PCR and Western blot technique to detect the expression of the HeLa and protein water in Siha cells after miR-634 HeLa and Siha cells Third mTOR gene expression in cervical cancer tissue and its role in research methods: 1. the difference expression of mTOR gene in cervical cancer tissues and normal tissues adjacent to cancer by Real-time PCR technique,.2. culture HeLa and Siha cells, construction of pSilencer/si-mTOR plasmids, and RNA interference. Technology closed the target gene mTOR.3. in HeLa and Siha cells using Western blot method to verify the validity of pSilencer/si-mTOR plasmid,.4.pSilencer/si-mTOR plasmid transfected to HeLa and Siha cells after HeLa and Siha cells, clone formation experiment, scratch test and Transwell experiment to detect the changes of cell growth characteristics. Research results: 1. application Real-time The PCR technique detected the expression level of the mTOR gene mRNA compared with the normal tissues adjacent to the carcinoma. The.2.WST-8 experiment was significantly increased in the cervical cancer tissue, the cloning experiment, the scratch test and the Transwell experiment showed that the proliferation, migration and invasion ability of HeLa and Siha cells decreased after the mTOR gene was closed. Conclusion 1.miR-634 can be suppressed. The proliferation, invasion and migration of HeLa and Siha cells play a role in the tumor suppressor gene,.2. will close the mTOR gene, and the proliferation ability of HeLa and Siha cells is weakened, and the colony formation and invasion ability also weakens the.3. in HeLa and Siha cells. The inhibition ability of the high expression miR-634 to the mTOR gene expression is enhanced and the mTOR gene is promoted. The effect of.4. on the specific mechanism of miR-634 in cervical cancer and the study of its target genes will help us to understand the development of cervical cancer and provide a new way for the diagnosis and treatment of cervical cancer.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33

【参考文献】

相关期刊论文 前1条

1 陈军莹;姚德生;贺婵娟;卢艳;欧婷瑜;;宫颈鳞癌MicroRNA差异表达的研究[J];实用医学杂志;2014年01期

，

本文编号：2096398