miR-138通过靶向hTERT抑制宫颈癌发生发展的机制研究

发布时间：2018-05-29 14:47

本文选题：宫颈癌 + microRNA　；参考：《吉林大学》2017年博士论文

【摘要】：目的:越来越多的证据表明,包括宫颈癌在内的多种危害较大的恶性肿瘤的发生、发展和转移与micro RNA的失调密切相关。从宫颈癌前病变发展到宫颈癌远处转移,疾病的每个阶段都有相应的micro RNA参与。micro RNA可以通过对癌基因、抑癌基因以及相关蛋白表达的表观遗传学调控来影响宫颈癌的发生和发展。据多个研究报道,micro RNA家族中的mi R-138参与了肾癌、非小细胞肺癌、黑色素瘤、神经胶质瘤、食管鳞状细胞癌、鼻咽癌和肝癌的发生和发展。但是mi R-138是否参与宫颈癌的发生和发展及其具体机制还未阐明。本研究旨在探讨mi R-138在宫颈癌发生、发展以及转移中的变化规律和作用靶点,为临床诊断和治疗宫颈癌提供理论依据。方法:本研究收集了2010年10月到2015年9月在吉林大学第一医院行宫颈癌手术治疗的59例宫颈癌患者的临床资料和标本。首先,通过RT-PCR的方法证明mi R-138在宫颈癌发生发展中的变化规律;其次,通过流式细胞术和细胞生物学技术等方法阐明了mi R-138对宫颈癌细胞增殖、克隆、凋亡、迁移和侵袭等细胞生物学行为的影响;并且通过生物信息学比对、RT-PCR和western blot等方法证实h TERT可能是mi R-138的直接调控靶点;最后,在体移植转染了mi R-138的宫颈癌He La细胞,建立裸鼠人宫颈癌转移瘤模型,通过评价小鼠体内移植瘤的大小和重量推断mi R-138在宫颈癌发展过程中的作用。结果:分为以下5部分:1.mi R-138在人源宫颈癌细胞系和宫颈癌组织中表达显著下调通过荧光实时定量PCR(RT-PCR)的方法分析mi R-138在人源宫颈癌细胞系He La和Si Ha的表达情况,结果显示,与对照细胞系即与人的永生化无HPV感染的表皮细胞系Ha Ca T细胞相比,mi R-138在宫颈癌细胞系He La细胞和Si Ha细胞中的表达水平明显下调,结果有统计学差异(p0.01),而与Si Ha细胞相比,mi R-138在He La细胞中的表达水平下调更加明显;通过RT-PCR的方法观察mi R-138在59例患者宫颈癌组织中的表达情况,结果显示,与癌旁正常宫颈组织相比,mi R-138在59例宫颈癌患者癌组织中的表达明显下调,mi R-138的相对表达值下调83.3%,相差约2.1倍,有统计学差异(p0.01)。mi R-138的表达水平与宫颈癌分期明显相关,宫颈癌分期越晚,mi R-138的表达水平越低,宫颈癌FIGO(International Federation of Gynecology and Obstetrics,国际妇产科联盟)分期Ib1-Ib2的21例患者mi R-138的相对表达值为:0.53±0.11,FIGO分期Ⅱa1-Ⅱa2的38例患者mi R-138的相对表达值为:0.35±0.06,有统计学差异(p0.05),mi R-138的表达水平还与宫颈癌是否发生淋巴转移密切相关,45例没有淋巴转移的患者mi R-138的相对表达值为:0.49?±?0.08,14例有淋巴转移的患者mi R-138的相对表达值为:0.21±0.06,有统计学差异(p0.01)。但是mi R-138的表达水平与宫颈癌患者年龄、肿块大小(是否为局部晚期)以及组织学分级无明显相关性,没有统计学差异。2.mi R-138可抑制宫颈癌He La细胞的增殖、克隆和抗凋亡能力通过脂质体在培养的宫颈癌细胞系He La中转染化学合成的mi R-138以及对照mi R-NC,通过RT-PCR的方法来检测转染后细胞内mi R-138的表达情况,结果显示,与对照组(转染mi R-NC)相比,mi R-138组(转染mi R-138)He La细胞mi R-138的表达水平明显上调,并有统计学差异(p0.01);MTT实验检测mi R-138对He La细胞增殖能力的影响,结果提示,与对照组(转染mi R-NC)相比,He La细胞系转染mi R-138后,He La细胞的增殖活力明显减弱,并有统计学差异(p0.01);在He La细胞系中分别转染mi R-138及对照mi R-NC后,检测He La细胞的克隆能力,通过细胞计数发现,与对照组相比,He La细胞系转染mi R-138后,He La细胞的克隆形成能力明显减弱,并有统计学差异(p0.01);通过流式细胞技术检测mi R-138对He La细胞凋亡的影响,结果证实,与对照组相比,He La细胞转染mi R-138后细胞凋亡明显增加,并有统计学差异(p0.01)。3.在宫颈癌细胞系He La中过表达mi R-138可明显抑制细胞的迁移与侵袭通过Transwell实验验证过表达mi R-138对宫颈癌细胞迁移及侵袭能力的影响。Transwell细胞体外迁移实验结果证实,与过表达对照mi R-NC相比,在He La细胞中过表达mi R-138可以明显抑制细胞的迁移,并有统计学差异(p0.01)。Transwell细胞体外侵袭实验结果证实,与过表达对照mi R-NC相比,在He La细胞中过表达mi R-138可以明显抑制细胞的侵袭能力,并有统计学差异(p0.01)。4.mi R-138可通过调控h TERT表达来抑制宫颈癌发生发展通过生物数据库Targetscan6.2和mi RWalk分析与mi R-138结合的靶基因,并通过文献比对和分析,推测人端粒酶逆转录酶(human Telomerase Reverse Transcriptase,h TERT)可能是mi R-138的直接作用靶点。通过荧光素酶报告实验证实,与过表达含h TERT3’端(3’UTR)突变位点的质粒相比,过表达h TERT质粒后荧光吸收值更高,并有统计学差异(p0.01);western blot实验从蛋白水平证实,转染mi R-138以后,He La细胞中h TERT的蛋白表达被明显抑制。5.mi R-138可在体抑制裸鼠人宫颈癌移植瘤的发展我们在He La细胞中转染mi R-138,并将过表达mi R-138的He La细胞移植到小鼠体内,构建裸鼠过表达mi R-138人宫颈癌细胞移植瘤模型,然后通过不同时间点观察小鼠体内移植瘤的大小在体评估mi R-138对宫颈癌发展的影响,结果提示,移植转染了mi R-138的He La细胞的小鼠,其体内移植瘤体积明显小于对照组,其移植瘤重量也明显轻于对照组,并有统计学差异(p0.01);最后我们将小鼠体内移植瘤取出并通过western blot实验在体检测h TERT的蛋白表达,实验结果提示,与对照组相比,移植过表达mi R-138的He La细胞后,移植瘤中h TERT的蛋白表达水平明显降低,并有统计学差异(p0.01)。结论与创新点:本研究首次通过体外和在体实验初步说明了mi R-138在宫颈癌发生、发展中的作用以及可能机制,即mi R-138可能通过与h TERT结合进而抑制h TERT的蛋白表达来抑制宫颈癌细胞的增殖、克隆,并抑制宫颈癌细胞的抗凋亡能力,减弱宫颈癌细胞的迁移与侵袭能力,从而减慢宫颈癌的发生、发展和转移。主要创新点:1.本研究表明,宫颈癌组织中mi R-138的表达水平变化可以实时提示宫颈癌的是否发生以及是否发生转移,通过检测宫颈癌组织总mi R-138的表达或许可监测宫颈癌的发生和是否发生转移,所以mi R-138未来有可能作为临床筛查和诊断宫颈癌并判断其预后的生物学标记物;2.通过在体实验证实,上调mi R-138的表达可以明显抑制裸鼠人宫颈癌移植瘤的发展,从而为临床治疗宫颈癌提供可参考的药物设计靶点和理论依据。
[Abstract]:Objective: more and more evidence shows that the development and metastasis of a variety of malignant tumors, including cervical cancer, are closely related to the maladjustment of micro RNA. From cervical precancerous lesions to distant metastasis of cervical cancer, each stage of the disease has a corresponding micro RNA involved in.Micro RNA through the oncogene and tumor suppressor It is reported that MI R-138 in the micro RNA family is involved in the occurrence and development of renal cancer, non small cell lung cancer, melanoma, glioma, glioma, esophageal squamous cell carcinoma, nasopharyngeal carcinoma and liver cancer, but whether mi R-138 is involved in cervical cancer, according to a number of reports. This study aims to explore the changes and targets of MI R-138 in the occurrence, development and metastasis of cervical cancer, and to provide a theoretical basis for the clinical diagnosis and treatment of cervical cancer. Methods: This study collected the operation of cervical cancer in No.1 Hospital of Jilin University from October 2010 to September 2015. The clinical data and specimens of 59 cases of cervical cancer were treated. First, the changes of MI R-138 in the occurrence and development of cervical cancer were proved by RT-PCR method. Secondly, the cell biological behavior of MI R-138 on the proliferation, Kuron, apoptosis, migration and invasion of cervical cancer cells was elucidated by flow cytometry and cell biology techniques. And through bioinformatics comparison, RT-PCR and Western blot and other methods confirmed that h TERT may be the direct target of MI R-138; finally, the MI R-138 cervical cancer He La cells were transfected in body transplantation, and the metastatic tumor model of human cervical cancer in nude mice was established, and the size and weight of the transplanted tumor in the mice was evaluated for cervical cancer. The following 5 parts: the expression of 1.mi R-138 in human cervical cancer cell lines and cervical cancer tissues was significantly reduced by fluorescence real-time quantitative PCR (RT-PCR) method to analyze the expression of MI R-138 in human cervical cancer cell line He La and Si Ha. Compared with the Ha Ca T cells infected by V, the expression level of MI R-138 in the He La cells and Si Ha cells in the cervical cancer cell line was obviously down, and the results were statistically different (P0.01). The expression level of MI R-138 in the cells was significantly lower than that in the Si cells. The expression in the cervical cancer tissue showed that the expression of MI R-138 in the cancer tissues of 59 patients with cervical cancer was significantly down, the relative expression value of MI R-138 decreased by 83.3%, and the difference was about 2.1 times, and the level of.Mi R-138 was significantly correlated with the stage of cervical cancer, the late stage of cervical cancer was, the later the stage of cervical cancer was, The lower the expression level of MI R-138, the relative expression value of MI R-138 in 21 cases of cervical cancer FIGO (International Federation of Gynecology and Obstetrics, international gynaecology and obstetrics alliance) was 0.53 + 0.11, and the relative value was 0.35 + 0.06. The expression level of MI R-138 in 45 patients without lymphatic metastasis was relative expression value: the relative expression value of MI R-138 in 0.49? 0.08,14 patients with lymphatic metastasis was 0.21 + 0.06, with statistical difference (P0.01). But the expression level of MI R-138 and the age of cervical cancer patients, mass There is no significant correlation between size (whether locally advanced) and histological classification. There is no statistical difference in.2.mi R-138 to inhibit the proliferation of He La cells in cervical cancer. The cloning and anti apoptotic ability can be transfected by chemically synthesized mi R-138 and control mi R-NC in the He La of the cultured cervical cancer cell line, and the conversion is detected by RT-PCR method. The expression of MI R-138 in the infected cells showed that compared with the control group (transfected mi R-NC), the expression level of MI R-138 in the MI R-138 group (transfected mi R-138) was obviously up and there was a statistical difference. After transfection of a cell line to MI R-138, the proliferation activity of He La cells was significantly weakened, and there was a statistical difference (P0.01). The cloning ability of the cells was detected in He La cell lines and after the control of MI R-NC, the cloning ability of the cells was detected by cell count, and the clone formation ability of the cells was compared with the control group. The effect of MI R-138 on the apoptosis of He La cells was detected by flow cytometry (P0.01). The results showed that compared with the control group, the apoptosis of He La cells was significantly increased after MI R-138 (MI R-138), and there was a statistical difference (P0.01).3. in the cervical cancer cell line. Migration and invasion test the effect of MI R-138 on the migration and invasion ability of cervical cancer cells by Transwell test, the results of in vitro migration of.Transwell cells confirmed that the overexpression of MI R-138 in He La cells could significantly inhibit cell migration in He La cells, and there was a statistically significant difference (P0.01).Transwell cell body. Compared with overexpression of MI R-NC, the overexpression of MI R-138 in He La cells could significantly inhibit the invasion of cells, and there was a statistically significant difference (P0.01).4.mi R-138 (P0.01).4.mi R-138 could be used to inhibit the development of cervical cancer by regulating h TERT expression. The target gene of the human telomerase reverse transcriptase (human Telomerase Reverse Transcriptase, H TERT) may be the direct target of MI R-138 through the literature comparison and analysis. Through the luciferase reporter experiment, it is proved that the fluorescence absorption of the plasmid is over expressed after the plasmid containing the H TERT3 'end (3' UTR) mutation site. The Western blot experiment showed that the protein expression of H TERT in He La cells was significantly inhibited after MI R-138, and.5.mi R-138 could inhibit the development of cervical cancer transplanted in nude mice after transfection of MI R-138. In the mice, the MI R-138 human cervical cancer cell transplantation tumor model was overexpressed in nude mice. Then the effect of MI R-138 on the development of cervical cancer was evaluated in vivo by observing the size of the transplanted tumor in the mice in vivo at different time points. The results suggested that the mice transplanted with He La cells of MI R-138 were significantly smaller than the control group, and the transplantation tumor volume was significantly smaller than that of the control group. The weight of the tumor was also significantly lighter than that in the control group (P0.01). Finally, we removed the transplanted tumor in the mice and tested the protein expression of H TERT through the Western blot test. The results showed that the protein expression level of H TERT in the transplanted tumor was significantly lower than that of the control group after the He La cells expressed mi R-138. Statistical difference (P0.01). Conclusions and innovation points: This study first demonstrated the role and possible mechanism of MI R-138 in the development of cervical cancer in vitro and in vivo, that is, MI R-138 may inhibit the proliferation, cloning, and inhibition of cervical cancer by combining with H TERT to inhibit the protein expression of H TERT. The anti apoptosis ability of the cells reduces the migration and invasiveness of cervical cancer cells and slows down the occurrence, development and metastasis of cervical cancer. 1. the main innovation points are: 1. this study shows that the changes in the expression level of MI R-138 in cervical cancer tissue can prompt the occurrence of cervical cancer in real time and whether it may occur or not, by detecting the total Mi of cervical cancer tissue The expression or permission of R-138 can monitor the occurrence and metastasis of cervical cancer, so mi R-138 may be used as a biological marker to screen and diagnose cervical cancer and determine its prognosis in the future. 2. the expression of MI R-138 can obviously inhibit the development of cervical cancer in nude mice by in vivo experiment, and thus for clinical treatment. It provides a reference point and theoretical basis for drug treatment of cervical cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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