miR-182靶向IGF1R抑制肾透明细胞癌增殖、侵袭和转移机制的研究

发布时间：2018-05-11 15:26

本文选题：肾透明细胞癌 + miR-182　；参考：《郑州大学》2016年博士论文

【摘要】：背景肾细胞癌(renal cell carcinoma,RCC)也称为肾腺癌或肾癌,是来源于肾实质小管上皮细胞的恶性肿瘤。据流行病学统计,肾细胞癌在所有类型的泌尿系统恶性肿瘤中排名第二位。最近几年来的研究表明,肾细胞癌的发病率较过去明显增高。流行病学研究表明,肾细胞癌约占成年人每年新发恶性肿瘤的3%,且其发病率在过去的二十年中呈不断上升趋势。肾透明细胞癌(clear cell renal cell carcinoma,CCRCC)为肾细胞癌中最为多见的病理类型,其发病率约占所有类型肾癌发病率的80~85%。现阶段临床上对肾癌的治疗仍是以根治性肾切除为主,对于没有累及肾周筋膜的肿瘤来说,采取手术切除的方法疗效最好,但对于肾癌晚期以及肾癌术后发生复发和转移的患者依然缺乏行之有效的治疗方法。尽管目前分子靶向治疗药物已经应用于临床上肾癌的治疗当中,且患者的预后亦有了较大改善,但肾癌患者的总体治疗效果依然不佳。过去的研究表明,机体内多种因素促进了肾透明细胞癌的发生以及恶性化进程,因此开展基础实验进行深入研究,探讨肾细胞癌的发病原因以及癌细胞内信号转导通路的改变以及癌基因/抑癌基因的变化情况,对于寻找出更具有针对性的关键靶点以及更加有效的临床治疗药物具有重要意义。mi R-182为一种mi RNA。目前已有研究证实,mi R-182在多种实体恶性肿瘤中的表达量发生异常增高或降低,从而提示其对肿瘤的发生和发展具有促癌或者抑癌基因的作用,但其在肾透明细胞癌中的表达情况以及作用机制仍尚未探明。胰岛素样生长因子(insulin-like growth factors,IGFS)是生长因子的重要成员之一,其在人类机体骨骼以及肌肉的生长和分化过程中发挥着十分重要的作用,而IGFS的大部分生物学活性均由IGF-1R介导产生。IGF-1R广泛表达于生物机体各种类型的细胞中,其信号转导途径与恶性肿瘤的发生和发展之间存在密切联系。IGF-1R经由多种信号转导通路介导了肿瘤细胞的恶性化增殖、侵袭以及转移过程,同时还介导了肿瘤血管的生成以及肿瘤细胞抗凋亡等作用。除此之外,IGF-1R对透明细胞癌的发生和发展亦具有重要影响。目的本研究通过荧光定量PCR检测mi R-182在肾透明细胞癌患者肿瘤组织中的表达,并探讨其与患者的重要临床病理参数的相关性,同时分析mi R-182的表达对肾透明细胞癌细胞增殖、侵袭及转移过程的影响,探究mi R-182信号活化或抑制对于肾透明细胞癌细胞中IGF-1R表达的调控作用,研究其对肾透明细胞癌细胞侵袭和转移的影响,旨在了解肾透明细胞癌的发生和发展机制,同时为肾透明细胞癌的临床诊断以及靶向治疗提供可靠依据。方法(1)采集2010年4月~2015年8月期间就诊于郑州大学第一附属医院泌尿外科行根治性肾切除手术57例肾透明细胞癌患者。其中男性患者为38例,女性患者为19例,年龄范围在50~79岁之间。肾透明细胞癌组织学分级根据Fuhrman分级系统进行划分:I级为15例,II期为26例,III~IV级16例。临床分期按照Robson法进行划分:I期22例、II期16例,III~IV期19例。按照是否发生转移进行划分:发生转移者为19例,无转移者为38例。此外选取10例于本院进行健康体检的志愿者外周血作为健康对照组。采用荧光实时定量PCR检测mi R-182在肾透明细胞癌组织及其相应癌旁正常组织、患者血浆及健康志愿者血浆中的表达,分析mi R-182与肾透明细胞癌患者临床病理参数的相关性。(2)设计并合成mi R-182 mimic、mi R-182 inhibitor和mi R-control,随后将三者分别转染人肾透明细胞癌Caki-1细胞,以此构建mi R-182过表达或抑制表达的Caki-1细胞系,并通过荧光实时定量PCR法检测转染后上述三组细胞中mi R-182的表达用以验证。随后采用MTT法检测转染后上述三组细胞的增殖活性;克隆形成实验检测生长情况;Transwell侵袭小室法检测细胞的迁移和侵袭能力。(3)运用生物信息学软件筛选mi R-182的靶基因。采用荧光定量PCR和western blot对分别检测转染mi R-182 control、mi R-182 mimic以及mi R-182 inhibitor的人透明细胞癌Caki-1细胞中IGF-1R m RNA和蛋白的表达情况。采用IGF-1R 3’UTR定点突变的方法构建含有荧光素酶报告基因的IGF-1R 3’UTR Mut(突变型)质粒载体,同时构建IGF-1R 3’UTR WT(野生型)质粒载体,将两者分别与mi R-182 mimic共转染Caki-1细胞。采用荧光素酶报告系统进行检测上述细胞中荧光素酶的表达量。使用si-IGF1R构建IGF-1R表达抑制的Caki-1细胞系,并用荧光定量PCR和western blot法分别检测细胞中IGF-1Rm RNA和蛋白的表达。将人透明细胞癌Caki-1细胞分为3组,采用Transwell小室法分别检测Control组、mi R-182 inhibitor组以及si-IGF1R+mi R-182 inhibitor组细胞的侵袭以及迁移能力。结果(1)荧光定量PCR检测结果表明,肾透明细胞癌组织中mi R-182的表达量明显低于其在癌旁相应正常组织中的表达(P0.05);mi R-182在肾透明细胞癌患者血浆中的表达明显低于健康志愿者(P0.05)。mi R-182与肾透明细胞癌患者临床病理参数分析结果表明,mi R-182的表达与肾透明细胞癌患者的组织学分级、临床分期以及转移有关,而与患者的性别和年龄因素无关。(2)荧光定量PCR检测结果表明,与control组相比,mi R-182 mimic组细胞中mi R-182的表达水平明显增高(P0.05),mi R-182 inhibitor组细胞中mi R-182的表达量明显降低(P0.05),表明mi R-182过表达/抑制表达Caki-1细胞系构建成功。MTT法检测结果表明,转染mi R-182 mimic的Caki-1细胞在48 h、72 h、96 h时的增殖率明显低于对照组(P0.05);转染mi R-182 inhibitor的Caki-1细胞在48 h、72 h、96 h时的增殖率明显高于对照组(P0.05)。克隆形成实验结果表明,mi R-182 mimic组Caki-1细胞的克隆形成数明显低于对照组(P0.05);mi R-182 inhibitor组Caki-1细胞的克隆形成数明显高于对照组(P0.05)。Transwell小室法检测结果表明,mi R-182 mimic组Caki-1细胞的迁移和侵袭细胞数均明显低于对照组(P0.05);而mi R-182 inhibitor组Caki-1细胞的迁移和侵袭细胞数均明显高于对照组(P0.05)。(3)生物信息学软件预测结果表明,IGF-1R为mi R-182的靶基因。转染mi R-182 mimic后,Caki-1细胞中IGF-1R m RNA和蛋白的表达量明显降低(P0.05);转染mi R-182 inhibitor后,Caki-1细胞中IGF-1R m RNA和蛋白的表达量明显增高(P0.05)。荧光素酶检测结果表明,与对照组相比,mi R-182 mimic与IGF1R 3’UTR WT共转染组的荧光素酶表达量明显降低(P0.05);而mi R-182 mimic和IGF1R 3’UTR Mut共转染组细胞中的荧光素酶表达量则与对照组相比无显著差异(P0.05)。si-IGF1R干扰表达后Caki-1细胞IGF-1R m RNA和蛋白的表达量明显降低。Transwell小室法检测结果表明,与对照组相比,mi R-182 inhibitor组细胞的迁移和侵袭细胞数明显增高(P0.05),而si-IGF1R+mi R-182 inhibitor组细胞的迁移和侵袭细胞数则明显降低(P0.05)。结论mi R-182在肾透明细胞癌组织以及患者外周血中呈低表达,且其表达量与肾透明细胞癌患者的组织学分级、临床分期以及有无转移密切相关。mi R-182能够通过直接抑制IGF-1R的表达来实现对肾透明细胞癌细胞增殖、侵袭以及转移的调控,从而抑制肾透明细胞癌的发生和发展。
[Abstract]:Renal cell carcinoma (RCC), also known as renal adenocarcinoma or renal carcinoma, is a malignant tumor derived from renal tubular epithelial cells. According to epidemiological statistics, renal cell carcinoma is ranked second in all types of urological malignancies. Recent studies have shown that the incidence of renal cell carcinoma is significantly higher than that in the past. Epidemiological studies have shown that renal cell carcinoma accounts for about 3% of new malignant tumors in adults, and its incidence is increasing in the past twenty years. Clear cell renal cell carcinoma (CCRCC) is the most common pathological type of renal cell carcinoma, and its incidence is about 80~85 in the incidence of all types of renal cancer. At this stage, the treatment of renal carcinoma is still mainly radical nephrectomy. For the tumor without involvement of the perirenal fascia, surgical resection is the best treatment. However, there is still a lack of effective treatment for patients with late renal cancer and recurrence and metastasis of renal carcinoma. Drugs have been used in the treatment of clinical renal cancer, and the prognosis of the patients has been improved greatly, but the overall treatment effect of renal cancer patients is still poor. Past studies have shown that many factors in the body promote the occurrence and malignant process of renal clear cell carcinoma. The cause of cell carcinoma and the change of signal transduction pathway in cancer cells and the change of oncogene / tumor suppressor gene are of great significance for finding more targeted key targets and more effective clinical drugs.Mi R-182 for a kind of MI RNA., MI R-182 is in a variety of malignant swelling. The expression of insulin-like growth factors (IGFS) is an important member of the growth factor. First, it plays a very important role in the growth and differentiation of human skeleton and muscle, and most of the biological activity of IGFS is mediated by IGF-1R to produce.IGF-1R widely expressed in various types of cells. The signal transduction pathway is closely related to the occurrence and development of malignant swollen tumor,.IGF-1 R mediated the malignant proliferation, invasion and metastasis of tumor cells via a variety of signal transduction pathways, and also mediates the formation of tumor vessels and the anti apoptosis effect of tumor cells. In addition, IGF-1R also plays an important role in the development and development of clear cell carcinoma. Objective this study was to detect mi R-182 by fluorescence quantitative PCR The expression in the tumor tissue of patients with renal clear cell carcinoma and its correlation with the important clinicopathological parameters of the patients, and the effect of the expression of MI R-182 on the proliferation, invasion and metastasis of renal clear cell carcinoma cells, and to explore the regulation of the activation or inhibition of the MI R-182 signal to the expression of IGF-1R in the renal clear cell carcinoma cells. The purpose of this study is to investigate the invasion and metastasis of renal clear cell carcinoma cells, to understand the mechanism of the occurrence and development of renal clear cell carcinoma, and to provide a reliable basis for the clinical diagnosis and target treatment of renal clear cell carcinoma. Method (1) collected in the Department of Urology, the First Affiliated Hospital of Zhengzhou University, April 2010, during the period of August ~2015. 57 cases of renal clear cell carcinoma were treated with radical nephrectomy, of which 38 cases were male and 19 female patients, age range was between 50~79 years. The histological grading of renal clear cell carcinoma was divided according to Fuhrman classification system: 15 cases of I grade, 26 cases of II stage, 16 cases of III~IV grade. The clinical stages were divided according to Robson method: 22 cases in I phase, II 16 cases and 19 cases of III~IV phase were divided into 19 cases and 38 cases without metastasis. In addition, the peripheral blood of 10 cases of healthy volunteers in our hospital was selected as the healthy control group. The fluorescence real-time quantitative PCR was used to detect mi R-182 in the tissues of renal hyaline cell carcinoma and the corresponding normal tissue adjacent to the cancer. Plasma and healthy volunteers were expressed in plasma, and the correlation between MI R-182 and the clinicopathological parameters of patients with renal clear cell carcinoma was analyzed. (2) mi R-182 mimic, MI R-182 inhibitor and MI R-control were designed and synthesized. Then the three persons were transfected into the Caki-1 cell of human renal clear cell carcinoma. Cell lines were detected by fluorescence real-time quantitative PCR method to detect the expression of MI R-182 in these three groups of cells after transfection. Then MTT method was used to detect the proliferation activity of the three groups of the above transfected cells; the clone formation test was used to detect the growth of the cells; Transwell invaded the cell to detect the cell migration and invasion ability. (3) use bioinformatics soft. The target gene of MI R-182 was screened by using the fluorescent quantitative PCR and Western blot to detect the expression of MI R-182 control, MI R-182 mimic and the human transparent cell carcinoma cell carcinoma cells. 1R 3 'UTR Mut (mutant) plasmid vector, simultaneously constructed IGF-1R 3' UTR WT (wild type) plasmid vector, and co transfected Caki-1 cells with MI R-182 mimic respectively. The luciferase expression was detected by the luciferase reporter system. The expression of IGF-1Rm RNA and protein in the cells was detected by PCR and Western blot. The Caki-1 cells of human transparent cell carcinoma were divided into 3 groups. The invasion and migration ability of Control group, MI R-182 inhibitor group and si-IGF1R+mi MI group were detected by Transwell chamber method. Results (1) the results of quantitative fluorescence detection showed that The expression of MI R-182 in the tissues of renal clear cell carcinoma was significantly lower than that in the normal tissues near the carcinoma (P0.05). The expression of MI R-182 in the plasma of patients with renal clear cell carcinoma was significantly lower than that of healthy volunteers (P0.05).Mi R-182 and renal clear cell carcinoma. The expression of MI R-182 and the transparency of renal transparency were revealed. The histological grade, clinical staging and metastasis of the patients were not related to the sex and age factors of the patients. (2) the results of fluorescence quantitative PCR showed that the expression level of MI R-182 in the MI R-182 mimic group was significantly higher than that in the control group (P0.05), and the expression of MI in MI R-182 inhibitor group was significantly reduced. .05) showed that MI R-182 overexpressed / suppressed expression of Caki-1 cell lines was successfully constructed by.MTT assay. The proliferation rate of Caki-1 cells transfected with MI R-182 mimic in 48 h, 72 h and 96 h was significantly lower than that of the control group, and the proliferation rate of the cells transfected in 48, 72, and 96 was significantly higher than that of the control group. The experimental results showed that the number of clone formation of Caki-1 cells in MI R-182 mimic group was significantly lower than that of the control group (P0.05), and the number of Caki-1 cells in the MI R-182 inhibitor group was significantly higher than that of the control group (P0.05).Transwell chamber method. 05), and the number of migration and invasion of Caki-1 cells in MI R-182 inhibitor group was significantly higher than that of the control group (P0.05). (3) the prediction results of bioinformatics software showed that IGF-1R was the target gene of MI R-182. The expression of IGF-1R m RNA and protein in the cells increased significantly (P0.05). The luciferase expression of MI R-182 mimic and IGF1R 3 'UTR WT co transfection group was significantly lower than that of the control group (P0.05), while the luciferase expression in the cells of the MI R-182 and the co transfected group was compared with the control group. The expression of IGF-1R m RNA and protein in Caki-1 cells decreased significantly after the expression of.Si-IGF1R interference without significant difference (P0.05), and the results of.Transwell compartment assay showed that the number of cell migration and invasion cells in MI R-182 inhibitor group increased significantly (P0.05) compared with the control group. The number of cells decreased significantly (P0.05). Conclusion the expression of MI R-182 in the tissues of renal clear cell carcinoma and the peripheral blood of the patients was low, and the expression of R-182 was closely related to the histological grade of the patients with clear cell carcinoma of the kidney, the clinical stages and the close correlation between the metastasis and the metastasis of the renal clear cell carcinoma cells by directly inhibiting the expression of IGF-1R. Regulation of proliferation, invasion and metastasis can inhibit the occurrence and development of renal clear cell carcinoma.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.11

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