miR-137在膀胱癌细胞中的表达及调控其增殖和侵袭的研究

发布时间：2018-03-01 20:14

本文关键词： 膀胱癌 miR-137 PAQR3 细胞增殖侵袭　出处：《哈尔滨工业大学》2016年博士论文　论文类型：学位论文

【摘要】：全世界膀胱癌的发病率位于全身恶性肿瘤的第五位,在泌尿系统恶性肿瘤中排在第二位。大约90%的膀胱癌患者的病理分型属于尿路上皮癌,其一般分为非肌层侵润性和肌层侵润性两种。大约80%左右的膀胱癌患者初期被诊断为非浸润性尿路上皮癌,虽然在后期积极的接受了膀胱内镜手术和膀胱灌注治疗,但在5年内复发的患者比例仍然占到50%-70%;并且有10%-30%的患者进一步发展成为肌层侵润性尿路上皮癌。浸润性尿路上皮癌具有较强的侵袭能力,患者的预后较差,5年生存率低,因此探索膀胱癌发生发展的分子机制,寻找可靠的生物标志物和有效的治疗靶点对提高膀胱癌患者的生存率显得尤为重要。微小核糖核酸(micro RNA,miRNA)是一种广泛表达的、大小为18-24nt的内源性非编码RNA,与靶基因的3?UTR特异性地结合从而抑制靶基因表达,在调节细胞的分化、增殖及凋亡中起重要作用。很多研究表明,miRNAs与多种肿瘤进展的有关,例如:胃癌、淋巴瘤、肝癌、肺癌、子宫内膜癌等等。miR-137是一个短链非编码的RNA分子,其主基因位于人染色体Lp22。有研究表明miR-137在膀胱癌中表达失调,但miR-137在膀胱癌中的作用还不清楚,尤其是如何影响膀胱癌的增殖和迁移还未见报道。因此,本文以miR-137为研究对象,分析了其在正常膀胱细胞和膀胱癌细胞系中,以及膀胱癌组织和邻近正常粘膜组织中的表达;通过生物信息学和荧光素酶报告实验鉴定其靶基因,并进一步分析了miR-137及其靶基因对膀胱癌细胞增殖、侵袭和迁移等生物学行为的影响,为阐明miR-137在膀胱癌发生中的作用机制奠定了基础。本论文使用高通量、高灵敏度和高特异性Agilent公司的miRNA芯片,所得的数据通过Agilent Feature Extraction 10.5和genespring分析研究了膀胱癌组织与癌旁正常膀胱组织的miRNA表达谱,从中选择在膀胱癌组织中表达上调的miR-137作为研究对象。同时,从生物信息学的角度,对miR-137的结构,表达特征进行了分析,并从表观遗传修饰甲基化的角度分析得出miR-137的表达与甲基化有一定的关系。进一步分析了miR-137在正常膀胱细胞系和膀胱癌细胞系,以及膀胱癌组织和邻近正常粘膜组织中的表达。实时定量PCR的结果表明,与正常的膀胱细胞系SVHUC-1相比,miR-137在膀胱癌细胞系中的表达显著上调;并且在所检测的50例临床病人的膀胱癌样本中,miR-137在肿瘤组织中的表达水平远高于邻近的正常粘膜组织,其中miR-137表达上调45例(45/50,90%),表达下调为5例(5/50,10%)。并将临床获取的50例膀胱癌组织按照pTNM和pM分组进一步进行分析,结果发现miR-137的表达随着膀胱癌的恶化、分期的进展而增高。分析了miR-137对膀胱癌细胞增殖、迁移及侵袭的影响。通过CCK-8细胞增殖实验结果表明,在膀胱癌细胞中过表达miR-137后促进了其增殖,而加入miR-137抑制剂后抑制了膀胱癌细胞的增殖。Transwell的实验结果表明,miR-137高表达促进膀胱癌细胞的侵袭和迁移,低表达抑制膀胱癌细胞的侵袭和迁移。通过生物信息学的方法预测并筛选了miR-137的靶基因,结果显示miR-137与膜蛋白受体基因PAQR3的3'UTR互补。定量RT-PCR的结果显示,在膀胱癌细胞T24中过表达miR-137后,PAQR3的表达降低;Western blot和荧光素酶的实验结果进一步证实,PAQR3是miR-137的一个靶基因。在T24细胞中转染PAQR3的siRNA后,PAQR3的蛋白表达显著降低;细胞增殖的实验结果显示,干扰PAQR3后,膀胱癌细胞T24的细胞增殖能力显著提高,这与miR-137过表达在膀胱癌细胞增殖中的影响相似。同时,在T24细胞中同时转染PAQR3过表达载体和miR-137mimics后,PAQR3表达恢复,进一步的实验表明PAQR3能拮抗miR-137对膀胱癌细胞系T24增殖和侵袭的促进作用。综上所述,miR-137在人膀胱癌的细胞和组织中的表达是显著上调的,过表达miR-137能够促进膀胱癌细胞的增殖,侵袭和迁移。在细胞水平上,miR-137可以靶向膜蛋白受体基因PAQR3,并对其产生负调控作用;并且PAQR3能拮抗miR-137对膀胱癌细胞系T24增殖和侵袭的促进作用。这些结果为进一步阐明miR-137/PAQR3在肿瘤发生中的功能研究奠定基础,并有望成为治疗膀胱癌的一种潜在策略。
[Abstract]:The incidence rate of bladder cancer in the world in all malignant tumors in fifth, urinary system malignant tumors in second. Approximately 90% of patients with bladder cancer's pathological type belongs to urothelial carcinoma, which is generally divided into non muscle invasive and muscle invasive bladder cancer patients in early two. About 80% of those diagnosed with non invasive urothelial carcinoma, although in the late positive received bladder endoscopic surgery and intravesical therapy, but the recurrence in 5 years the proportion of patients still accounted for 50%-70%; and 10%-30% were further developed into muscle invasive urothelial carcinoma with invasion. Strong ability of invasion of urothelial carcinoma with poor prognosis, 5 years survival rate is low, so the exploration of molecular mechanism of the occurrence and development of bladder cancer, looking for reliable biomarkers and effective therapeutic target to improve the survival rate of patients with bladder cancer It is particularly important. The micro ribonucleic acid (micro RNA miRNA) is a widely expressed and endogenous 18-24nt the size of the non target gene encoding RNA, and 3? UTR bind specifically to inhibit target gene expression in regulating cell differentiation, proliferation and apoptosis play an important role. Many studies show that the progress of miRNAs with a variety of tumors such as gastric cancer, lymphoma, liver cancer, lung cancer, endometrial cancer,.MiR-137 is a short chain of non encoding RNA molecules, the main gene is located on human chromosome Lp22. studies showed that the expression of miR-137 in bladder cancer disorders, but the role of miR-137 in bladder cancer is unclear. Especially how to influence the proliferation and migration of bladder cancer has not been reported. Therefore, this paper takes miR-137 as the research object, analyzes its cells in normal bladder and bladder cancer cell lines, and bladder cancer tissues and adjacent normal mucosa The expression; bioinformatics and luciferase reporter experiments for the identification of target genes, and further analysis of miR-137 and its target gene on the proliferation of bladder cancer cells, the influence of biological behavior of invasion and migration etc., laid the foundation for elucidating the mechanism of miR-137 in bladder cancer. The use of high throughput, high sensitivity miRNA chip and the high specificity of Agilent, the data obtained by Agilent Feature Extraction 10.5 and genespring of bladder cancer and adjacent normal bladder tissue miRNA expression profiles in bladder cancer tissues and up-regulated miR-137 as the research object from it. At the same time, from the perspective of bioinformatics, the structure of miR-137 the expression characteristics were analyzed, and the concept of genetic modification of methylation analysis from the angle of the table there is a certain relationship between miR-137 expression and methylation. Further analysis The miR-137 cells in the normal bladder and bladder cancer cell lines, and the expression of bladder cancer tissues and adjacent normal mucosa tissues. The real-time quantitative PCR results showed that compared with normal bladder cell line SVHUC-1, up regulate the expression of miR-137 in bladder cancer cell lines; and in 50 patients detected the bladder cancer samples, the expression level of miR-137 in tumor tissues is much higher than that of normal mucosa adjacent, the expression of miR-137 (45/50,90%), 45 cases of up-regulated expression in 5 cases (5/50,10%). And 50 cases of bladder cancer tissues from clinical pTNM and pM group according to the further analysis, it is found that miR-137 with the deterioration of expression of bladder cancer, staging progress increased. Analysis of miR-137 on the proliferation of bladder cancer cells and the effects of migration and invasion of CCK-8 cell proliferation. The experimental results show that over expression in bladder cancer cells MiR-137 promoted the proliferation, and after adding miR-137 inhibitor inhibited the proliferation of bladder cancer cell.Transwell. The experimental results show that the high expression of miR-137 promotes migration and invasion of bladder cancer cells, low expression inhibited the invasion and migration of bladder cancer cells. The prediction method of bioinformatics and screening the target gene of miR-137. The results show complementary protein receptor miR-137 and membrane PAQR3 gene 3'UTR. Quantitative RT-PCR results showed that over expression of miR-137 in bladder cancer T24 cells, decreased the expression of PAQR3; the experimental results Western blot and luciferase further confirmed that PAQR3 is a target gene of miR-137. SiRNA in T24 cells after PAQR3 dye transfer, the expression of PAQR3 protein significantly decreased cell proliferation; the experimental results show that the interference of PAQR3, cell proliferation of bladder cancer T24 cells significantly increased, and the expression of miR-137 in bladder Effects of the proliferation of cancer cells in similar. At the same time, at the same time in T24 cells transfected with PAQR3 expression vector and the expression of PAQR3 miR-137mimics after recovery, further experiments showed that PAQR3 could promote the inhibition of miR-137 on proliferation and invasion of human bladder cancer cell line T24. In summary, the expression of miR-137 in human bladder cancer cells and tissues is up-regulated, overexpression of miR-137 could promote the proliferation of bladder cancer cells, invasion and migration. At the cellular level, miR-137 can target membrane protein receptor gene PAQR3, and the negative regulation effect; and PAQR3 can promote the inhibition of miR-137 on proliferation and invasion of human bladder cancer cell line T24. These results lay further clarify the function of miR-137/PAQR3 in tumorigenesis, and is expected to become a potential strategy for the treatment of bladder cancer.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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