本文關(guān)鍵詞： 膀胱癌 miR-137 PAQR3 細(xì)胞增殖 侵襲　出處：《哈爾濱工業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：全世界膀胱癌的發(fā)病率位于全身惡性腫瘤的第五位,在泌尿系統(tǒng)惡性腫瘤中排在第二位。大約90%的膀胱癌患者的病理分型屬于尿路上皮癌,其一般分為非肌層侵潤(rùn)性和肌層侵潤(rùn)性兩種。大約80%左右的膀胱癌患者初期被診斷為非浸潤(rùn)性尿路上皮癌,雖然在后期積極的接受了膀胱內(nèi)鏡手術(shù)和膀胱灌注治療,但在5年內(nèi)復(fù)發(fā)的患者比例仍然占到50%-70%;并且有10%-30%的患者進(jìn)一步發(fā)展成為肌層侵潤(rùn)性尿路上皮癌。浸潤(rùn)性尿路上皮癌具有較強(qiáng)的侵襲能力,患者的預(yù)后較差,5年生存率低,因此探索膀胱癌發(fā)生發(fā)展的分子機(jī)制,尋找可靠的生物標(biāo)志物和有效的治療靶點(diǎn)對(duì)提高膀胱癌患者的生存率顯得尤為重要。微小核糖核酸(micro RNA,miRNA)是一種廣泛表達(dá)的、大小為18-24nt的內(nèi)源性非編碼RNA,與靶基因的3?UTR特異性地結(jié)合從而抑制靶基因表達(dá),在調(diào)節(jié)細(xì)胞的分化、增殖及凋亡中起重要作用。很多研究表明,miRNAs與多種腫瘤進(jìn)展的有關(guān),例如:胃癌、淋巴瘤、肝癌、肺癌、子宮內(nèi)膜癌等等。miR-137是一個(gè)短鏈非編碼的RNA分子,其主基因位于人染色體Lp22。有研究表明miR-137在膀胱癌中表達(dá)失調(diào),但miR-137在膀胱癌中的作用還不清楚,尤其是如何影響膀胱癌的增殖和遷移還未見報(bào)道。因此,本文以miR-137為研究對(duì)象,分析了其在正常膀胱細(xì)胞和膀胱癌細(xì)胞系中,以及膀胱癌組織和鄰近正常粘膜組織中的表達(dá);通過生物信息學(xué)和熒光素酶報(bào)告實(shí)驗(yàn)鑒定其靶基因,并進(jìn)一步分析了miR-137及其靶基因?qū)Π螂装┘?xì)胞增殖、侵襲和遷移等生物學(xué)行為的影響,為闡明miR-137在膀胱癌發(fā)生中的作用機(jī)制奠定了基礎(chǔ)。本論文使用高通量、高靈敏度和高特異性Agilent公司的miRNA芯片,所得的數(shù)據(jù)通過Agilent Feature Extraction 10.5和genespring分析研究了膀胱癌組織與癌旁正常膀胱組織的miRNA表達(dá)譜,從中選擇在膀胱癌組織中表達(dá)上調(diào)的miR-137作為研究對(duì)象。同時(shí),從生物信息學(xué)的角度,對(duì)miR-137的結(jié)構(gòu),表達(dá)特征進(jìn)行了分析,并從表觀遺傳修飾甲基化的角度分析得出miR-137的表達(dá)與甲基化有一定的關(guān)系。進(jìn)一步分析了miR-137在正常膀胱細(xì)胞系和膀胱癌細(xì)胞系,以及膀胱癌組織和鄰近正常粘膜組織中的表達(dá)。實(shí)時(shí)定量PCR的結(jié)果表明,與正常的膀胱細(xì)胞系SVHUC-1相比,miR-137在膀胱癌細(xì)胞系中的表達(dá)顯著上調(diào);并且在所檢測(cè)的50例臨床病人的膀胱癌樣本中,miR-137在腫瘤組織中的表達(dá)水平遠(yuǎn)高于鄰近的正常粘膜組織,其中miR-137表達(dá)上調(diào)45例(45/50,90%),表達(dá)下調(diào)為5例(5/50,10%)。并將臨床獲取的50例膀胱癌組織按照pTNM和pM分組進(jìn)一步進(jìn)行分析,結(jié)果發(fā)現(xiàn)miR-137的表達(dá)隨著膀胱癌的惡化、分期的進(jìn)展而增高。分析了miR-137對(duì)膀胱癌細(xì)胞增殖、遷移及侵襲的影響。通過CCK-8細(xì)胞增殖實(shí)驗(yàn)結(jié)果表明,在膀胱癌細(xì)胞中過表達(dá)miR-137后促進(jìn)了其增殖,而加入miR-137抑制劑后抑制了膀胱癌細(xì)胞的增殖。Transwell的實(shí)驗(yàn)結(jié)果表明,miR-137高表達(dá)促進(jìn)膀胱癌細(xì)胞的侵襲和遷移,低表達(dá)抑制膀胱癌細(xì)胞的侵襲和遷移。通過生物信息學(xué)的方法預(yù)測(cè)并篩選了miR-137的靶基因,結(jié)果顯示miR-137與膜蛋白受體基因PAQR3的3'UTR互補(bǔ)。定量RT-PCR的結(jié)果顯示,在膀胱癌細(xì)胞T24中過表達(dá)miR-137后,PAQR3的表達(dá)降低;Western blot和熒光素酶的實(shí)驗(yàn)結(jié)果進(jìn)一步證實(shí),PAQR3是miR-137的一個(gè)靶基因。在T24細(xì)胞中轉(zhuǎn)染PAQR3的siRNA后,PAQR3的蛋白表達(dá)顯著降低;細(xì)胞增殖的實(shí)驗(yàn)結(jié)果顯示,干擾PAQR3后,膀胱癌細(xì)胞T24的細(xì)胞增殖能力顯著提高,這與miR-137過表達(dá)在膀胱癌細(xì)胞增殖中的影響相似。同時(shí),在T24細(xì)胞中同時(shí)轉(zhuǎn)染PAQR3過表達(dá)載體和miR-137mimics后,PAQR3表達(dá)恢復(fù),進(jìn)一步的實(shí)驗(yàn)表明PAQR3能拮抗miR-137對(duì)膀胱癌細(xì)胞系T24增殖和侵襲的促進(jìn)作用。綜上所述,miR-137在人膀胱癌的細(xì)胞和組織中的表達(dá)是顯著上調(diào)的,過表達(dá)miR-137能夠促進(jìn)膀胱癌細(xì)胞的增殖,侵襲和遷移。在細(xì)胞水平上,miR-137可以靶向膜蛋白受體基因PAQR3,并對(duì)其產(chǎn)生負(fù)調(diào)控作用;并且PAQR3能拮抗miR-137對(duì)膀胱癌細(xì)胞系T24增殖和侵襲的促進(jìn)作用。這些結(jié)果為進(jìn)一步闡明miR-137/PAQR3在腫瘤發(fā)生中的功能研究奠定基礎(chǔ),并有望成為治療膀胱癌的一種潛在策略。
[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.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.14

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