本文關(guān)鍵詞： 膀胱癌 微小RNA 遷移與侵襲 c-Met　出處：《浙江大學(xué)》2015年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景：膀胱癌是泌尿系統(tǒng)最常見(jiàn)的惡性腫瘤,也是全身十大常見(jiàn)腫瘤之一。占我國(guó)泌尿生殖系腫瘤發(fā)病率的第一位,在西方其發(fā)病率僅次于前列腺癌,居第2位。然而,目前的研究仍然對(duì)膀胱癌的發(fā)生以及進(jìn)展的機(jī)制缺乏足夠深入的理解。現(xiàn)在可以明確的是,膀胱癌發(fā)生及進(jìn)展是一個(gè)多因素涉及、多基因參與、多步驟形成以及多途徑變化的過(guò)程,異�；蛐偷幕A(chǔ)在外在高危因素的作用下逐漸積累,最終出現(xiàn)表型的惡形。miRNA是近年來(lái)研究比較熱點(diǎn)的一類(lèi)由內(nèi)源基因編碼的非編碼RNA單鏈分子,長(zhǎng)度約為20-24個(gè)核苷酸。人們已經(jīng)認(rèn)識(shí)到這些普遍存在的小分子在真核基因表達(dá)調(diào)控中有著廣泛的作用。miRNA的水平在不同組織、不同發(fā)育階段中有顯著差異,呈現(xiàn)具有分化的位相性和時(shí)序性。對(duì)于各類(lèi)疾病,尤其是腫瘤而言,miRNA也出現(xiàn)了時(shí)間和空間上的表達(dá)異常,本研究希望在膀胱癌中尋找這樣的異常表達(dá),并發(fā)現(xiàn)其在基因調(diào)控中扮演的作用。 目的：在膀胱癌患者手術(shù)切除的標(biāo)本組織中采用Real Time PCR的方法檢測(cè)miR-101的表達(dá)情況；體外實(shí)驗(yàn)中根據(jù)一系列實(shí)驗(yàn)方法鑒定miR-101在膀胱癌T24細(xì)胞株中的生物學(xué)功能。并且,通過(guò)生物信息學(xué)對(duì)miR-101作用靶點(diǎn)進(jìn)行預(yù)測(cè),并行生物學(xué)實(shí)驗(yàn)驗(yàn)證。 方法：1.在20對(duì)配對(duì)的膀胱癌組織標(biāo)本檢測(cè)miR-101的相對(duì)表達(dá)量,對(duì)膀胱癌組織及對(duì)照的正常粘膜組織中miR-101的表達(dá)差異進(jìn)行統(tǒng)計(jì),并分析不同病例級(jí)別與分期的膀胱癌中miR-101的表達(dá)情況； 2.使用對(duì)膀胱癌T24細(xì)胞株轉(zhuǎn)染miR-101mimic使其過(guò)表達(dá)的方法進(jìn)行一系列功能實(shí)驗(yàn)。通過(guò)CCK-8、平板克隆形成、流式細(xì)胞周期和凋亡檢測(cè)、劃痕愈合實(shí)驗(yàn)和Transwell小室法等手段來(lái)分析上調(diào)miR-101的表達(dá)對(duì)細(xì)胞增殖及遷移侵襲能力帶來(lái)的影響； 3.使用在線(xiàn)軟件對(duì)miR-101進(jìn)行靶基因預(yù)測(cè),然后利用Real Time PCR, Western Blot等驗(yàn)證靶基因mRNA以及蛋白表達(dá)量是否相應(yīng)變化,并利用熒光素酶雙報(bào)告系統(tǒng)對(duì)miR-101的靶序列進(jìn)行鑒定,使用拯救實(shí)驗(yàn)進(jìn)一步驗(yàn)證miR-101的作用靶基因。 結(jié)果：1.收集的20例膀胱癌癌組織標(biāo)本中,有19例為miR-101相對(duì)低表達(dá),占95%。癌組織中miR-101表達(dá)量平均約癌旁組織的1/2。肌層浸潤(rùn)性膀胱癌與非肌層浸潤(rùn)性膀胱癌以及高級(jí)別膀胱癌與低級(jí)別膀胱癌在miR-101的相對(duì)表達(dá)量的平均值有差異,但未能有統(tǒng)計(jì)學(xué)上的顯著性意義。 2.膀胱癌T24細(xì)胞株和正常的膀胱移行上皮細(xì)胞株SV-HUC-1相比,miR-101也表現(xiàn)為低表達(dá)。 3.體外轉(zhuǎn)染miR-101mimic至膀胱癌T24細(xì)胞株使miR-101過(guò)表達(dá),50nM濃度可以抑制細(xì)胞增殖活力。體外平板克隆形成實(shí)驗(yàn)未能明顯抑制細(xì)胞克隆形成。劃痕愈合實(shí)驗(yàn)和Transwell小室等實(shí)驗(yàn)顯示miR-101過(guò)表達(dá)可以減弱T24細(xì)胞的遷移和侵襲能力,但對(duì)細(xì)胞周期和凋亡無(wú)明顯影響。 4.通過(guò)在線(xiàn)軟件的分析,預(yù)測(cè)c-Met可能是miR-101的靶基因,同時(shí)通過(guò)Real Time PCR和Western Blot實(shí)驗(yàn)檢測(cè)到c-Met在過(guò)表達(dá)miR-101后mRNA水平和蛋白水平都有下調(diào)。接下來(lái)的熒光素酶雙報(bào)告系統(tǒng)分析證實(shí)c-Met是miR-101的直接作用靶點(diǎn),其3'-UTR中存在miR-101調(diào)控的作用序列。 結(jié)論：1.膀胱癌組織中miR-101與癌旁對(duì)照組織相比,相對(duì)表達(dá)量較低。但是本研究未能提示miR-101表達(dá)量高低與膀胱癌的惡性程度相關(guān)。 2.體外轉(zhuǎn)染miR-101mimic使miR-101過(guò)表達(dá)削弱了膀胱癌T24細(xì)胞株的增殖能力,但對(duì)克隆形成及細(xì)胞周期凋亡無(wú)明顯影響。 3.miR-101的直接靶基因是c-Met,并影響下游ERK1/2蛋白的磷酸化水平。
[Abstract]:Background: bladder cancer is the most common malignant tumor of urinary system, the body is also one of the ten most common tumor. Our first urogenital tumor incidence in the west, its incidence after prostate cancer, ranking second. However, the current study is still on the occurrence of bladder cancer and the mechanism of the lack of progress have a deep understanding. It is now clear that the occurrence and progression of bladder cancer is related to multiple factors, multiple genes, multiple steps and multiple ways to change the formation process, abnormal genotypes accumulated in the external risk factors under the action of ultimate evil shape.MiRNA phenotype is comparative research in recent years focus of a class from non RNA single molecule encoding endogenous gene encoding, the length is about 20-24 nucleotides. It has been recognized that these ubiquitous small molecule regulation is widely expressed in eukaryotic gene The effect of the level of.MiRNA in different tissues, there is significant difference in different developmental stages, showing the spatial and temporal differentiation. For various diseases, especially tumor, the expression of miRNA also appeared on time and space anomalies, this study hopes to find the abnormal expression in bladder cancer, and found it plays a role in gene regulation.
Objective: to detect the expression of miR-101 by Real PCR Time in patients with bladder cancer tissues; in vitro according to a series of experimental methods for identification of miR-101 in bladder cancer cell line T24 in biological function. Moreover, by means of bioinformatics to predict the targets of miR-101, parallel biological experiments.
Methods: 1.. In 20 pairs of paired bladder cancer tissues, the relative expression of miR-101 was detected. The difference of miR-101 expression between bladder cancer tissues and normal mucosa tissues was statistically analyzed, and the expression of miR-101 in different grade and stage of bladder cancer was analyzed.
Methods 2. of bladder carcinoma cell line T24 transfected with miR-101mimic and its over expression of a series of functional experiment. Through CCK-8, colony formation, flow cytometry cell cycle and apoptosis detection, expression means wound healing assay and Transwell chamber method to analyze the regulation of miR-101 brings on the invasion ability of cell proliferation and migration;
3. the use of online software to predict target gene of miR-101, and then use the Real Time PCR, Western Blot mRNA and protein expression of target genes to verify whether the amount of the corresponding changes, and the target sequence of miR-101 was identified by dual luciferase system, using the save target experiment to further validate the miR-101.
Results: 20 cases of carcinoma of bladder cancer specimens collected in 1., there were 19 cases of low miR-101 expression, 95%. miR-101 expression in carcinomas of 1/2. muscle layer averaged about cancer tissues of invasive bladder cancer and non muscle invasive bladder cancer and high-grade bladder cancer and bladder cancer at a relatively low level expression of miR-101 with different average values, but not statistically significant.
2. the T24 cell line of bladder cancer and the normal bladder transitional cell line SV-HUC-1, miR-101 also showed low expression.
3. in vitro transfection of miR-101mimic to bladder cancer cell line T24. Overexpression of miR-101, the concentration of 50nM can inhibit cell proliferation in vitro. Colony formation assay failed to inhibit the formation of cell clones. Wound healing assay and Transwell chamber experiment showed that overexpression of miR-101 can reduce the migration and invasion of T24 cells, but had no obvious effect on cell cycle and apoptosis.
4. through the analysis of online software, c-Met may predict the target gene of miR-101, while Time PCR and Western Real by Blot assay after overexpression of miR-101 to c-Met in mRNA and protein level were down regulated. The dual luciferase system analysis confirmed that c-Met is a direct target of miR-101, miR-101 regulatory sequences the 3'-UTR.
Conclusion: 1., the expression of miR-101 in bladder cancer tissue is lower than that in adjacent tissues. However, this study failed to indicate that the expression level of miR-101 is correlated with the malignancy of bladder cancer.
2. in vitro transfection of miR-101mimic caused miR-101 overexpression to weaken the proliferation ability of bladder cancer T24 cell line, but had no significant effect on clonogenic formation and cell cycle apoptosis.
The direct target gene of 3.miR-101 is c-Met, which affects the phosphorylation level of the downstream ERK1/2 protein.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R737.14

【共引文獻(xiàn)】

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