本文選題：微小RNA　切入點(diǎn)：膀胱癌　出處：《浙江大學(xué)》2014年博士論文

【摘要】：[背景] 膀胱癌是人類最常見(jiàn)的惡性腫瘤之一,但其發(fā)病機(jī)制復(fù)雜,涉及許多基因表達(dá)、功能異常及多種信號(hào)通路的改變。當(dāng)前,膀胱癌發(fā)生、發(fā)展的分子遺傳學(xué)機(jī)制尚不清楚。 MicroRNA是在真核生物中發(fā)現(xiàn)的一種內(nèi)源性的具有調(diào)控功能的非編碼單鏈小分子RNA,大小約21-25個(gè)核苷酸,通常在轉(zhuǎn)錄后水平調(diào)控基因表達(dá)。許多研究表明,microRNA參與腫瘤細(xì)胞的分化、增殖、凋亡、侵襲、轉(zhuǎn)移及等各個(gè)環(huán)節(jié)。microRNA在膀胱癌細(xì)胞中也存在的異常表達(dá),其可能在腫瘤發(fā)生與發(fā)展過(guò)程中起著重要的作用。 miR-520b是最近發(fā)現(xiàn)的一種在腫瘤中異常表達(dá)的重要microRNA.初步研究發(fā)現(xiàn),其在多種惡性腫瘤(包括膀胱癌在內(nèi))中表達(dá)顯著下調(diào),我們推測(cè)其對(duì)膀胱癌可能有抑癌作用。 [目的] 本論文探討了miR-520b在膀胱癌發(fā)生、發(fā)展中的生物學(xué)功能,并對(duì)其相關(guān)的分子作用機(jī)制進(jìn)行深入的研究。 [方法] 1。在18對(duì)配對(duì)的組織標(biāo)本中進(jìn)行miR-520b的相對(duì)表達(dá)量檢測(cè),并統(tǒng)計(jì)分析miR-520b在膀胱癌及癌旁膀胱粘膜組織中的表達(dá)差異,以及tniR-520b表達(dá)量與膀胱癌不同組織病理分期及分級(jí)之間的關(guān)系；RT-PCR驗(yàn)證]miR-520b在膀胱癌細(xì)胞系(T24、UC3)與永生化人正常膀胱粘膜上皮細(xì)胞株SV-HUC-1的表達(dá)差異。 2.通過(guò)體外轉(zhuǎn)染miR-520b mimic的方式來(lái)進(jìn)行獲得性功能實(shí)驗(yàn)。上調(diào)miR-520b在膀胱癌細(xì)胞系T24、UC3中的表達(dá),以CCK8、平板克隆形成、流式細(xì)胞周期檢測(cè)等體外實(shí)驗(yàn)驗(yàn)證miR-520b對(duì)膀胱癌細(xì)胞增殖的影響。以劃痕實(shí)驗(yàn)以及Transwell小室遷移和侵襲來(lái)分析miR-520b過(guò)表達(dá)對(duì)細(xì)胞遷移、侵襲的影響； 3.運(yùn)用生物信息學(xué)方法,預(yù)測(cè)miR-520b可能的下游靶基因,并對(duì)預(yù)測(cè)的靶基因進(jìn)行功能聚類分析,結(jié)合文獻(xiàn)、預(yù)實(shí)驗(yàn)驗(yàn)證等方法,初步判斷Cyclin D1可能是miR-520b的靶基因之一。利用Western Blot等進(jìn)行靶蛋白表達(dá)量的驗(yàn)證,利用qRT-PCR、雙熒光素酶報(bào)告系統(tǒng)對(duì)miR-520b的作用的靶序列進(jìn)行鑒定。 [結(jié)果] 1.收集的18例膀胱癌組織標(biāo)本中,有16例為miR-520b相對(duì)低表達(dá),占88.9%。癌組織中miR-520b表達(dá)量平均約癌旁組織的56%。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-520b的相對(duì)表達(dá)量的平均值有差異,但在統(tǒng)計(jì)學(xué)上無(wú)顯著性意義。 2.膀胱癌細(xì)胞株T24和UC3細(xì)胞同樣存在miR-520b低表達(dá),與永生化的膀胱移行上皮細(xì)胞株SV-HUC-1相比,T24細(xì)胞和UC3細(xì)胞中]miR-520b的表達(dá)量均約為SV細(xì)胞的1／2。 3.通過(guò)對(duì)T24細(xì)胞和UC3細(xì)胞轉(zhuǎn)染miR-520b mimic來(lái)過(guò)表達(dá)細(xì)胞中的miR-520b,可以抑制其生長(zhǎng)。其抑制率呈現(xiàn)濃度依賴性,在50nM miR-520b mimic處理48小時(shí)后,T24細(xì)胞和UC3細(xì)胞的抑制率分別達(dá)到31.8%和34.0%。流式細(xì)胞周期檢測(cè)發(fā)現(xiàn)miR-520b mimic處理可以誘導(dǎo)T24細(xì)胞和UC3發(fā)生G1期阻滯,50nM miR-520b mimic作用48小時(shí)后,T24細(xì)胞和UC3細(xì)胞G1期的比例分別增加12%和19%。而50nM miR-520b mimic作用48小時(shí)后,流式細(xì)胞凋亡檢測(cè)提示miR-520b過(guò)表達(dá)并不能誘導(dǎo)細(xì)胞凋亡。miR-520b過(guò)表達(dá)可以抑制T24細(xì)胞和UC3細(xì)胞體內(nèi)外克隆形成能力。體外平板克隆形成實(shí)驗(yàn)顯示,miR-520b過(guò)表達(dá)使T24細(xì)胞和UC3的克隆形成率分別下降7.5%(從18%降到10.5%)和10.6%(從21.8%降到11.2%)。劃痕實(shí)驗(yàn)、Transwell小室實(shí)驗(yàn)提示miR-520b過(guò)表達(dá)不能抑制T24細(xì)胞和UC3細(xì)胞的侵襲和遷移。 4.我們?cè)赥24細(xì)胞和UC3細(xì)胞中檢測(cè)到Cyclin D1、CDK4、pRb和E2F3在miR-520b過(guò)表達(dá)后在蛋白水平有下調(diào)。進(jìn)一步行RT-PCR分析和雙熒光素酶報(bào)告系統(tǒng)分析證實(shí)Cyclin D1是miR-520b的直接作用靶點(diǎn),其3-UTR中存在miR-520b調(diào)控的作用序列。 [結(jié)論] 1與正常組織和細(xì)胞相比,膀胱癌組織和細(xì)胞中miR-520b的表達(dá)量呈現(xiàn)顯著下調(diào)。但是本研究未能提示miR-520b表達(dá)量高低與膀胱癌的分級(jí)、分期相關(guān)。 2.過(guò)表達(dá)miR-520b使得膀胱癌細(xì)胞T24和UC3細(xì)胞發(fā)生細(xì)胞周期G1期阻滯及增殖顯著抑制。同時(shí),miR-520b的過(guò)表達(dá)還削弱了T24細(xì)胞和UC3細(xì)胞的克隆形成能力。 3. Cyclin D1是確認(rèn)的microRNA-520b在膀胱癌細(xì)胞中的直接調(diào)控靶基因。
[Abstract]:[background]
Bladder cancer is one of the most common malignant tumors in humans, but its pathogenesis is complex. It involves many gene expression, dysfunction and multiple signaling pathways. Currently, the molecular genetic mechanism of bladder cancer occurrence and development is not clear.
MicroRNA is a kind of endogenous found in eukaryotes with regulatory function of non encoding small single stranded RNA, the size of about 21-25 nucleotides, usually expressed in post transcriptional regulation genes. Many studies indicate that microRNA is involved in tumor cell differentiation, proliferation, apoptosis, invasion, metastasis and abnormal expression of.MicroRNA links etc. there are also in bladder cancer cells and its possible in tumor genesis and development plays an important role in the process.
MiR-520b is a recently discovered microRNA., which is abnormally expressed in cancer. Its preliminary study shows that its expression is down regulated in many kinds of malignancies, including bladder cancer. We speculate that it may have a tumor suppressor effect on bladder cancer.
[Objective]
In this paper, the biological functions of miR-520b in the development of bladder cancer were discussed, and the mechanism of its molecular action was studied.
[method]
The relative expression of 1. miR-520b in 18 paired tissue samples were determined and analyzed the differential expression of miR-520b in bladder cancer and adjacent bladder mucosa tissues, and the expression of tniR-520b between different pathological tissue volume and bladder cancer staging and grading of the relationship; RT-PCR verification of]miR-520b in bladder cancer cell lines (T24. UC3) differential expression of immortalized human normal bladder epithelial cell line SV-HUC-1.
2. by in vitro transfection of miR-520b mimic to gain of function experiments. The upregulation of miR-520b in bladder cancer cell line T24, the expression of UC3, in CCK8, colony formation, effect of FCM in vitro experiments of miR-520b on the proliferation of bladder cancer cells. The scratch test and Transwell chamber migration and invasion. Analysis of miR-520b over expression on cell migration, invasion and influence;
3.榪愮敤鐢熺墿淇℃伅瀛︽柟娉，

本文編號(hào)：1678558