本文選題：小分子核糖核酸　切入點(diǎn)：miR-4295　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：膀胱癌(Bladder cancer)是常見的在世界范圍內(nèi)第九大惡性實(shí)體腫瘤,是我國泌尿系統(tǒng)腫瘤發(fā)病率第一位的惡性腫瘤。小分子核糖核酸(Micro RNA)已被證明參與腫瘤的發(fā)生發(fā)展過程,已成為細(xì)胞生物學(xué)調(diào)節(jié)的關(guān)鍵因子。而且,在正常組織與腫瘤組織中的Micro RNA表達(dá)水平存在明顯差異。最近研究發(fā)現(xiàn),mi R-4295在非小細(xì)胞肺癌中是一種新的致癌Micro RNA。但是mi R-4295在膀胱癌中的表達(dá)及其生物學(xué)功能還未見報(bào)道。此外,研究表明B細(xì)胞易位基因(BTG)可以抑制細(xì)胞的增殖、轉(zhuǎn)移和血管生成,調(diào)節(jié)多種細(xì)胞的細(xì)胞周期和分化。而且生物信息學(xué)研究表明BTG是mi R-4295的靶基因之一。因此,本研究首先檢測mi R-4295與膀胱癌的相關(guān)性,然后驗(yàn)證mi R-4295是否通過其靶基因BTG1促進(jìn)膀胱癌增殖和遷移,為膀胱癌提供了新的可能的治療靶點(diǎn)。方法:(1)采用RT-PCR檢測25例確診膀胱癌及其配對癌周正常組織,體外培養(yǎng)膀胱癌細(xì)胞中mi R-4295和BTG1的m RNA表達(dá)水平,pearson統(tǒng)計(jì)學(xué)方法分析兩者表達(dá)水平之間的相關(guān)性。(2)利用mi R-4295 inhibitor或mi R-4295mimics分析抑制或者增加細(xì)胞中mi R-4295表達(dá)對BTG1表達(dá)水平的影響。突變BTG13’UTR,利用熒光素酶表達(dá)質(zhì)粒探討3’UTR區(qū)對mi R-4295調(diào)控BTG1表達(dá)的影響。(3)利用mi R-4295 inhibitors或mi R-4295mimics觀察抑制或者增加細(xì)胞中mi R-4295表達(dá)對膀胱癌細(xì)胞增殖和遷移的影響。構(gòu)建BTG1高表達(dá)質(zhì)粒,明確BTG1蛋白在mi R-4295引起的膀胱癌細(xì)胞增殖和遷移改變中的作用。WST-8法,克隆形成實(shí)驗(yàn),細(xì)胞周期檢測細(xì)胞增殖率的改變,Transwell小室試驗(yàn)檢測膀胱癌細(xì)胞遷移。結(jié)果:(1)膀胱癌組織中及膀胱癌細(xì)胞系中mi R-4295的m RNA表達(dá)水平明顯高于癌旁組織及正常人輸尿管上皮細(xì)胞,而BTG1的m RNA表達(dá)水平明顯低于癌旁組織和正常人輸尿管上皮細(xì)胞,兩者之間的相關(guān)性呈現(xiàn)出明顯的負(fù)相關(guān)。(2)使用mi R-4295 mimics進(jìn)行過表達(dá)后,與對照組相比,膀胱癌細(xì)胞的OD490值明顯增加,呈時(shí)間依賴性,克隆形成數(shù)量超過mi R-4295 control對照組1.5倍以上,細(xì)胞周期s期比例增加,遷移細(xì)胞數(shù)量顯著高于對照組。(3)使用mi R-4295 inhibitors抑制表達(dá)后,與對照組相比,膀胱癌細(xì)胞的OD490值明顯減少,呈時(shí)間依賴性,克隆形成數(shù)量減少,細(xì)胞周期S期比例下降,遷移細(xì)胞數(shù)量顯著低于對照組。(4)T24和Hbc膀胱癌細(xì)胞中,mi R-4295mimics均可以明顯降低野生型BTG1的熒光素酶活性,而3’UTR突變型BTG1的熒光素酶活性與對照組比較無顯著變化。(5)mi R-4295 mimics和BTG1高表達(dá)質(zhì)粒共轉(zhuǎn)染組的生長曲線低于對照組,OD490值明顯下降,呈時(shí)間依賴性,克隆形成數(shù)量減少,細(xì)胞周期S期比例下降,遷移細(xì)胞數(shù)量顯著低于對照組。BTG1高表達(dá)可逆轉(zhuǎn)mi R-4295誘導(dǎo)膀胱癌細(xì)胞增殖作用。結(jié)論:mi R-4295在膀胱癌組織和細(xì)胞中的表達(dá)量顯著增加,表現(xiàn)出致癌Micro RNA活性,抑制mi R-4295可削弱膀胱癌細(xì)胞的增殖和遷移能力,BTG1被確定為mi R-4295在膀胱癌細(xì)胞中的直接調(diào)控靶基因。
[Abstract]:Bladder cancer (Bladder cancer) is common in the world within the scope of Article Nine malignant solid tumor, is China's first incidence rate of tumor in urinary system malignant tumors. MicroRNAs (Micro RNA) has been proved to be involved in the occurrence and development of tumor, has become a key factor in the regulation of cell biology. Moreover, there are significant differences in the expression level of Micro RNA in normal tissues and tumor tissues. Recent studies found that MI R-4295 in non small cell lung cancer is a kind of new Micro RNA. but mi R-4295 expression of cancer and its biological function in bladder cancer has not been reported. In addition, studies have shown that B cells can inhibit the translocation gene (BTG) cell proliferation, metastasis and angiogenesis, regulate various cell cycle and differentiation. And bioinformatics research showed that BTG is a target gene of MI R-4295. Therefore, this study first detected mi R-429 5 Correlation with bladder cancer, and then verify whether the MI R-4295 and its target gene BTG1 promote the proliferation and migration of bladder cancer, may provide new therapeutic targets for bladder cancer. Methods: (1) detected by RT-PCR in 25 cases of bladder cancer and its adjacent normal tissues, m RNA mi R-4295 cultured in vitro and BTG1 expression in bladder cancer cells, the correlation between Pearson expression level between the two was analyzed. (2) using mi R-4295 inhibitor or MI R-4295mimics or MI in the cells of inhibition of increased expression of R-4295 effect on BTG1. The mutation BTG13 'UTR, the luciferase expression plasmid of 3 UTR region of MI R-4295 regulates the expression of BTG1. (3) using mi R-4295 inhibitors or MI R-4295mimics effect or influence on the proliferation and migration of bladder cancer cells increased in cells expressing mi R-4295. Construction of the high expression of BTG1 Effect of.WST-8 plasmid, clear BTG1 protein in MI R-4295 induced bladder cancer cell proliferation and migration changes, clone formation assay, cell cycle change detection cell proliferation rate, detection of bladder cancer cell Transwell cell migration test. Results: (1) the expression level of M RNA in bladder cancer and bladder cancer cell lines mi R-4295 was significantly higher than that in adjacent tissues and normal ureter epithelial cells, m and RNA BTG1 expression level was significantly lower than that of adjacent tissues and normal ureter epithelial cells, the correlation between the two showing a significant negative correlation. (2) using mi R-4295 mimics expression, compared with the control group, the bladder cancer the OD490 cells were significantly increased in a time-dependent manner, MI R-4295 control clone number more than 1.5 times of the control group, the S phase of the cell cycle increased, the number of cells migration was significantly higher than the control group. (3). The expression of MI R-4295 inhibited by inhibitors, compared with the control group, the bladder cancer cell OD490 was significantly reduced, time-dependent clone formation decreased, cell cycle S migration decreased the proportion of cell number was significantly lower than the control group. (4) T24 and Hbc in bladder cancer cells, MI can significantly reduce the R-4295mimics wild type BTG1 luciferase activity, and luciferase activity 3 UTR mutant BTG1 compared with the control group showed no significant changes. (5) the high expression of MI R-4295 and mimics BTG1 growth curve of plasmid co transfection group than the control group, the OD490 value decreased and time-dependent decrease in the number of clonogenic cell cycle, S during the period of decline in the proportion of migrating cells number was significantly lower than the control group with high expression of.BTG1 could reverse mi R-4295 induced bladder cancer cell proliferation. Conclusion: Mi R-4295 in bladder cancer cell and the expression increased significantly, table The carcinogenic activity of Micro RNA is inhibited. Mi R-4295 inhibits the proliferation and migration of bladder cancer cells. BTG1 is identified as a direct target gene of MI R-4295 in bladder cancer cells.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.14

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