miR-4295靶向BTG1调节膀胱癌细胞增殖和迁移

发布时间：2018-03-20 08:06

本文选题：小分子核糖核酸　切入点：miR-4295　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：膀胱癌(Bladder cancer)是常见的在世界范围内第九大恶性实体肿瘤,是我国泌尿系统肿瘤发病率第一位的恶性肿瘤。小分子核糖核酸(Micro RNA)已被证明参与肿瘤的发生发展过程,已成为细胞生物学调节的关键因子。而且,在正常组织与肿瘤组织中的Micro RNA表达水平存在明显差异。最近研究发现,mi R-4295在非小细胞肺癌中是一种新的致癌Micro RNA。但是mi R-4295在膀胱癌中的表达及其生物学功能还未见报道。此外,研究表明B细胞易位基因(BTG)可以抑制细胞的增殖、转移和血管生成,调节多种细胞的细胞周期和分化。而且生物信息学研究表明BTG是mi R-4295的靶基因之一。因此,本研究首先检测mi R-4295与膀胱癌的相关性,然后验证mi R-4295是否通过其靶基因BTG1促进膀胱癌增殖和迁移,为膀胱癌提供了新的可能的治疗靶点。方法:(1)采用RT-PCR检测25例确诊膀胱癌及其配对癌周正常组织,体外培养膀胱癌细胞中mi R-4295和BTG1的m RNA表达水平,pearson统计学方法分析两者表达水平之间的相关性。(2)利用mi R-4295 inhibitor或mi R-4295mimics分析抑制或者增加细胞中mi R-4295表达对BTG1表达水平的影响。突变BTG13’UTR,利用荧光素酶表达质粒探讨3’UTR区对mi R-4295调控BTG1表达的影响。(3)利用mi R-4295 inhibitors或mi R-4295mimics观察抑制或者增加细胞中mi R-4295表达对膀胱癌细胞增殖和迁移的影响。构建BTG1高表达质粒,明确BTG1蛋白在mi R-4295引起的膀胱癌细胞增殖和迁移改变中的作用。WST-8法,克隆形成实验,细胞周期检测细胞增殖率的改变,Transwell小室试验检测膀胱癌细胞迁移。结果:(1)膀胱癌组织中及膀胱癌细胞系中mi R-4295的m RNA表达水平明显高于癌旁组织及正常人输尿管上皮细胞,而BTG1的m RNA表达水平明显低于癌旁组织和正常人输尿管上皮细胞,两者之间的相关性呈现出明显的负相关。(2)使用mi R-4295 mimics进行过表达后,与对照组相比,膀胱癌细胞的OD490值明显增加,呈时间依赖性,克隆形成数量超过mi R-4295 control对照组1.5倍以上,细胞周期s期比例增加,迁移细胞数量显著高于对照组。(3)使用mi R-4295 inhibitors抑制表达后,与对照组相比,膀胱癌细胞的OD490值明显减少,呈时间依赖性,克隆形成数量减少,细胞周期S期比例下降,迁移细胞数量显著低于对照组。(4)T24和Hbc膀胱癌细胞中,mi R-4295mimics均可以明显降低野生型BTG1的荧光素酶活性,而3’UTR突变型BTG1的荧光素酶活性与对照组比较无显著变化。(5)mi R-4295 mimics和BTG1高表达质粒共转染组的生长曲线低于对照组,OD490值明显下降,呈时间依赖性,克隆形成数量减少,细胞周期S期比例下降,迁移细胞数量显著低于对照组。BTG1高表达可逆转mi R-4295诱导膀胱癌细胞增殖作用。结论:mi R-4295在膀胱癌组织和细胞中的表达量显著增加,表现出致癌Micro RNA活性,抑制mi R-4295可削弱膀胱癌细胞的增殖和迁移能力,BTG1被确定为mi R-4295在膀胱癌细胞中的直接调控靶基因。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.14

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