MicroRNA-146a对人胶质瘤细胞U251耐药株的作用及其与Notch1基因相关性的研究

发布时间：2018-11-08 20:14

【摘要】：目的:1.研究micro RNA-146a对人神经胶质瘤U251细胞耐药株的作用。2.探讨Notch1基因与micro RNA-146a在人胶质瘤耐药性中的相关性。方法:1.通过慢病毒包装、转染、筛选,分别获得高表达NICD的U251细胞株及其空载plvx的U251细胞株,并通过Western Blotting及免疫荧光染色进行鉴定。2.micro RNA芯片检测和筛选NICD高表达和空载plvx的U251细胞及对照U251细胞中差异性表达的micro RNAs。3.RT-q PCR检测鉴定NICD高表达和空载plvx的U251细胞及对照U251细胞中mi RNA-146a的表达情况。4.懫用替莫唑胺(TMZ)浓度递增的作用方式诱导U251细胞,初始诱导剂量0.25μg/ml(0.2875μM),最高诱导剂量为20μg/ml(103.0μM),获得稳定的对TMZ耐药的U251细胞,命名为U251TR,用CCK-8法检测U251,U251TR的细胞增殖抑制率,计算半数抑制浓度IC50及耐药指数(RF)。5.RT-q PCR检测U251TR及其对照U251细胞中mi RNA-146a的表达情况。6.用脂质体介导的转染方法将mi R-146a-mimics转入U251TR,称为转染组细胞(U251TR-mimics),荧光显微法检测转染组转染效率,RT-q PCR检测U251TR及U251TR-mimics中mi R-146a的表达情况,CCK-8法检测U251TR-mimics细胞增殖抑制率,计算IC50及RF。7.Western Blotting检测U251,U251TR及U251TR-mimics细胞中Notch1基因的表达情况。 8.通过生物信息学预测靶向调控Notch1的mi RNA-146a,然后通过双荧光素酶报告基因实验及Western blotting实验对mi R-146a进行靶标的验证。结果:1.成功建立了高表达NICD和空载plvx的U251细胞株。2.Micro RNA芯片结果显示NICD高表达的U251细胞中mi RNA-146a与对照组相比明显降低(P0.05)。3.成功建立了对TMZ耐药的U251TR,U251TR细胞IC50(320.68μM)是未经诱导U251细胞IC50(30.54μM)的10.5倍(P0.05),其耐药指数(RF)约为11。4.RT-q PCR结果显示,mi R-146a在NICD高表达组细胞及其对照组细胞中的表达情况与micro RNA芯片结果一致;RT-q PCR显示耐药株U251TR中mi R-146a相对表达量低于U251(P0.05)。5.转染组mi R-146a-mimics的转染效率在90%以上;转染组细胞U251TR-mimics IC50(160.79μM)是未经诱导的U251细胞IC50(30.54μM)的5.26倍(P0.05),其耐药指数(RF)约为5。6.双荧光素酶报告基因实验和Western blotting实验均显示mi R-146a-5p作用后,荧光素酶活性及Notch1下调明显(P0.05)。结论:成功构建了对TMZ耐药的U251TR细胞,其耐药指数(RF)约为10,U251TR细胞中mi R-146a相对表达量低于U251细胞;将mi R-146a-mimics转入U251TR细胞后,其耐药指数下降。双荧光素酶报告基因和Western blotting结果均显示mi R-146a靶向调控Notch1。mi RNA-146a可能通过Notch1信号通路在人胶质瘤细胞U251耐药形成机制中发挥一定的作用。
[Abstract]:Objective: 1. To study the effect of micro RNA-146a on human glioma cell line U251. 2. To investigate the relationship between Notch1 gene and micro RNA-146a in human glioma resistance. Methods: 1. U251 cell lines with high expression of NICD and U251 cell lines with no plvx were obtained by packaging, transfection and screening of lentivirus. Western Blotting and immunofluorescence staining were used to identify the high expression of NICD in U251 cells with high NICD expression and non-loaded U251 cells and U251 cells without plvx and control U251 cells. Micro RNAs.3.RT-q PCR detection was used to identify the high expression of NICD in U251 cells. The expression of mi RNA-146a in U251 cells and control U251 cells without plvx. 4. U251 cells were induced by the increasing concentration of temozolidomide (TMZ). The initial induction dose was 0.25 渭 g/ml (0.2875 渭 M),) and the maximum inducing dose was 20 渭 g/ml (103.0 渭 M),) to obtain stable TMZ resistant U251 cells named U251 TR. CCK-8 assay was used to detect the proliferation inhibition rate of U251TR cells. The expression of mi RNA-146a in U251TR and its control U251 cells was detected by calculating the half inhibitory concentration (IC50) and drug resistance index (RF). 5.RT-q PCR). The transfection efficiency of mi R-146a-mimics was detected by fluorescence microscopy, and the expression of mi R-146a in U251TR and U251TR-mimics was detected by RT-q PCR. CCK-8 assay was used to detect the proliferation inhibition rate of U251TR-mimics cells, and the expression of Notch1 gene in U251TR and U251TR-mimics cells was calculated by IC50 and RF.7.Western Blotting. 8. Mi RNA-146a, targeting Notch1 was predicted by bioinformatics. Then the target of mi R-146a was verified by double luciferase reporter gene experiment and Western blotting experiment. The result is 1: 1. U251 cell lines with high expression of NICD and no plvx were successfully established. The results of 2.Micro RNA microarray showed that mi RNA-146a in U251 cells with high NICD expression was significantly lower than that in control group (P0.05). The IC50 (320.68 渭 M) of U251 TR-U251TR cells resistant to TMZ was 10. 5 times as much as that of uninduced U251 cells IC50 (30.54 渭 M) (P0.05). The drug resistance index (RF) of U251 TR- U251TR cells was about the same as that of 11.4.RT-q PCR. The expression of mi R-146a in the cells with high expression of NICD and its control group was consistent with the results of micro RNA microarray. RT-q PCR showed that the relative expression of mi R-146a in U251TR was lower than that in U251 (P0.05). The transfection efficiency of mi R-146a-mimics in transfection group was more than 90%, and the transfection group U251TR-mimics IC50 (160.79 渭 M) was 5.26 times as high as the uninduced U251 cell IC50 (30.54 渭 M) (P0.05), and its drug resistance index (RF) was about 5.6. Double luciferase reporter gene experiment and Western blotting assay showed that luciferase activity and Notch1 decreased significantly after mi R-146a-5p treatment (P0.05). Conclusion: U251TR cells resistant to TMZ were successfully constructed, and the relative expression of mi R-146a in U251TR cells was lower than that in U251 cells, and the drug resistance index decreased after mi R-146a-mimics was transferred to U251TR cells. The results of double luciferase reporter gene and Western blotting showed that mi R-146a could play a role in the formation of drug resistance in human glioma cell line U251 through Notch1 signaling pathway.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.41

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