miR-1和miR-184对SH-SY5Y细胞系多巴胺转运体（DAT）表达的调节作用

发布时间：2018-03-18 19:02

本文选题：miR-1　切入点：miR-184　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：检测niR-1、miR-184及多巴胺转运体(dopamine transporter, DAT)在多巴胺能细胞系SH-SY5Y中的表达,探讨miR-1、miR-184在SH-SY5Y细胞系中对DAT基因表达的影响及其作用机制。方法：采用microRNA.org、miRBase、TargetScan.等在线数据库预测和文献参阅相结合的方法,选择可能作用于DAT基因的候选miRNAs,并化学合成候选miRNAs模拟物(miRNAs mimics和inhibitor)转染到SH-SY5Y细胞中。分别于转染后12h和24h后,提取NC-mimics组、NC-inhibitor组、miRNAs mimics组和miRNAsinhibitor组细胞总RNA和蛋白；采用实时荧光定量RT-PCR检测各组细胞内的候选niRNA、DAT mRNA表达水平；采用Western Blot检测各组细胞内DAT蛋白表达水平。结果：(1)通过生物信息学预测结合参考文献挑选出的候选miRNAs为miR-1、miR-184;(2)miRNAs表达水平：miR-1mimics12h组和miR-1mimics24h组的miR-1表达量显著高于对照组(Ps0.001),miR-1inhibitor12h组、niR-1inhibitor24h组与对照组相比均没有显著差异(Ps0.05)；miR-184mimics24h处理组中miR-184的表达量与对照组相比均有显著差异(P0.01),miR-184inhibitor12h、miR-184mimics12hmiR-184inhibitor24h处理组与对照组相比均没有显著差异(Ps0.05)。 (3) DAT mRNA表达水平：miR-1mimics12h处理组中DATmRNA的表达量与对照组相比有显著差异(P0.05), miR-1mimics24h组、niR-1inhibitor12h组和niR-1inhibitor24h组DAT mRNA的表达量与对照组相比均没有显著差异(Ps0.05)；miR-184mimics24h组DAT mRNA的表达量与对照组相比有显著差异(P0.05),miR-184mimics12h组、miR-184inhibitor12h组和miR-184inhibitor24h组DAT mRNA的表达量与对照组相比均没有显著差异(Ps0.05)。 (4)DAT蛋白表达水平：niR-1mimics12h处理组中DAT蛋白的表达量与对照组相比有显著差异(P0.05), miR-1mimics24h组、miR-1inhibitor12h组和miR-1inhibitor24h处理组中DAT蛋白的表达量与对照组相比均没有显著差异(Ps0.05)；miR-184mimics24h处理组中DAT蛋白的表达量与对照组相比有显著差异(P0.05),miR-184mimics12h组、miR-184inhibitor12h组和miR-184inhibitor24h处理组中DAT蛋白的表达量与对照组相比均没有显著差异(Ps0.05)；结论：miR-1、miR-184参与SH-SY5Y细胞中DAT基因表达的调控,过表达的miR-1和miR-184均能抑制SH-SY5Y细胞中DAT蛋白的表达。
[Abstract]:Aim: to detect the expression of niR-1mmiR-184 and dopamine transporter (DAT) in dopaminergic cell line SH-SY5Y, and to investigate the effect of miR-1 miR-184 on the expression of DAT gene in SH-SY5Y cell line and its mechanism. Methods: using the method of microRNA.orgfmiRBasetTargetScan. and other online database prediction and literature review, we selected the candidate miRNAss that might act on the DAT gene, and chemically synthesized the candidate miRNAs mimics, miRNAs mimics and inhibitors, into SH-SY5Y cells. After 12 h and 24 h, respectively, the candidate miRNs were transfected into SH-SY5Y cells. The total RNA and protein were extracted from NC-mimics group NC-inhibitor mimics group and miRNAsinhibitor group. The expression level of candidate niRNA-DAT mRNA was detected by real-time fluorescence quantitative RT-PCR, and the expression level of DAT protein was detected by Western Blot. Results (1) the expression level of miR-1miR-1844 miRNAs selected by bioinformatics prediction combined with references was significantly higher in the miR-1 mimics12h group and miR-1mimics24h group than in the control group (Ps0.001) MiR-1 inhibitory or12h group. There was no significant difference between the control group and the control group. There was no significant difference in the expression of miR-1 between the control group and the control group. There was no significant difference in the expression of miR-184 between the control group and the control group. There was no significant difference in the expression of miR-184 between the control group and the control group. (3) the expression level of DATmRNA in the control group was significantly higher than that in the control group (P 0.05). The expression of DATmRNA in the miR-1mimics24h group and the niR-1inhibitor24h group was not significantly different from that in the control group. There was no significant difference in the expression of DATmRNA between the control group and the control group. There was no significant difference in the expression of DATmRNA between the 24 h group and the control group. There was no significant difference in the expression of DAT mRNA between the two groups (P 0.05) and the control group (P 0.05). There was no significant difference in the expression of DAT mRNA between the control group and the control group (P 0.05), and there was no significant difference in the expression of DAT mRNA between the control group and the control group (P 0.05). The expression of DAT protein in the control group was significantly different from that in the control group. There was no significant difference in the expression of DAT protein in the miR-1mimics24h group and the miR-1inhibitor24h treatment group compared with the control group. There was no significant difference in the expression of DAT protein between the control group and the control group. There was significant difference in the expression of DAT protein between the control group and the control group. There was no significant difference in the expression of DAT protein between the control group and the control group. Conclusion 1: miR-1 miR-184 is involved in the regulation of DAT gene expression in SH-SY5Y cells. Overexpression of both miR-1 and miR-184 can inhibit the expression of DAT protein in SH-SY5Y cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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