本文選題：miR-1　切入點(diǎn)：miR-184　出處：《中南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：檢測niR-1、miR-184及多巴胺轉(zhuǎn)運(yùn)體(dopamine transporter, DAT)在多巴胺能細(xì)胞系SH-SY5Y中的表達(dá),探討miR-1、miR-184在SH-SY5Y細(xì)胞系中對DAT基因表達(dá)的影響及其作用機(jī)制。 方法：采用microRNA.org、miRBase、TargetScan.等在線數(shù)據(jù)庫預(yù)測和文獻(xiàn)參閱相結(jié)合的方法,選擇可能作用于DAT基因的候選miRNAs,并化學(xué)合成候選miRNAs模擬物(miRNAs mimics和inhibitor)轉(zhuǎn)染到SH-SY5Y細(xì)胞中。分別于轉(zhuǎn)染后12h和24h后,提取NC-mimics組、NC-inhibitor組、miRNAs mimics組和miRNAsinhibitor組細(xì)胞總RNA和蛋白；采用實時熒光定量RT-PCR檢測各組細(xì)胞內(nèi)的候選niRNA、DAT mRNA表達(dá)水平；采用Western Blot檢測各組細(xì)胞內(nèi)DAT蛋白表達(dá)水平。 結(jié)果：(1)通過生物信息學(xué)預(yù)測結(jié)合參考文獻(xiàn)挑選出的候選miRNAs為miR-1、miR-184;(2)miRNAs表達(dá)水平：miR-1mimics12h組和miR-1mimics24h組的miR-1表達(dá)量顯著高于對照組(Ps0.001),miR-1inhibitor12h組、niR-1inhibitor24h組與對照組相比均沒有顯著差異(Ps0.05)；miR-184mimics24h處理組中miR-184的表達(dá)量與對照組相比均有顯著差異(P0.01),miR-184inhibitor12h、miR-184mimics12hmiR-184inhibitor24h處理組與對照組相比均沒有顯著差異(Ps0.05)。 (3) DAT mRNA表達(dá)水平：miR-1mimics12h處理組中DATmRNA的表達(dá)量與對照組相比有顯著差異(P0.05), miR-1mimics24h組、niR-1inhibitor12h組和niR-1inhibitor24h組DAT mRNA的表達(dá)量與對照組相比均沒有顯著差異(Ps0.05)；miR-184mimics24h組DAT mRNA的表達(dá)量與對照組相比有顯著差異(P0.05),miR-184mimics12h組、miR-184inhibitor12h組和miR-184inhibitor24h組DAT mRNA的表達(dá)量與對照組相比均沒有顯著差異(Ps0.05)。 (4)DAT蛋白表達(dá)水平：niR-1mimics12h處理組中DAT蛋白的表達(dá)量與對照組相比有顯著差異(P0.05), miR-1mimics24h組、miR-1inhibitor12h組和miR-1inhibitor24h處理組中DAT蛋白的表達(dá)量與對照組相比均沒有顯著差異(Ps0.05)；miR-184mimics24h處理組中DAT蛋白的表達(dá)量與對照組相比有顯著差異(P0.05),miR-184mimics12h組、miR-184inhibitor12h組和miR-184inhibitor24h處理組中DAT蛋白的表達(dá)量與對照組相比均沒有顯著差異(Ps0.05)； 結(jié)論：miR-1、miR-184參與SH-SY5Y細(xì)胞中DAT基因表達(dá)的調(diào)控,過表達(dá)的miR-1和miR-184均能抑制SH-SY5Y細(xì)胞中DAT蛋白的表達(dá)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

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本文編號：1630888