尿路上皮癌相关基因1对3T3-L1脂肪细胞糖代谢的作用及机制研究

发布时间：2018-01-27 15:09

本文关键词： 长链非编码RNA 尿路上皮癌相关基因1 过氧化物酶增殖物激活受体γ 3T3-L1脂肪细胞糖代谢　出处：《武汉大学》2016年博士论文　论文类型：学位论文

【摘要】：目的观察长链非编码RNA(Long noncoding RNA, lncRNA)尿路上皮癌相关基因1(urothelial carcinoma associated 1, UCA1)对3T3-L1脂肪细胞Akt信号通路及糖代谢的影响,并探索其中可能的机制。方法检测地塞米松和胰岛素等药物诱导3T3-L1前脂肪细胞分化成为成熟的脂肪细胞,通过荧光实时定量PCR检测其中UCA1的RNA水平变化。构建UCA1过表达质粒并筛选有效的UCA1特异性siRNA,通过转染UCA1过表达质粒或UCAl特异性siRNA干预3T3-L1脂肪细胞中UCA1的水平,利用蛋白免疫印迹法(Western blot)检测Akt信号通路相关蛋白磷酸化水平的变化,通过双荧光素酶报告系统检测过氧化物酶增殖物激活受体y(peroxisome proliferater activated receptor y, PPARγ)的转录活性,利用荧光实时定量PCR检测PPARy的mRNA水平,通过2-脱氧-3H-D-葡萄糖掺入法检测细胞的葡萄糖摄取能力。利用SPSS12.0统计学软件进行统计分析。实验数据以均数±标准差表示,两组之间比较用t检验,多组之间比较采用单因素方差分析。结果随着3T3-L1前脂肪细胞诱导分化为成熟的脂肪细胞,UCA1的nRNA水平逐渐升高,第六天时为诱导前的3.48±0.09倍,t=12.093,P0.01；过表达UCA1可增加3T3-L1脂肪细胞中Akt及FOXO1的磷酸化水平：p-AKT(S473):Control vs UCA1, 1103±42 vs 2338±59, t=16.390, P0.01; p-AKT(T308):Control vs UCA1,1524±34 vs 3565±45, t=13.025, P0.01; p-FOXO1:Control vs UCA1,912±21 vs 2190±31, t=10.544,P0.01；而利用特异性siRNA沉默UCA1,可降低细胞中Akt及FOXO1的磷酸化水平：p-AKT(S473):si-scramble vs si-UCAl,1090±22 vs 540±34, t=11.045, P0.01; p-AKT(T308):si-scramble vs si-UCA1,1409±26 vs 805±18, t=9.527, P0.01; p-FOXO1: si-scramble vs si-UCAl,2444±29 vs 459±31, t=16.899, P0.01;未给予胰岛素刺激时,过表达UCA1可增加细胞的葡萄糖摄取能力至2.21±0.09倍,t=5.922,P0.05,沉默UCA1可使细胞的葡萄糖摄取能力降低66%以上,t=-5.557,P0.05；而给予胰岛素刺激时,过表达UCA1可增加细胞的葡萄糖摄取能力至3.25±0.12倍,t=5.709,P0.05,沉默UCA1可使细胞的葡萄糖摄取能力降低77%以上, t=6.567, P0.05。UCA1可促进PPARy的表达。结论UCA1可激活3T3-L1脂肪细胞Akt信号通路,促进PPARγ的转录水平和蛋白表达,并升高细胞的葡萄糖摄取能力。
[Abstract]:Objective to observe the long chain noncoding RNA(Long noncoding RNA. (1) urothelial carcinoma associated 1. The effect of UCA1 on Akt signaling pathway and glucose metabolism in 3T3-L1 adipocytes. Methods Dexamethasone and insulin induced 3T3-L1 preadipocytes to differentiate into mature adipocytes. The RNA level of UCA1 was detected by real-time quantitative PCR. The UCA1 overexpression plasmid was constructed and the effective UCA1 specific siRNA was screened. The level of UCA1 in 3T3-L1 adipocytes was inhibited by transfection of UCA1 overexpression plasmid or UCAl specific siRNA. Western blots were used to detect the phosphorylation level of Akt signaling pathway. Detection of peroxidase proliferator activated receptor y1by double luciferase reporting system. Peroxisome proliferater activated receptor y. The transcriptional activity of PPAR 纬 was measured by real-time quantitative PCR. The mRNA level of PPARy was detected by fluorescence quantitative PCR. The glucose uptake capacity of the cells was measured by 2-deoxy-3H-D- glucose incorporation method. The results were statistically analyzed by SPSS12.0 software. The experimental data were expressed as mean 卤standard deviation. T test was used in the comparison between the two groups and univariate analysis of variance was used in the comparison between the two groups. Results the preadipocytes of 3T3-L1 were induced to differentiate into mature adipocytes with 3T3-L1 preadipocytes. The level of nRNA in UCA1 increased gradually, which was 3.48 卤0.09 times of that before induction at 6th days. Overexpression of UCA1 increased the phosphorylation level of Akt and FOXO1 in 3T3-L1 adipocytes. 1103 卤42 vs 2338 卤59, t0. 390, P0.01; P-AKT T308: control vs UCA 1 524 卤34 vs 3565 卤45, t0. 025, P 0. 01; P-FOXO1: control vs UCA1C 912 卤21 vs 2190 卤31, t0. 544% P0.01; UCA1 was silenced by specific siRNA. It can reduce the phosphorylation level of Akt and FOXO1 in the cells:: p-AKT S473N: si-scramble vs si-UCAl. 1090 卤22 vs 540 卤34, t0. 045, P0.01; P-AKT T308: si-scramble vs si-UCA _ 1 1409 卤26 vs 805 卤18, t _ (9.527), P _ (0.01); P-FOXO1: si-scramble vs si-UCAlN 2444 卤29 vs 459 卤31, taut 16.899, P0.01; Without insulin stimulation, overexpression of UCA1 could increase the glucose uptake of the cells to 2.21 卤0.09 times. Silencing UCA1 decreased the glucose uptake of cells by more than 66%. However, when stimulated by insulin, overexpression of UCA1 increased the glucose uptake of the cells to 3.25 卤0.12 times (P < 0.05). Silencing UCA1 could decrease glucose uptake by more than 77% and tr 6.567. Conclusion UCA1 can activate the Akt signaling pathway of 3T3-L1 adipocytes and promote the transcription level and protein expression of PPAR 纬. And increase the glucose uptake of cells.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.1

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