本文關鍵詞： 長鏈非編碼RNA 尿路上皮癌相關基因1 過氧化物酶增殖物激活受體γ 3T3-L1脂肪細胞 糖代謝　出處：《武漢大學》2016年博士論文　論文類型：學位論文

【摘要】：目的觀察長鏈非編碼RNA(Long noncoding RNA, lncRNA)尿路上皮癌相關基因1(urothelial carcinoma associated 1, UCA1)對3T3-L1脂肪細胞Akt信號通路及糖代謝的影響,并探索其中可能的機制。方法檢測地塞米松和胰島素等藥物誘導3T3-L1前脂肪細胞分化成為成熟的脂肪細胞,通過熒光實時定量PCR檢測其中UCA1的RNA水平變化。構建UCA1過表達質粒并篩選有效的UCA1特異性siRNA,通過轉染UCA1過表達質�；騏CAl特異性siRNA干預3T3-L1脂肪細胞中UCA1的水平,利用蛋白免疫印跡法(Western blot)檢測Akt信號通路相關蛋白磷酸化水平的變化,通過雙熒光素酶報告系統(tǒng)檢測過氧化物酶增殖物激活受體y(peroxisome proliferater activated receptor y, PPARγ)的轉錄活性,利用熒光實時定量PCR檢測PPARy的mRNA水平,通過2-脫氧-3H-D-葡萄糖摻入法檢測細胞的葡萄糖攝取能力。利用SPSS12.0統(tǒng)計學軟件進行統(tǒng)計分析。實驗數(shù)據(jù)以均數(shù)±標準差表示,兩組之間比較用t檢驗,多組之間比較采用單因素方差分析。結果隨著3T3-L1前脂肪細胞誘導分化為成熟的脂肪細胞,UCA1的nRNA水平逐漸升高,第六天時為誘導前的3.48±0.09倍,t=12.093,P0.01；過表達UCA1可增加3T3-L1脂肪細胞中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；而利用特異性siRNA沉默UCA1,可降低細胞中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;未給予胰島素刺激時,過表達UCA1可增加細胞的葡萄糖攝取能力至2.21±0.09倍,t=5.922,P0.05,沉默UCA1可使細胞的葡萄糖攝取能力降低66%以上,t=-5.557,P0.05；而給予胰島素刺激時,過表達UCA1可增加細胞的葡萄糖攝取能力至3.25±0.12倍,t=5.709,P0.05,沉默UCA1可使細胞的葡萄糖攝取能力降低77%以上, t=6.567, P0.05。UCA1可促進PPARy的表達。結論UCA1可激活3T3-L1脂肪細胞Akt信號通路,促進PPARγ的轉錄水平和蛋白表達,并升高細胞的葡萄糖攝取能力。
[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 緯 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 鹵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 鹵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 鹵42 vs 2338 鹵59, t0. 390, P0.01; P-AKT T308: control vs UCA 1 524 鹵34 vs 3565 鹵45, t0. 025, P 0. 01; P-FOXO1: control vs UCA1C 912 鹵21 vs 2190 鹵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 鹵22 vs 540 鹵34, t0. 045, P0.01; P-AKT T308: si-scramble vs si-UCA _ 1 1409 鹵26 vs 805 鹵18, t _ (9.527), P _ (0.01); P-FOXO1: si-scramble vs si-UCAlN 2444 鹵29 vs 459 鹵31, taut 16.899, P0.01; Without insulin stimulation, overexpression of UCA1 could increase the glucose uptake of the cells to 2.21 鹵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 鹵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 緯. And increase the glucose uptake of cells.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R587.1

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