本文選題：miR--p + 細(xì)胞自噬��； 參考：《中國免疫學(xué)雜志》2016年11期

【摘要】：目的:研究miR-142-3p對自噬相關(guān)基因ATG4c的靶向調(diào)控作用,探究miR-142-3p影響RAW264.7細(xì)胞自噬途徑的作用機(jī)制。方法:生物信息學(xué)軟件分析miR-142-3p的靶基因為ATG4c,構(gòu)建pMIR-Report-ATG4c和pMIR-Report-ATG4c mut重組質(zhì)粒,雙熒光素酶報告系統(tǒng)、qRT-PCR、Western blot驗證miR-142-3p與ATG4c的靶向作用;將做不同處理的RAW264.7細(xì)胞分為4組:正常細(xì)胞作為對照、50ng/ml雷帕霉素作用2h、EBSS饑餓作用12 h、10 nmol/L的3-甲基腺嘌呤(3-MA)作用12h后,實時熒光定量PCR(qRT-PCR)檢測miR-142-3p不同干預(yù)組中的相對表達(dá)情況;將miR-142-3p mimics、miR-142-3p inhibitor及miR-142-3p control分別轉(zhuǎn)染到RAW264.7細(xì)胞中,檢測miR-142-3p和LC3Ⅱ的相對表達(dá)。結(jié)果:雙熒光素酶報告系統(tǒng)、qRT-PCR、Western blot驗證miR-142-3p通過靶向作用于ATG4c的3'-UTR抑制其表達(dá);與對照組相比,雷帕霉素和饑餓處理的RAW264.7細(xì)胞miR-142-3p明顯上調(diào),而3-MA處理組miR-142-3p明顯下調(diào);與miR-142-3p control組相比,轉(zhuǎn)染miR-142-3p mimics組中LC3Ⅱ蛋白表達(dá)顯著下調(diào),而miR-142-3p inhibitor組中表達(dá)顯著上調(diào)。結(jié)論:miR-142-3p通過靶向調(diào)控自噬相關(guān)基因ATG4c,參與RAW264.7小鼠巨噬細(xì)胞自噬的調(diào)控。
[Abstract]:Aim: to investigate the targeted regulation of miR-142-3p on autophagy related gene ATG4c and to explore the mechanism of miR-142-3p affecting autophagy pathway in RAW264.7 cells. Methods: the target base of miR-142-3p was analyzed by bioinformatics software. The recombinant plasmids of pMIR-Report-ATG4c and pMIR-Report-ATG4c mut were constructed. The double luciferase report system was used to detect the targeting effect of miR-142-3p and ATG4c by Western blot. The RAW264.7 cells treated with different treatments were divided into four groups: normal cells were treated with 50ng / ml rapamycin for 2 h and then treated with 3-methyladenine 3-MAfor 12 h or 10 nmol/L. The relative expression of miR-142-3p in different intervention groups was detected by real-time quantitative PCR qRT-PCRR. MiR-142-3p mimicstmiR-142-3p inhibitor and miR-142-3p control were transfected into RAW264.7 cells respectively to detect the relative expression of miR-142-3p and LC3 鈪，

本文編號：1814771