Lnc RNA-GAS5对BCG感染小鼠巨噬细胞后细胞坏死的调控作用

Regulation of LncRNA-GAS5 on necrosis of macrophage induced by Bacillus Calmette-Guérin

【背景】LncRNA-GAS5是由Gas5基因编码的功能性LncRNA分子,对细胞极化、细胞凋亡、坏死和自噬等多种生物学过程具有重要的调控作用。【目的】探讨LncRNA-GAS5对卡介苗(Bacillus Calmette-Guérin,BCG)诱导的小鼠巨噬细胞RAW264.7坏死的调控作用。【方法】构建LncRNA-GAS5的过表达及干扰载体,分别转染巨噬细胞RAW264.7后使用BCG感染,采用噻唑蓝比色法(MTT)检测细胞存活率,通过透射电镜观察细胞坏死形态,利用碘化丙啶(Propidine iodide,PI)单染法流式细胞仪检测细胞坏死率,通过实时荧光定量PCR和Western blot法研究坏死相关调控因子RIP1、RIP3和MLKL的表达水平。【结果】BCG感染巨噬细胞后,LncRNA-GAS5表达水平显著上调;同时,采用LncRNA-GAS5过表达载体单独转染或结合BCG感染后,巨噬细胞存活率均下降且细胞坏死率显著增加。同时坏死关键调控因子RIP1、RIP3和MLKL的mRNA及蛋白表达水平均显著上调(P<0.001),而GAS5干扰载体可有效抑制这一效果。【结论】LncRNA-GAS5通过上调坏死相关因子RIP1、RIP3及MLKL的表达促进BCG感染巨噬细胞后诱导的细胞坏死,研究结果为进一步探讨LncRNA-GAS5对BCG感染巨噬细胞后细胞坏死调控的分子机制奠定了基础。

[Background]LncRNA-GAS5,a functional LncRNA that is encoded by Gas5 gene,plays an important role in the regulation of cell polarization,apoptosis,necrosis and autophagy.[Objective]To investigate the regulatory function of LncRNA-GAS5 on necrosis of macrophage RAW 264.7 induced by Bacillus Calmette-Guérin(BCG)infection.[Methods]The overexpression plasmid and interference plasmid of LncRNA-GAS5 were constructed and transfected into macrophage RAW264.7.Furthermore,the both transfected macrophage were infected by BCG.The cell viability was detected by MTT assay.The morphological changes of necrosis were observed with transmission electron microscope and the necrosis rate was analyzed by flow cytometry.The expression of RIP1,RIP3 and MLKL in mRNA and protein level were determined by qRT-PCR and Western blot.[Results]The expression of LncRNA-GAS5 in macrophage RAW264.7 significantly increased after BCG infection.The cell viability decreased and cell necrosis rate increased significantly when macrophage was transfected with the LncRNA-GAS5 overexpression plasmid alone or combined with BCG infection.Meanwhile,the m RNA and protein expression levels of RIP1,RIP3 and MLKL were upregulated dramatically and this process can be inhibited by LncRNA-GAS5 interference plasmid.[Conclusion]LncRNA-GAS5 can promote necrosis of RAW264.7 infected with BCG by up-regulating necrosis associated factors such as RIP1,RIP3,and MLKL.The results lay foundation on further insight into the molecular mechanisms of macrophage necrosis induced by BCG infection.