Long‑chain non‑coding RNA GAS5 promotes cell autophagy by modulating the miR‑181c‑5p/ATG5 and miR‑1192/ATG12 axes.

Tao Xu, Xiangrong Xu, Yuankui Chu, Dan Jiang, Guangxian Xu

International journal of molecular medicine 2021 Dec

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The main aim of the present study was to explore the role of long‑chain non‑coding RNA (lncRNA) growth arrest‑specific transcript 5 (GAS5) in macrophage autophagy. Firstly, the expression of lncRNA GAS5 during cell starvation or following treatment with 3‑methyladenine was determined using reverse transcription‑quantitative PCR (RT‑qPCR). Additionally, fluorescent in situ hybridization (FISH) assay was utilized to determine the localization of the expression of lncRNA GAS5 in RAW264.7 cells. In vitro cell models were established through the transfection of LV5‑lncRNA GAS5 (LV5‑GAS5) or LV3‑shRNA‑lnc GAS5 (sh‑GAS5), in order to overexpress or knockdown lncRNA GAS5 expression in RAW264.7 cells. The potential target microRNAs (miRNAs/miRs) of lncRNA GAS5 were analyzed using bioinformatics. The formation of autophagic bodies was detected with the use of laser confocal and transmission electron microscopy. Dual‑luciferase reporter assay was performed to determine the target specificities of miR‑181c‑5p or miR‑1192 to lncRNA GAS5 and autophagy‑related gene (ATG) or ATG12. The mRNA levels of miR181c‑5p, miR‑1192, as well as ATG5 and ATG12 were detected using RT‑qPCR. The protein levels of microtubule‑associated proteins 1A/1B light chain 3B (LC3), p62, ATG5 and ATG12 were measured using western blot analysis. It was revealed that lncRNA GAS5 expression in RAW264.7 macrophages increased significantly during starvation‑induced autophagy, and that lncRNA GAS5 overexpression was able to markedly promote the formation of autophagic bodies. Bioinformatics analysis demonstrated that miR‑181c‑5p and miR‑1192 were potential targets of lncRNA GAS5, which was further confirmed by RT‑qPCR, western blot analysis and the dual‑luciferase reporter assay. Finally, it was confirmed that lncRNA GAS5 promoted autophagy by sponging miR‑181c‑5p and miR‑1192, and upregulating the expression levels of the key autophagic regulators, ATG5 and ATG12. On the whole, the present study demonstrates that total, lncRNA GAS5 promotes macrophage autophagy by targeting the miR‑181c‑5p/ATG5 and miR‑1192/ATG12 axes.

Citation

Tao Xu, Xiangrong Xu, Yuankui Chu, Dan Jiang, Guangxian Xu. Long‑chain non‑coding RNA GAS5 promotes cell autophagy by modulating the miR‑181c‑5p/ATG5 and miR‑1192/ATG12 axes. International journal of molecular medicine. 2021 Dec;48(6)