Mechanism Of Long Noncoding RNA Gas5 Regulating Cell Autophagy Through Competitively Binding To MiR-181c-5p

Objective To explore the molecular mechanism of long-chain non-coding RNA Gas5 regulating RAW264.7 cell autophagy through competitively binding miRNAs and further regulating mi RNAs` autophagic target gene expression.Methods Autophagy inhibited and autophagy promted model were established with RAW264.7 cells treated with 3-methyladenine(3-MA)or starvation,respectively.The expression of lncRNA Gas5 in each group was detected by quantitative Real-time PCR(qRT-PCR).To clarify the regulatory effect of lnc RNA Gas5 on autophagy in RAW264.7 cells,we established lncRNA Gas5 overexpression and knockout cell models,and then the formation and quantity of autophagosome,autophagy marker protein LC3 expression were detected by confocal laser scanning microscopy,transmission electron microscopy and western blot,respectively.Bioinformatic analysis was used to study the target miRNAs and associated autophagy-related target genes of lncRNA Gas5.MiR-181c-5p and its target gene Atg5 were chosen as our targets.Gas5-wt/mut and Atg5 3'UTR-wt/mut recombination luciferase reporter vectors were constructed to verify the targeted regulation of miR-181c-5p on lncRNA Gas5 and Atg5 by detecting the changes of luciferase activity.The effect of lncRNA Gas5 overexpression and knockout on the expression of miR-181c-5p and Atg5 genes were detected by qRT-PCR and western blot.MiR-181c-5p mimics/inhibitors were transfected into RAW264.7 cells whose lncRNA Gas5 was overexpressed(Gas5)or knocked out(sh-Gas5),and then autophagy-related genes Atg5,LC3 and p62 were detected by western blot.Finally,the Atg5 3'UTR recombinant vector and miR-181c-5p mimic were co-transfected into the RAW264.7 cells whose lncRNA Gas5 was overexpressed,subsequently,relative change in luciferase activity was detected and calculated to investigate whether lncRNA Gas5 affects Atg5 expression via regulating miR-181c-5p directly.Results 1.qRT-PCR results showed that lncRNA Gas5 expression was significantly up-regulated in starvation-induced autophagy and decreased in 3-MA-treated autophagy inhibition group comparing with control group(P<0.05).Western blot results showed that lncRNA Gas5 overexpression promoted LC3 II protein expression,accelerated the degradation of p62 protein(P<0.05),and vice versa(P<0.05).Laser confocal and TEM results also showed that lncRNA Gas5 overexpression could promote the formation of autophagic bodies(P<0.05).These results indicated that lncRNA Gas5 could affect the autophagy key proteins in RAW264.7 cells,and then play a positive regulatory role on autophagy.2.Bioinformatic results showed that mir-181c-5p had the same binding sites for lncRNA Gas5 and Atg5.The results of dual luciferase reporter assay showed that miR-181c-5p over-expression could significantly decrease the luciferase activity of Atg5 3'UTR-wt and Gas5-wt(P<0.05),while this suppression was aborted when the binding sites were mutated.These results indicated that miR-181c-5p could target both lncRNA Gas5 and Atg5.3.Quantitative detection of miR-181c-5p and Atg5 mRNA expression in Gas5 and shGas5 groups showed that Gas5 overexpression could significantly downregulate miR-181c-5p expression and upregualte Atg5 expression(P<0.05).In contrast,mi R-181c-5p expression was significantly upregulated and Atg5 expression was significantly downregulated when Gas5 was decreased.Western blot results showed that comparing with the control group,the expressions of Atg5 and LC3 II in Gas5 group were significantly increased,while both of them were significantly decreased in sh-Gas5 group,indicating that lncRNA Gas5 had a positive effect on the expression of Atg5.4.Comparing with the control group,were increased in Gas5-treated group,but they were decreased in miR-181c-5p mimics group.Meanwhile,we found Atg5 and LC3 II expression had no significant difference in Gas5/mi R-181c-5p mimics co-transfection group,but they were decreased apparently in sh-Gas5/miR-181c-5p mimics co-transfection group.The expression of p62 in each group was opposite to the expression of LC3 II.The results of dual luciferase reporter assay showed that the luciferase activity of Atg5 3'UTR-wt was significantly decreased by miR-181c-5p overexpression,however,lncRNA Gas5 overexpression could abort this impression(P<0.05).These results indicated that the regulatory effect of lncRNA Gas5 on Atg5 was achieved by inhibiting the action of miR-181c-5p.Conclusion LncRNA Gas5 aborted the suppression of miR-181c-5p on its targeting gene Atg5 by competitively binding to miR-181c-5p,and then positively regulates autophagy.