Dexamethasone can attenuate the inflammatory response in asthma via regulation of the lncH19/miR-324-3p cascade
Authors: Chen Ye , Zhang Chao , Xiao Changxue , Li Xiao-dong , Hu Zhi-li , Xu Feng 
Published: June 2020

Abstract
Objective: To investigate lncRNAs and their roles in regulating the inflammatory response under treatment of Dexamethasone (Dex) in asthma.Methods: IL-1 beta (10 ng/ml) and LPS (1 μg/ml) was used to induce an inflammatory cell model with A549 cells, and the results showed that IL-1 beta performed better against LPS. Dex with different concentration was used to attenuate inflammation by IL-1 beta, and its effect was assessed by RT-PCR to detect the inflammatory related mRNA, including IKbeta-alpha, IKKbeta, IL-6, IL-8, and TNF-alpha and ELISA to detect the inflammatory cytokines TNF-alpha, IL-6 and IL-8. RT-PCR was used to quantify levels of lncRNAs, including lncMALAT1, lncHotair, lncH19, and lncNeat1. lncH19 was most closely correlated with the inflammatory response, which was induced by IL-1beta and attenuated by Dex, Among the lncRNAs, the level of lncH19 exhibited the highest increase following treatment with 1 μM and 10 μM Dex. Therefore, lncH19 was selected for further function study. lncH19 expression was inhibited by shRNA transduced by lentivirus. Cell assays for cell proliferation and apoptosis as well as RT-PCR, western blot, and ELISA for inflammatory related genes were conducted to confirm the functions of lncH19. Predicted target miRNAs of lncH19 included the following: hsa-miR-346, hsa-miR-324-3p, hsa-miR-18a-3p, hsa-miR-18b-5p, hsa-miR-146b-3p, hsa-miR-19b-3p, and hsa-miR-19a-3p. Following estimation by RT-PCR, hsa-miR-346, hsa-miR-18a-3p, hsa-miR-324-3p showed consistent patterns in A549 NC and A549 shlncH19. miRNA inhibitor was transfected into A549 NC and A549 shlncH19 cells, and expression levels were determined by RT-PCR. hsa-miR-324-3p was inhibited the most relative to hsa-miR-346 and hsa-miR-18a-3p and was subjected to further function study. RT-PCR, ELISA and Western Blot for inflammatory related genes detection were conducted to validate the functions of the target hsa-miR-324-3p. Results: Dex with 1μM and 10 μM were shown to be effective in attenuating the inflammatory response. During this process, lncH19 significantly increased in expression (P < 0.05). Dex with 1μM was for further study. Under IL-1 beta treatment with or without Dex, the inhibition of lncH19 lead to an increase cell proliferation, a decrease in cell apoptosis, an increase in the protein level of inflammatory-related genes, and the phosphorylation of P65, ICAM-1, VCAM-1, and inflammatory cytokines. Following prediction of the targets of lncH19 and validation by RT-PCR, miR-346, miR-18a-3p, and miR-324-3p were found to be negatively correlated to lncH19. Additionally, Dex increased the expression of lncH19, but the expression of the aforementioned miRNAs was reduced. Among miRNAs, miR-324-3p was the most markedly down-regulated following treatment of miRNA inhibitors. The MTS assay and cell apoptosis assay showed that the miR-324-3p inhibitor inhibited cell proliferation and induced cell apoptosis, thereby significantly attenuating the inflammatory response, which reversed the effect of lncH19 in regulating cell proliferation and the secretion of inflammatory cytokines (P < 0.05). Therefore, lncH19 might regulate miR-324-3p during Dex treatment in inflammatory response of asthma.Conclusion: Dex can attenuate the inflammatory response in asthma via regulation of the lncH19/miR-324-3p cascade.