To investigate lncRNAs and their roles in regulating the pulmonary inflammatory response under dexamethasone (Dex) treatment. IL-1β (10 ng/mL) and LPS (1 μg/mL) was used to construct inflammatory cell models with A549 cells; IL-1β performed better against LPS. Different concentrations of Dex were used to attenuate the inflammation induced by IL-1β, and its effect was assessed via RT-PCR to detect inflammatory cytokine-related mRNA levels, including those of IKβ-α, IKKβ, IL-6, IL-8, and TNF-α. Furthermore, ELISA was used to detect the levels of the inflammatory cytokines TNF-α, IL-6, and IL-8. RT-PCR was used to quantify the levels of lncRNAs, including lncMALAT1, lncHotair, lncH19, and lncNeat1. LncH19 was most closely associated with the inflammatory response, which was induced by IL-1β and attenuated by Dex. Among the lncRNAs, the level of lncH19 showed the highest increase following treatment with 1 and 10 μM Dex. Therefore, lncH19 was selected for further functional studies. LncH19 expression was inhibited by shRNA transduced with lentivirus. Cell assays for cell proliferation and apoptosis as well as RT-PCR, western blot, and ELISA for inflammatory genes were conducted to confirm the functions of lncH19. The predicted target miRNAs of lncH19 were 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 via RT-PCR, hsa-miR-346, hsa-miR-18a-3p, and hsa-miR-324-3p showed consistent patterns in A549 NC and A549 shlncH19. An miRNA inhibitor was transfected into A549 NC and A549 shlncH19 cells, and the expression levels were determined via RT-PCR. hsa-miR-324-3p was inhibited the most compared with hsa-miR-346 and hsa-miR-18a-3p and was subjected to further functional studies. RT-PCR, ELISA, and western blotting for inflammatory gene detection were conducted to validate the functions of the target hsa-miR-324-3p. Treatment with 1 and 10 μM Dex could effectively attenuate the inflammatory response. During this process, lncH19 expression significantly increased (P < 0.05). Therefore, treatment with 1 μM Dex was used for further study. Under IL-1β treatment with or without Dex, lncH19 inhibition led to an increase in cell proliferation; a decrease in cell apoptosis; an increase in the protein levels of inflammatory genes; phosphorylation of P65, ICAM-1, and VCAM-1; and increase inflammatory cytokines. Prediction of the targets of lncH19 and validation via RT-PCR revealed that miR-346, miR-18a-3p, and miR-324-3p negatively correlate with lncH19. Additionally, Dex increased the lncH19 expression but reduced that of the miRNAs. Among the miRNAs, miR-324-3p was the most markedly downregulated miRNA 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 in pulmonary inflammatory response under Dex treatment. Dex can attenuate the pulmonary inflammatory response by regulating the lncH19/miR-324-3p cascade.

中文翻译：

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地塞米松可通过调节lncH19 / miR-324-3p级联反应减轻肺部炎症反应

调查lncRNAs及其在地塞米松（Dex）治疗下调节肺部炎症反应中的作用。用IL-1β（10 ng / mL）和LPS（1μg/ mL）构建具有A549细胞的炎性细胞模型。IL-1β对LPS表现更好。使用不同浓度的Dex来减轻IL-1β诱导的炎症，并通过RT-PCR评估其作用，以检测炎症细胞因子相关的mRNA水平，包括IKβ-α，IKKβ，IL-6，IL-8的水平。和TNF-α。此外，ELISA被用于检测炎性细胞因子TNF-α，IL-6和IL-8的水平。RT-PCR用于定量检测lncRNA的水平，包括lncMALAT1，lncHotair，lncH19和lncNeat1。LncH19与炎症反应最密切相关，后者由IL-1β诱导并由Dex减弱。在lncRNA中，在用1和10μMDex处理后，lncH19的水平显示出最高的增加。因此，选择lncH19进行进一步的功能研究。LncH19表达被慢病毒转导的shRNA抑制。进行了用于细胞增殖和凋亡的细胞测定，以及用于炎症基因的RT-PCR，western印迹和ELISA，以确认lncH19的功能。lncH19的预测靶标miRNA是hsa-miR-346，hsa-miR-324-3p，hsa-miR-18a-3p，hsa-miR-18b-5p，hsa-miR-146b-3p，hsa-miR-19b -3p和hsa-miR-19a-3p。通过RT-PCR进行估计后，hsa-miR-346，hsa-miR-18a-3p和hsa-miR-324-3p在A549 NC和A549 shlncH19中显示出一致的模式。将miRNA抑制剂转染到A549 NC和A549 shlncH19细胞中，并通过RT-PCR确定表达水平。与hsa-miR-346和hsa-miR-18a-3p相比，hsa-miR-324-3p被抑制得最多，并进行了进一步的功能研究。进行了RT-PCR，ELISA和Western blotting来检测炎症基因，以验证目标hsa-miR-324-3p的功能。用1和10μMDex进行治疗可以有效地减轻炎症反应。在此过程中，lncH19表达显着增加（P <0.05）。因此，使用1μMDex进行治疗需要进一步研究。在有或没有Dex的IL-1β治疗下，lncH19抑制作用导致细胞增殖增加。细胞凋亡减少；炎性基因蛋白质水平的增加；P65，ICAM-1和VCAM-1的磷酸化；并增加炎症细胞因子。对lncH19靶标的预测并通过RT-PCR验证表明，miR-346，miR-18a-3p和miR-324-3p与lncH19负相关。另外，Dex增加了lncH19表达，但降低了miRNA的表达。在miRNA中，miR-324-3p是经miRNA抑制剂处理后最显着下调的miRNA。MTS分析和细胞凋亡分析表明，miR-324-3p抑制剂抑制细胞增殖并诱导细胞凋亡，从而显着减弱炎症反应，从而逆转了lncH19在调节细胞增殖和炎症细胞因子分泌中的作用（P < 0.05）。因此，lnxH19可能在Dex治疗下调节miR-324-3p的肺部炎症反应。Dex可以通过调节lncH19 / miR-324-3p级联来减轻肺部炎症反应。Dex增加了lncH19表达，但降低了miRNA的表达。在miRNA中，miR-324-3p是经miRNA抑制剂处理后最显着下调的miRNA。MTS分析和细胞凋亡分析表明，miR-324-3p抑制剂抑制细胞增殖并诱导细胞凋亡，从而显着减弱炎症反应，从而逆转了lncH19在调节细胞增殖和炎性细胞因子分泌中的作用（P < 0.05）。因此，lnxH19可能在Dex治疗下调节miR-324-3p的肺部炎症反应。Dex可以通过调节lncH19 / miR-324-3p级联来减轻肺部炎症反应。Dex增加了lncH19表达，但降低了miRNA的表达。在miRNA中，miR-324-3p是经miRNA抑制剂处理后最显着下调的miRNA。MTS分析和细胞凋亡分析表明，miR-324-3p抑制剂抑制细胞增殖并诱导细胞凋亡，从而显着减弱炎症反应，从而逆转了lncH19在调节细胞增殖和炎性细胞因子分泌中的作用（P < 0.05）。因此，lnxH19可能在Dex治疗下调节miR-324-3p的肺部炎症反应。Dex可以通过调节lncH19 / miR-324-3p级联来减轻肺部炎症反应。治疗miRNA抑制剂后，miR-324-3p最显着下调。MTS分析和细胞凋亡分析表明，miR-324-3p抑制剂抑制细胞增殖并诱导细胞凋亡，从而显着减弱炎症反应，从而逆转了lncH19在调节细胞增殖和炎性细胞因子分泌中的作用（P < 0.05）。因此，lnxH19可能在Dex治疗下调节miR-324-3p的肺部炎症反应。Dex可以通过调节lncH19 / miR-324-3p级联来减轻肺部炎症反应。治疗miRNA抑制剂后，miR-324-3p最显着下调。MTS分析和细胞凋亡分析表明，miR-324-3p抑制剂抑制细胞增殖并诱导细胞凋亡，从而显着减弱炎症反应，从而逆转了lncH19在调节细胞增殖和炎性细胞因子分泌中的作用（P < 0.05）。因此，lnxH19可能在Dex治疗下调节miR-324-3p的肺部炎症反应。Dex可以通过调节lncH19 / miR-324-3p级联来减轻肺部炎症反应。从而逆转了lncH19调节细胞增殖和炎性细胞因子分泌的作用（P <0.05）。因此，lnxH19可能在Dex治疗下调节miR-324-3p的肺部炎症反应。Dex可以通过调节lncH19 / miR-324-3p级联来减轻肺部炎症反应。从而逆转了lncH19调节细胞增殖和炎性细胞因子分泌的作用（P <0.05）。因此，lnxH19可能在Dex治疗下调节miR-324-3p的肺部炎症反应。Dex可以通过调节lncH19 / miR-324-3p级联来减轻肺部炎症反应。