Dex attenuates the inflammatory response
To investigate the effect of Dex, we induced the pulmonary inflammatory cell model with IL-1β and LPS. A549 cells were applied for the assay and treated with IL-1β at 10 ng/mL or LPS at 1 μg/mL in DMEM culture medium. Cells incubated with the reagents after 8, 16 and 24 hours were collected, and total RNA was isolated. RT-PCR was conducted to confirm the expression of the inflammatory related mRNA, including IKβ-α, IKKβ, IL-6, IL-8, and TNF-α. Generally, the results showed that IL-1β performed better against LPS, as the mRNA levels of most of the inflammatory cytokines, aside from IL-6 and TNF-α, were significantly up-regulated at 8hours; in addition, mRNA levels of all other cytokines were 2- to 4-fold greater at 24 hours (Figure 1A). The secretary cytokines in the cell culture medium, including IL-6, IL-8 and TNF-α were determined by ELISA. The results were consistent in showing that IL-1β was generally more effective, and the effect can be observed at 8 hours (Figure 1B). Therefore, we decided to use IL-1β for 24 hours incubation to induce the pulmonary inflammation cell model.
Following the same principle, we used the cell model to characterize the effect of Dex. We incubated A549 cells with IL-1β and Dex at different concentrations, ranging from 10 nM to 10000 nM at 24 hours. As before, RT-PCR and ELISA were used to evaluate the effect of Dex. mRNA levels of inflammatory related genes, including IKβ-α, IKKβ, IL-6, IL-8, and TNF-α, significantly increased following treatment with IL-1β (P < 0.05). After adding Dex, the mRNA level was reduced to baseline levels, indicating that supplementation with Dex can attenuate the inflammatory response (Figure 2A). With respect to the secretary inflammatory cytokines, the concentration of IL-6, IL-8, and TNF-α increased significantly following treatment with IL-1β relative to the control group. However, when cells were supplemented with Dex, the concentration of cytokines decreased, and Dex at both 1 uM and 10 uM significantly decreased in concentration (Figure 2B). Therefore, these observations suggest that Dex plays a role in attenuating the inflammatory response.
Verification of the lncRNAs involved in inflammation attenuation
Based on our preliminary findings, we targeted four lncRNAs, lncMALAT1, lncHotair, lncH19, and lncNeat1 to determine which of these lncRNAs are involved in processes underlying inflammation attenuation [17-20]. RT-PCR was conducted to determine lncRNA levels following treatment with IL-1beta and Dex. The results show that the four candidate lncRNAs were down-regulated following treatment with IL-1beta; however, lncRNA levels increased following supplementation with Dex (Figure 3). In general, lncRNA levels can be most clearly observed by the treatment of Dex at 1 μM and 10 μM (P < 0.05). Among the lncRNAs, the level of lncH19 exhibited the highest increase, approximately 2-fold, following treatment with 1 μM and 10 μM Dex. Therefore, lncH19 and Dex with 1 μM was selected for further function validation.
To verify the functions of lncH19, the corresponding shRNA was designed and infected to A549 cells by lentivirus. The performance of the specific shRNA was assessed by RT-PCR. The expression of lncH19 showed a 50% decreased after the specific shRNA transfection (Figure 4A). The cells were then treated with IL-1β and Dex (1 μM) to investigate the effect of decreasing the expression of lncH19. Cell proliferation was assessed via the MTS assay. Treatment of 1 μM Dex clearly inhibited cell proliferation, which might only be a side effect. When the expression of lncH19 was induced, cell proliferation increased relative to IL-1β-treated cells lacking lncH19 inhibition with or without Dex (Figure 4B). We then further validated the functions of lncH19 via the flow cytometry cell apoptosis assay. Compared to the percentage of apoptotic cells with and without Dex treatment, apoptotic cells increased approximately 2-fold when Dex was applied. Cell apoptosis was inhibited and reduced by 30–50% relative to IL-1β-treated samples lacking lncH19 inhibition with or without Dex (Figure 4C & 4D). Western blotting was conducted to determine the protein level of the genes that might be involved in the inflammatory response, including P65, p-P65, ICAM-1 and VCAM-1. The protein level of P65 did not change. For the rest of the genes, supplementation of Dex clearly decreased protein levels, but inhibition of lncH19 can also increase protein levels (Figure 4E). Therefore, given the consistency of these results with those obtained via flow cytometry and the MTS assay, the proteins p-P65, ICAM-1, and VCAM-1 are likely involved in the regulation of Dex. The cell culture supernatant was then collected, and the concentration of inflammatory cytokines, including IL-6, IL-8, and TNF-α was determined. Cytokines decreased in concentration by approximately 50% following the addition of Dex. Moreover, the inhibition of lncH19 increased the concentration of inflammatory cytokines (Figure 4F). Therefore, Dex can attenuate the inflammatory response, and lncH19 plays an important role in inflammatory processes.
Validation of candidate miRNAs regulated by lncH19
Miranda v3.3a has been used to predict the target miRNAs of lncH19. miRNAs are presented in Table 2. Because expression of lncH19 was up-regulated following addition of Dex and because up-regulation of lncRNA tends to result in the downregulation of miRNAs, we searched for down-regulated miRNAs following addition of Dex. MiR-346, miR-18a-3p, and miR-324-3p were down-regulated and were therefore chosen for further validation (Figure 5). The corresponding miRNA inhibitors were designed and transfected to A549 cells. The performance of the inhibitors was assessed via RT-PCR. For both miR-18a-3p and miR-324-3p, the corresponding inhibitors clearly decreased the expression of miRNAs by ~50%. The inhibitor of miR-324-3p was significantly more effective relative to that of miR-18a-3p (Figure 6A). Therefore, we further investigated the functions of miR-324-3p and divided into four groups, the first group, shNC+IL-1 beta+Dex+miRNA NC, abbreviated as NC+miRNA NC, the second group, shNC+IL-1 beta+Dex+miR-324-3p inhibitor, abbreviated as NC+miR-324-3p inhibitor, the third group, shlncH19+IL-1 beta+Dex+miRNA NC, abbreviated as shlncH19+miRNA NC, the fourth group, shlncH19+IL-1 beta+Dex+miR-324-3p inhibitor, abbreviated as shlncH19+miR-324-3p inhibitor. An MTS assay was conducted to assess cell proliferation. By comparing the NC+miRNA NC group to NC+miR-324-3p inhibitor group, cell proliferation increased significantly at 72 hours (P < 0.05). When cells were infected with shRNA targeting lncH19, cell proliferation increased from an OD value of 1.5 to 2.0 relative to NC+miRNA NC group. Cell proliferation was lower in shlncH19+miR-324-3p inhibitor, group compare to shlncH19+miRNA NC (Figure 6B). Therefore, the evidence suggests that the expression of lncH19 inhibited cell proliferation, but the expression of miR-324-3p promoted cell proliferation. We also conducted cell apoptosis assays via flow cytometry to determine the functions of miR-324-3p. The inhibition of lncH19 reduced apoptotic cells by ~30%, but inhibition of miR-324-3p clearly enhanced cell apoptosis relative to the NC+miRNA NC group (Figure 6C). mRNA levels of IKβ-α, IKKβ, IL-6, IL-8 and TNF-α were all up-regulated when lncH19 was inhibited but down-regulated when miR-324-3p was simultaneously inhibited relative to the NC+miRNA NC group (Figure 6D). The cell culture supernatant was collected to determine the concentration of the cytokines IL-6, IL-8 and TNF-α. The concentration of cytokines increased significantly when lncH19 was inhibited by shRNA relative to the NC+miRNA NC group. However, the concentration of cytokines decreased significantly when the expression of miR-324-3p was simultaneously inhibited, indicating that the inflammatory response was suppressed (Figure 6E). Western blotting was performed to determine the protein levels of inflammation-related genes, including p-P65, P65, ICAM-1 and VCAM-1. There was no significant change in the protein level of P65. Inhibition of lncH19 increased the protein levels of p-P65, ICAM-1 and VCAM-1 relative to the NC+miRNA NC group. However, protein levels were decreased when miR-324-3p was down-regulated by an inhibitor simultaneously. These findings suggest that miR-324-3p is involved in lncH19 regulation. lncH19 might target and inhibit the expression of miR-324-3p and thereby regulate inflammation.