Dex attenuates the inflammatory response
To investigate the effects of Dex, we constructed a pulmonary inflammatory cell model using IL-1β and LPS. A549 cells were used for the assay and were treated with 10 ng/mL IL-1β or 1 μg/mL LPS in DMEM. The cells incubated with the reagents were collected after 8, 16, and 24 h, and the total RNA was isolated. RT-PCR was conducted to confirm the mRNA expression levels of IKβ-α, IKKβ, IL-6, IL-8, and TNF-α. Generally, the results showed that IL-1β performed better against LPS because the mRNA levels of most of the inflammatory cytokines, except for IL-6 and TNF-α, were significantly upregulated at 8 h; in addition, the mRNA levels of all other cytokines were two- to four-fold greater at 24 h (Figure 1A). The secretary cytokines in the cell culture medium, including IL-6, IL-8, and TNF-α were determined via ELISA. The results were consistent with that of RT-PCR and showed that IL-1β was generally more effective and that an effect can be observed at 8 h (Figure 1B). Therefore, we decided to use IL-1β with 24-h incubation to construct the pulmonary inflammatory cell model.
Following the same principle, we used the cell model to characterize the effects of Dex treatment. We incubated A549 cells with IL-1β and Dex at different concentrations, ranging from 10 nM to 10000 nM, for 24 h. As mentioned above, RT-PCR and ELISA were used to evaluate the effects of Dex. The mRNA expression levels of IKβ-α, IKKβ, IL-6, IL-8, and TNF-α, significantly increased following IL-1β treatment (P < 0.05). After adding Dex, the mRNA levels decreased to their baseline levels, indicating that Dex supplementation can attenuate the inflammatory response (Figure 2A). With respect to the levels of secretary inflammatory cytokines, compared with the control group, the levels of IL-6, IL-8, and TNF-α significantly increased following IL-1β treatment. However, when cells were supplemented with Dex, the levels of cytokines decreased; both 1 and 10 µM Dex significantly decreased the levels (Figure 2B). Taken together, these observations suggest that Dex plays a role in attenuating the inflammatory response.
Verification of the lncRNAs involved in the attenuation of the inflammatory response
Based on our preliminary findings, we targeted four lncRNAs, namely lncMALAT1, lncHotair, lncH19, and lncNeat1, to determine which of these lncRNAs are involved in processes underlying inflammation attenuation [16-20]. RT-PCR was conducted to determine lncRNA levels following treatment with IL-1β and Dex. The results showed that the four candidate lncRNAs were downregulated following IL-1β treatment; however, lncRNA levels increased after Dex supplementation (Figure 3). In general, lncRNA levels were most clearly observed after treatment with 1 and 10µM Dex (P < 0.05). Among the lncRNAs, the level of lncH19 exhibited the highest increase, approximately two-fold, following treatment with 1 and 10 μM Dex. Therefore, lncH19 and 1 μM Dex were selected for further functional validation.
To verify the functions of lncH19, the corresponding shRNA was designed and infected into A549 cells using lentivirus. The performance of the specific shRNA was assessed via RT-PCR. LncH19 expression showed a 50% decrease after transfection with the specific shRNA (Figure 4A). The cells were then treated with IL-1β and Dex (1 μM) to investigate the effect of decreased lncH19 expression. Cell proliferation was assessed via the MTS assay. Treatment with 1 μM Dex clearly inhibited cell proliferation, which might only be a side effect. When lncH19 expression was inhibited, cell proliferation increased relative to the group of normal expression of lncH19 (Figure 4B). We then further validated the functions of lncH19 via the cell apoptosis assay using flow cytometry. Compared with the percentage of apoptotic cells without Dex treatment, apoptotic cells increased approximately two-fold when Dex was supplemented. Cell apoptosis was inhibited and reduced by 30%–50% when lncH19 was inhibited under IL-1β and Dex treatment compared with normal expression of lncH19 under IL-1β and Dex treatment (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, Dex supplementation clearly decreased protein levels; however, lncH19 inhibition could 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 Dex regulation. The cell culture supernatant was then collected and the levels of the inflammatory cytokines, including IL-6, IL-8, and TNF-α, were determined. Cytokines decreased in concentration by approximately 50% following Dex addition. However, lncH19 inhibition increased the levels of inflammatory cytokines (Figure 4F). Therefore, Dex can attenuate the inflammatory response, and lncH19 plays an important role in the inflammatory response.
Validation of the candidate miRNAs regulated by lncH19
Miranda v3.3a was used to predict the target miRNAs of lncH19. The miRNAs are listed in Table 2. Because lncH19 expression was upregulated following Dex addition and because the upregulation of lncRNA tends to result in the downregulation of miRNAs, we searched for downregulated miRNAs following Dex addition. We observed that miR-346, miR-18a-3p, and miR-324-3p were downregulated; therefore, they were chosen for further validation (Figure 5). The corresponding miRNA inhibitors were designed and transfected into 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 approximately 50%. The inhibitor of miR-324-3p was significantly more effective compared to that of miR-18a-3p (Figure 6A). Therefore, we further investigated the functions of miR-324-3p and divided the cells into four groups: shNC + IL-1β + Dex + miRNA NC (NC + miRNA NC), shNC + IL-1β + Dex + miR-324-3p inhibitor (NC + miR-324-3p inhibitor), shlncH19 + IL-1β + Dex + miRNA NC (shlncH19 + miRNA NC), and shlncH19 + IL-1β + Dex + miR-324-3p inhibitor (shlncH19 + miR-324-3p inhibitor). The MTS assay was conducted to assess cell proliferation.Compared with the NC + miR-324-3p inhibitor group, the cell proliferation of the NC + miRNA NC group significantly increased at 72 h (P < 0.05). Cell proliferation of the shlncH19 + miRNA NC group increased from an OD value of 1.5 to 2.0 compared with in the NC + miRNA NC group. Cell proliferation was lower in the shlncH19 + miR-324-3p inhibitor group than in the shlncH19 + miRNA NC group (Figure 6B). Therefore, these results suggest that lncH19 expression inhibits cell proliferation and miR-324-3p expression promotes cell proliferation. We also conducted the cell apoptosis assays using flow cytometry to determine the functions of miR-324-3p. lncH19 inhibition reduced the number of apoptotic cells by ~30%; however, miR-324-3p inhibition clearly enhanced cell apoptosis compared with in the NC + miRNA NC group (Figure 6C). Compared with the NC + miRNA NC group, the mRNA levels of IKβ-α, IKKβ, IL-6, IL-8, and TNF-α were upregulated when lncH19 was inhibited but downregulated when miR-324-3p was simultaneously inhibited (Figure 6D). The cell culture supernatant was collected to determine the levels of the cytokines IL-6, IL-8, and TNF-α. The levels of the cytokines significantly increased when lncH19 was inhibited by shRNA relative to the NC + miRNA NC group. However, the levels of cytokines significantly decreased 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 inflammatory genes, including p-P65, P65, ICAM-1, and VCAM-1. There was no significant change in the protein level of P65. lncH19 inhibition 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 downregulated by an inhibitor simultaneously. These findings suggest that miR-324-3p is involved in lncH19 regulation. lncH19 might target and inhibit miR-324-3p expression and thus regulate inflammation.