The YAP/HIF-1α/miR-182/EGR2 Axis is Implicated in Asthma Severity by Control of Th17 Cell Differentiation

Background: Asthma is a heterogeneous chronic inammatory disease of the airways, with reversible airow limitations and airway remodeling. T helper 17 (Th17) cells play an important role in the pathogenesis of allergic asthma. However, there is hitherto little data about signaling pathways controlling Th17 cell differentiation in asthma. The aim of this study was to ascertain whether the YAP/HIF-1α/miR-182/EGR2 axis underpins Th17 cell differentiation and asthma severity. Methods: The study included 29 pediatric patients with asthma, 22 healthy volunteers, ovalbumin (OVA)-induced murine asthma models, and mouse naive CD4 + T. The subpopulation of Th17 cells was examined by ow cytometry. The level of IL-17A was determined by ELISA method. ChIP-qPCR assay and dual-luciferase reporter gene assay were performed to examine interaction between HIF-1α and miR-182, miR-182 and EGR2. Results: YAP, HIF-1α, and miR-182 were found to be up-regulated but EGR2 was down-regulated in human and mouse peripheral blood mononuclear cells (PBMCs) in the context of asthma. Abundant expression of YAP and HIF-1α promoted miR-182 expression and then inhibited EGR2, a target of miR-182, thus enhancing Th17 differentiation and deteriorating asthma and lipid metabolism dysfunction. In addition, in vivo ndings revealed that over-expression of EGR2 undermined the promoting effect of the YAP/HIF-1α/miR-182 axis on asthma and lipid metabolism dysfunction. Conclusion: These results shed light on that the activation of the YAP/HIF-1α/miR-182/EGR2 axis may promote Th17 cell differentiation, exacerbate asthma development, and aggravate lipid metabolism dysfunction, providing a potential therapeutic target in asthma. Collectively, these ndings suggested that miR-182 accelerated the differentiation of CD4 + T cells into cells The aforementioned ndings suggested that EGR2 over-expression could inhibit miR-182-induced Th17 cell differentiation. In this part of the experiments, we investigated whether EGR2 inhibited the Th17 cell differentiation evoked by the YAP/HIF-1α axis. RT-qPCR and Western blot analysis (Supplementary Fig. 4A, B) displayed that the YAP expression was increased in CD4 + T cells after YAP over-expression alone or both over-expression of YAP and EGR2 (p < 0.05). The HIF-1α expression was also increased after HIF-1α overexpression alone or both over-expression of HIF-1α and EGR2 (p < 0.05). In addition, cells with both overexpression of YAP and EGR2 exhibited higher EGR2 expressions relative to those with YAP over-expression alone, and the cells with both over-expression of HIF-1α and EGR2 also exhibited higher EGR2 expressions compared to those with HIF-1α over-expression alone (p < 0.05). Flow cytometry results (Fig. 5A) depicted that YAP or HIF-1α over-expression promoted Th17 cell proportion, which was inhibited by EGR2 over-expression (p < 0.05). ± two multiple using one-way analysis of variance (ANOVA) with post-hoc analysis of YAP and HIF-1α proteins in Th17 cells; C, Western blot analysis of HIF-1α protein in Th17 cells after YAP overexpression or knockdown; D, Western blot analysis of YAP and HIF-1α proteins in Th17 cells following different treatments; E, Th17 cell proportion after different treatments detected by ow cytometry; F, IL-17A serum level in cell supernatant after different treatments by ELISA; G, RORγt mRNA expression in cells after different treatments detected by RT-qPCR; H, Western blot analysis of RORγt protein in cells after different treatments. Comparisons between two groups were conducted by unpaired t test, and those among multiple groups were conducted by one-way ANOVA with Tukey’s post hoc test; the experiments were repeated 3 times; * p < 0.05, compared with the CD4+ T cells, or cells treated with sh-NC, oe-NC, or sh-NC + oe-NC; # p < 0.05, compared with the cells treated with oe-YAP + sh-NC.

indicated that YAP1 targeted the HIF1A gene (Fig. 1E). Based on the aforementioned in silico analysis and previous literatures, we presumed that YAP1 (YAP) may affect the development of asthma through regulation of HIF1A (HIF-1α), and consequently designed the current study in order to validate our theories.
The results of hematoxylin-eosin staining illustrated that the OVA-induced asthma mice presented with airway remodeling (p < 0.05; Fig. 1F) and increased thickening of the airway smooth muscles, airway wall, and airway epithelium mucosa relative to the sham-operated mice (p < 0.05; Fig. 1G). These ndings veri ed the successful establishment of OVA-induced murine asthma models. Next, the proportion of Th17 cells was measured in human PBMCs and mouse spleen cells by means of ow cytometry, which revealed an increased proportion of Th17 cells in both asthma patients and mice (p < 0.05; Fig. 1H). Meanwhile, detection results of ELISA assay displayed that the serum levels of IL-17A were up-regulated in both asthma patients (p < 0.05; Fig. 1I) and asthma mice (p < 0.05; Fig. 1J). In addition, RT-qPCR and Western blot analysis determined that RORγt mRNA and protein expressions were elevated in PBMCs of asthma patients (p < 0.05; Fig. 1K, L) and spleen cells of asthma mice (p < 0.05; Fig. 1M, N).
Additionally, results from RT-qPCR and Western blot analysis revealed that YAP and HIF-1α expressions were both augmented in asthma patients (p < 0.05; Fig. 1O, P). Similar trends were observed in YAP and HIF-1α expressions in OVA-induced asthma mice (p < 0.05; Fig. 1Q, R). There is a signi cant association between asthma and the serum level of HDL-C and thus, HDL-C was employed as a marker to assess the degree of asthma in the current study [20]. As depicted in Fig. 1S, T, HDL-C serum levels were elevated in both asthma patients and mice (p < 0.05). Evidence further suggests that HIF-1α can promote the expression of miR-182 [12]. Meanwhile, the miR-183/96/182 cluster is also known to be signi cantly up-regulated as a result of Th17 differentiation [13]. Therefore, we speculated that the HIF-1α signaling pathway might promote Th17 differentiation by regulating miR-182. Subsequently, RT-qPCR was conducted to detect the expressions of miR-96/182/183 in human PBMCs and mouse spleen cells, which showed an upward trend in the miR-96/182/183 expressions in asthma patients and mice, with a more pronounced increase in the miR-182 expression (p < 0.05; Fig. 1U, V). Collectively, these results suggested that miR-182, YAP and HIF-1α were upregulated, while HDL-C was decreased in asthma.
YAP and HIF-1α promote the differentiation of CD4 + T Cells into Th17 Cells In order to further verify whether YAP and HIF-1α were also up-regulated in Th17 cells, we rst induced the differentiation of CD4 + T cells into Th17 cells in vitro. Next, RT-qPCR and Western blot analysis were applied to determine the expressions of YAP and HIF-1α in the differentiated TH17 cells. As illustrated in Fig. 2A, B, the mRNA and protein expressions of YAP and HIF-1α were increased in Th17 cells compared to the CD4 + T cells (p < 0.05).
Additionally, the infection e ciency of YAP/HIF-1α over-expression or knockdown in the TH17 cells was evaluated and con rmed by means of RT-qPCR and Western blot analysis ( Supplementary Fig. 1A, B), wherein sh-YAP-1 and sh-HIF-1α-1 exhibited superior e ciency and were consequently used for further experimentation (p < 0.05). Moreover, Western blot analysis demonstrated that the protein expression of HIF-1α was increased in cells with YAP over-expression, while being decreased in YAP knockdown cells (p < 0.05; To further investigate whether YAP/HIF-1α promoted the differentiation of CD4 + T cells into Th17 cells, Western blot analysis was applied to detect the protein expressions of YAP and HIF-1α in Th17 cells with different treatments. As shown in Fig. 2D, cells treated with YAP over-expression or with a combination of YAP over-expression and HIF-1α silencing exhibited increased YAP protein expressions (p < 0.05). However, no signi cant changes were noted in the YAP protein expression between HIF-1α over-expression cells and HIF-1α knockdown cells (p > 0.05). After YAP was over-expressed or HIF-1α was over-expressed, elevated HIF-1α expressions were found in the Th17 cells (p < 0.05). Meanwhile, opposite trends were observed in the Th17 cells following YAP knockdown or/and HIF-1α knockdown treatment (p < 0.05).
After that, the Th17 cell proportion was measured by means of ow cytometry, the results of which (Fig. 2E) displayed that YAP or HIF-1α over-expression brought about promoted Th17 cell proportion, while YAP or HIF-1α silencing resulted in reduced Th17 cell proportion (p < 0.05). In addition, augmented Th17 cell proportion caused by YAP over-expression was inhibited by HIF-1α silencing (p < 0.05).
Furthermore, the results of ELISA (Fig. 2F), RT-qPCR and Western blot analysis (Fig. 2G, H) revealed that IL-17A levels and RORγt expressions were both elevated as a result of YAP or HIF-1α over-expression, while being reduced in the absence of YAP or HIF-1α (p < 0.05). Additionally, increased IL-17A levels and RORγt expressions brought about YAP over-expression were found to be inhibited by HIF-1α silencing (p < 0.05).
All the aforementioned ndings highlighted that the differentiation of CD4 + T cells into Th17 cells was promoted by over-expressed YAP and HIF-1α.
HIF-1α promotes the differentiation of CD4 + T Cells into Th17 Cells by up-regulating miR-182 In the following experiments, we aimed to uncover the underlying mechanism of HIF-1α in Th17 cell differentiation. RT-qPCR detected that the expression of miR-182 was up-regulated in Th17 cells relative to CD4 + T cells (p < 0.05; Fig. 3A). In addition, Western blot analysis demonstrated that the protein expression of HIF-1α was elevated after treatment with HIF-1α over-expression in HEK293T cells with miR-182-wt or miR-182-mut (p < 0.05). Dual luciferase reporter assay revealed that HIF-1α over-expression brought about a signi cantly increase in the luciferase activity of miR-182-wt in HEK293T cells (p < 0.05; Fig. 3B). Moreover, the ChIP assay illustrated the binding of HIF-1α to the miR-182 promoter (Fig. 3C): compared to the IgG control, the enrichment of HIF-1α in the miR-182 promoter was notably increased, and was further promoted after HIF-1α over-expression (p < 0.05). Furthermore, the results of RT-qPCR demonstrated that the expression of miR-182 was increased in cells treated with oe-HIF-1α or oe-YAP, while being decreased in cells treated with sh-HIF-1α or sh-YAP (p < 0.05; Fig. 3D). These results indicated that HIF-1α promoted the expression of miR-182 in HEK293T cells.
Furthermore, the results of RT-qPCR and Western blot analysis illustrated that RORγt expressions were diminished in cells upon miR-182 down-regulation or HIF-1α down-regulation, while being elevated in cells with treated with miR-182 up-regulation or HIF-1α up-regulation. Meanwhile, dual treatment with oe-HIF-1α and miR-182 inhibitor inhibited the RORγt expression, while treatment with both sh-HIF-1α and miR-182 mimic brought about increased RORγt levels (p < 0.05; Fig. 3G, H).
The aforementioned ndings highlighted that HIF-1α up-regulated the miR-182 expression, and consequently promoted differentiation of CD4 + T cells into Th17 cells. miR-182 promotes the differentiation of CD4 + T Cells into Th17 Cells by inhibiting EGR2 In accordance with the cutoff criteria (|logFC| > 1, and p < 0.05), 27 DEGs were initially obtained from the GSE64913 dataset (epithelial cells from central airways and from peripheral airways), which included 42 normal samples and 28 asthma samples (Fig. 4A, Supplementary Table 3). Next, 18165 downstream genes of miR-182 were identi ed from the mirDIP database (score class: medium; http://ophid.utoronto.ca/mirDIP/), which were then analyzed with the human transcription factors in the Cistrome database by means of a Venn diagram. Subsequently, EGR2 was found to be a key downstream transcription factor for miR-182 (Fig. 4B, Supplementary Table 4). EGR2 has also been reported to exert an inhibitory role on Th17 cell differentiation [19]. Therefore, we evaluated the expression patterns of EGR2 in human PBMCs (Fig. 4C, D) and mouse spleen cells (Fig. 4E, F) using RT-qPCR and Western blot assays. The results demonstrated that EGR2 expression was down-regulated in asthma (p < 0.05). As a result, we speculated that miR-182 might promote Th17 cell differentiation via inhibition of EGR2.
Subsequently, we aimed to elucidate and verify the relationship between miR-182 and EGR2. The online website (http://starbase.sysu.edu.cn/) predicted the presence of binding sites between miR-182 and EGR2 3'UTR ( Fig. 4G). In addition, RT-qPCR analysis revealed that miR-182 expressions were increased in HEK293T cells co-transfected with miR-182 mimic and EGR2-wt or EGR2-mut (p < 0.05). Furthermore, dual luciferase reporter assay demonstrated that the luciferase activity of EGR2-wt was reduced (p < 0.05), while that of EGR2-mut showed no changes in HEK293T cells following miR-182 mimic transfection (p > 0.05; Fig. 4H). The results of RT-qPCR and Western blot analysis shown in Fig. 4I, J showed the diminished expressions of EGR2 in miR-182 mimic-treated cells and elevated EGR2 expressions in miR-182 inhibitor-treated cells (p < 0.05). These results indicated that miR-182 targeted EGR2 and inhibited its expression.
After that, we attempted to elaborate whether miR-182 induced Th17 cell differentiation via EGR2 inhibition. The results of RT-qPCR and Western blot analysis showed that EGR2 expression was increased upon EGR2 over-expression, while being decreased following sh-EGR2-1 and sh-EGR2-2 treatment (p < 0.05; Supplementary Fig. 3A, B). Due to the higher silencing e ciency of EGR2-1 compared to EGR2-2, sh-EGR2-1 was chosen for subsequent experimentation.
Furthermore, RT-qPCR and Western blot analysis ( Supplementary Fig. 3C, D) showed that cells treated with oe-EGR2 or both miR-182 mimic and oe-EGR2 presented with elevated expression of EGR2, while those treated with sh-EGR2 exhibited reduced EGR2 (p < 0.05). Moreover, cells with miR-182 mimic showed increased miR-182 expression and decreased EGR2 expression (p < 0.05). However, compared to cells treated with miR-182 mimic, miR-182 expressions were not notably different from the cells treated with both miR-182 mimic and oe-EGR2 (p > 0.05). In addition, cells with miR-182 inhibitor exhibited reduced miR-182 expressions and elevated EGR2 expressions (p < 0.05). However, when compared to the cells treated with miR-182 inhibitor, miR-182 expression was not notably different from the cells co-treated with miR-182 inhibitor and sh-EGR2 (p > 0.05), while EGR2 was decreased (p < 0.05).
In addition, ow cytometry was applied to examine the Th17 cell proportion in treated cells. As illustrated in Fig. 4K, EGR2 over-expression or miR-182 inhibitor inhibited the Th17 cell proportion, while EGR2 knockdown or miR-182 mimic promoted the Th17 cell proportion (p < 0.05). When compared with miR-182 mimic treatment alone, the combination treatment of miR-182 mimic and EGR2 over-expression resulted in decreased Th17 cell proportion (p < 0.05). Meanwhile, when compared with miR-182 inhibitor treatment alone, the combination treatment of miR-182 inhibitor and EGR2 silencing resulted in increased Th17 cell proportion (p < 0.05).
Finally, ELISA was performed to detect IL-17A level (p < 0.05; Fig. 4L) and RT-qPCR and Western blot were conducted to determine the RORγt expression patterns (p < 0.05; Fig. 4M, N). IL-17A levels and RORγt expressions were both decreased in cells with miR-182 down-regulation or EGR2 up-regulation, and elevated in cells with miR-182 up-regulation or EGR2 down-regulation (p < 0.05). When compared with miR-182 mimic treatment, the combination treatment of miR-182 mimic and EGR2 over-expression led to reduced IL-17A levels and RORγt expressions (p < 0.05). When compared with miR-182 inhibitor treatment, the combination treatment of miR-182 inhibitor and EGR2 silencing exhibited elevated IL-17A levels and RORγt expressions (p < 0.05).
Collectively, these ndings suggested that miR-182 accelerated the differentiation of CD4 + T cells into Th17 cells by inhibiting the expression of EGR2.

EGR2 inhibits the Th17 cell differentiation induced by YAP/HIF-1α/miR-182
The aforementioned ndings suggested that EGR2 over-expression could inhibit miR-182-induced Th17 cell differentiation. In this part of the experiments, we investigated whether EGR2 inhibited the Th17 cell differentiation evoked by the YAP/HIF-1α axis. RT-qPCR and Western blot analysis ( Supplementary Fig. 4A, B) displayed that the YAP expression was increased in CD4 + T cells after YAP over-expression alone or both over-expression of YAP and EGR2 (p < 0.05). The HIF-1α expression was also increased after HIF-1α overexpression alone or both over-expression of HIF-1α and EGR2 (p < 0.05). In addition, cells with both overexpression of YAP and EGR2 exhibited higher EGR2 expressions relative to those with YAP over-expression alone, and the cells with both over-expression of HIF-1α and EGR2 also exhibited higher EGR2 expressions compared to those with HIF-1α over-expression alone (p < 0.05). Flow cytometry results ( Fig. 5A) depicted that YAP or HIF-1α over-expression promoted Th17 cell proportion, which was inhibited by EGR2 over-expression (p < 0.05).
Finally, Fig. 5B illustrates the detection results of IL-17A levels by ELISA and Fig. 5C, D displays the detection results of RORγt expression patterns by RT-qPCR and Western blot analysis. IL-17A levels and RORγt expressions were both elevated in cells with YAP or HIF-1α over-expression, while EGR2 over-expression reversed these trends (p < 0.05).
These data demonstrated that the differentiation of CD4 + T cells into Th17 cells induced by the YAP/HIF-1α/miR-182 axis might be inhibited by over-expression of EGR2.
Over-expression of EGR2 alleviates asthma and lipid metabolism dysfunction by inhibiting the YAP/HIF-1α/miR-182 axis in vivo Next, we sought to verify the aforementioned results in vivo. RT-qPCR was performed to detect the miR-182 expression patterns, and RT-qPCR and Western blot analysis were used to determine the protein expression patterns of YAP, HIF-1α, and EGR2 in mice ( Supplementary Fig. 5A, B). Asthma mice were found to present with elevated YAP and HIF-1α expressions, but reduced EGR2 expressions (p < 0.05). Mice following YAP overexpression treatment or dual treatment with YAP over-expression and EGR2 over-expression exhibited increased expressions of YAP (p < 0.05). In addition, the treatments with YAP over-expression alone, HIF-1α over-expression alone, both over-expression of YAP and EGR2, as well as both over-expression of HIF-1α and EGR2 resulted in increased expressions of miR-182 and HIF-1α (p < 0.05). However, YAP over-expression, HIF-1α over-expression, or miR-182 over-expression resulted in decreased EGR2 expressions, which could be rescued by EGR2 over-expression (p < 0.05). Moreover, when compared with the mice treated with mimic-NC and oe-NC, the expression of miR-182 was still higher in the mice treated with both EGR2 over-expression and miR-182 mimic (p < 0.05).
After that, the Th17 cell proportion in mouse spleen was measured by means of ow cytometry. The results are displayed in Fig. 6A, which revealed an increased Th17 cell proportion in the asthma mice and the mice treated with YAP over-expression, HIF-1α over-expression, or miR-182 over-expression, while being inhibited by EGR2 over-expression (p < 0.05). In addition, ELISA was performed to detect the IL-17A levels (Fig. 6B) and RT-qPCR and Western blot were conducted to determine the RORγt expression patterns (Fig. 6C, D). IL-17A levels and RORγt expressions were elevated in the asthma mice and the mice with YAP over-expression, HIF-1α overexpression, or miR-182 over-expression, which was observed to be inhibited by EGR2 over-expression (p < 0.05).
The above data indicated that the over-expression of EGR2 alleviated the exacerbated asthma and lipid metabolism dysfunction evoked by YAP/HIF-1α/miR-182 signaling in vivo.

Discussion
Asthma is a common disease affecting millions of people worldwide characterized by upper airway in ammation and chronic nature [25]. Although asthma can affect people of all ages, a vast majority of asthma patients begin to experience the symptoms in childhood. Therefore, it would be wise to elucidate and control pediatric asthma to avoid any detrimental changes to the long-term quality of life. In the current study, we aimed to investigate the potential molecular mechanism in the development of asthma, and found that the YAP/HIF-1α factors could potentially augment Th17 cell differentiation, consequently exacerbating asthma and lipid metabolism dysfunction via miR-182-mediated EGR2 inhibition.
One of the important ndings of the current study is that the factors YAP1 and HIF-1α were both increased in pediatric asthma. A previous study also noted that YAP was up-regulated in the bronchial airway smooth muscles of chronic asthma mouse models [7]. Meanwhile, YAP can also bind to HIF-1α in the nucleus and sustain HIF-1α protein stability in conditions of hypoxic stress in hepatocellular carcinoma cells [10]. Meanwhile, silencing YAP has been documented to markedly down-regulate the protein expression of HIF-1α, while inhibition of the YAP/HIF-1α signaling aids in the prevention of angiogenesis of liver sinusoidal endothelial cells [26]. These previous ndings in conjunction with our results suggested a positive relationship between YAP and HIF-1α. Other studies have also illustrated that de ciency of HIF-1α can reduce eosinophil in ltration, goblet cell hyperplasia, and levels of cytokines IL-4, IL-5, and IL-13 in the lungs of OVAinduced asthma models [11], further highlighting the importance of the elevated levels of HIF-1α in asthma.
Moreover, HIF-1α has been reported to facilitate the differentiation of Th17 cells [27], and may serve as an important signaling molecule for the induction of asthma through Th17 cells [14,15]. HIF-1α can also cause asthma by means of airway smooth muscle remodeling [28,29], which is in line with our ndings. Whereas, YAP is known to function as an ampli er of the regulatory T cells Treg-reinforcing pathway, holding signi cant potential as an anticancer immunotherapeutic target [8]. In addition to that, loss of YAP in T cells is known to result in enhanced T-cell activation, differentiation, and function, which translates in vivo to an improved ability for T cells to in ltrate and repress the development of tumors [30]. The aforementioned ndings and results explained the promoting role of YAP/HIF-1α in enhancing differentiation of Th17 cells.
Lipid metabolism dysfunction is another potential issue faced by patients plagued by asthma. Accordingly, reductions in good cholesterol levels of HDL-C have been found in asthmatic children [20]. In addition to reduced HDL-C, abnormally high LDL-C and triglycerides are also a common occurrence in pediatric asthma patients [31]. More importantly, the reduction in HDL-C levels has been linked to an increased number of Th17 cells [19]. The signi cance of this is re ected by the fact that Th17 cells are associated with the production of IL-17, a highly pro-in ammatory cytokine [32]. Moreover, Th17 cells also produce other pro-in ammatory cytokines such as IL-6 and tumor necrosis factor-α, which play trivial role in the in ammatory cascade awakened in the state of asthma [33]. Thus, Th17 cells are rightfully believed to increased immune hyperresponsiveness in an enhanced in ammatory state during asthma [34]. Ni et al., have demonstrated that loss of YAP results in dysfunctional Treg cells failing to inhibit anti-tumor immunity or elicit tumor growth in mice [8]. Additionally, YAP de ciency in T cells enhances T-cell activation, differentiation, and function, as well as improving T-cell responses in cancer [30]. By contrast, the results obtained from the present study revealed that YAP could potentiate Th17 cell differentiation both in PBMCs and asthma mice. This discrepancy may be the difference of the laboratory environment, study subjects and the detection methods used. However, a previous study found signi cant overexpression and activation of YAP-1 in PBMCs collected from a total of 152 hepatocellular carcinoma cases, and that YAP-1 shares a positive correlation to the percentage of Treg cells; speci cally, YAP-1 overexpression in hepatocellular carcinoma T cells induces immunosuppression by promoting Treg cell differentiation [35]. This nding is consistent with the results in this work. It indicates that the role of YAP in Th17 generation could be bilateral, no matter of overexpression or deletion.
Another focus of the current study was microRNAs (miRNAs), which are small non-coding RNA molecules that can modulate gene expression posttranscriptionally by interacting with the 3'-UTR of speci c target mRNAs [36]. Herein, we identi ed that miR-182 could bind to the 3'-UTR of EGR2 and inhibit its expression.
Accumulating evidence has shown that EGR2 exerts an inhibitory role on Th17 cell differentiation by negatively regulating Batf [18]. In addition, a previous study found up-regulated expressions of in ammatory transcription factors, such as RORγt and Bhlhe40, in EGR2/3 de cient T cells under tolerogenic conditions [37]. Furthermore, EGR2 has the potential to retard the development of chronic rhinosinusitis induced by miR-150-5p in dendritic cells [38]. On the other hand, previous studies have highlighted that miR-182 may promote asthma by stimulating in ammation [39]. Our results also agree with the idea that miRs, like miR-182, miR-30 [40] and miR-221 [41], serve as potential candidates for the treatment of asthma [42]. In line with our ndings, miR-182 is also known to be signi cantly up-regulated upon Th17 differentiation [13]. Therefore, we reasoned that miR-182 allowed Th17 cell differentiation in pediatric asthma by targeting EGR2. Moreover, miR-182 is capable of elevating the HIF-1α expression. and subsequently promoting breast cancer cell proliferation and invasion [43], indicating a positive correlation between miR-182 and HIF-1α. Whereas, HIF-1α-de cient mice are known to exhibit elevated metabolic rate, hyperventilation, and improved glucose and lipid homeostasis [44]. Hence, based on the aforementioned information, we asserted the hypothesis that ampli ed EGR2 eliminated Th17 cell differentiation, asthma and lipid metabolism dysfunction driven by YAP/HIF-1α/miR-182 signaling.

Conclusions
In conclusion, ndings in the current study demonstrated that YAP/HIF-1α enhanced Th17 cell differentiation, consequently exacerbating asthma and lipid metabolism dysfunction via miR-182-mediated EGR2 inhibition (Fig. 7). Thus, the YAP/HIF-1α/miR-182/EGR2 signaling may serve as a novel biomarker for asthma diagnosis and prognosis. However, there are a few notable limitations to our study. First, only OVA was used to stimulate asthma in mice in this study. While human asthma can stem from different causes and exhibit variable forms and severity, animal models can be used to mimic one of more features of the human variation of the disease [45,46]. Therefore, different animal models should be used to con rm the results uncovered in our study in the future to prevent over-generalization to human situations. Moreover, further studies with different cell lines, different disease models and larger cohorts are essential to validate these ndings and expand the translational potential of this direction to realize the full potential of the YAP/HIF-1α/miR-182/EGR2 signaling axis.

Ethics Statement
The current study was approved by the Ethics Committee of The First A liated Hospital of Nanchang University (approval number: 201908029) and performed in strict accordance with the Declaration of Helsinki. Signed consents were obtained from all participants prior to the study. Animal experiments were strictly designed and performed according to the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health, and extensive efforts were made to minimize the suffering of the animals used in the study.

Study Subjects
A total of 29 pediatric patients with asthma (aged < 18 years old; 14 males & 15 females) and 22 healthy volunteers from the First A liated Hospital of Nanchang University from June 2018 to December 2018 were enrolled in the current study, and peripheral blood mononuclear cells (PBMCs) were collected in order to extract RNA and protein content.

Ovalbumin (OVA)-Induced Murine Asthma Model
A total of 120 BALB/C mice (aged: 6-8 weeks; weighing 16-20 g) were purchased from the Experimental Animal Center of Nanchang University, amongst which 12 mice where used as normal control, 96 mice were subjected to adenovirus infection, while the remaining 12 mice were used for asthma model establishment. The asthma models were constructed according to a previously reported method [47], with some additional adjustments. Firstly, the mice were subjected to intraperitoneal injections with 0.2 mL of OVA/aluminum hydroxide on days 0, 7, and 14. After that, starting from day 21, the mice were exposed to 1% OVA inhalation (30 mL) for 30 min once per day, for a total of 7 days. Every time prior to OVA inhalation, each mouse was intraperitoneally injected with 0.2 mL of normal saline. Normal control mice were subjected to matching procedures, with the exception of 30 min of OVA inhalation treatment. Meanwhile, for the mice subjected to adenovirus infection, before administration of OVA and normal saline, the mice were intratracheally injected with phosphate buffer saline (PBS) or adenovirus harboring over-expression plasmids for YAP, EGR2, and HIF- Spleen specimens from normal mice were collected and digested using Collagenase D for 30 min at room temperature, followed with treatment using 5 mM of ethylene diamine tetra-acetic acid (EDTA) for 5 min to harvest spleen cells. Next, naive CD4 + T cells were isolated from the harvested spleen cells with the help of CD4 + CD62L + T cell isolation kits (130-106-643, Miltenyi Biotech, Germany), with a purity of 95%.

Hematoxylin-eosin Staining
Lung tissue sections were xed with 4% paraformaldehyde at room temperature, and then subjected to hematoxylin-eosin staining (hematoxylin for 60 s and eosin for 3 min) for airway lesion observation. Each section was observed under an optical microscope (BX63, Tokyo, Japan) in a double-blinded manner, followed by measuring the thickness of the airway smooth muscle (µm), airway wall (µm), and airway epithelium mucosa (µm). The experiment was repeated 3 times.

Detection of Th17 Cell Proportion
After 24 h of infection, naive CD4 + T cells were differentiated by the addition of Th17 cell differentiation medium (500 ng/mL phorbol 12-myristate 13-acetate and ionomycin in complete medium) for 72 h. Next, the cells were collected and rinsed once with PBS. Cell suspension of human PBMCs, mouse lung and spleen was then obtained. Incubation was subsequently conducted with uorescein isothiocyanate (FITC)conjugated anti-human CD4 antibody at 4℃ for 30 min. Thereafter, the cells were further incubated with mouse IL-17/IL-17A PE-conjugated antibody (IC7211P, R&D Systems, USA) at room temperature under dark conditions for 30 min. Finally, the proportion of Th17 cells was detected using a CytoFLEX ow cytometer (Beckman, USA). The experiment was repeated 3 times [49].

RNA Isolation and Analysis
For detection of miR and mRNA expressions, total RNA content was extracted from tissues and cells using the TRIzol reagent (6096020, Thermo Fisher Scienti c, USA). After that, the obtained RNA was reverse transcribed into complementary DNA (cDNA) with the use of the TaqMan™ MicroRNA Reverse Transcription Kit (4366596, Thermo Fisher Scienti c) for miR-182, and High-Capacity cDNA Reverse Transcription Kit (4368813, Thermo Fisher Scienti c) for mRNAs. Then, RT-qPCR was performed with a RT-qPCR kit (11732020, Thermo Fisher Scienti c) on a Real-Time PCR system (CFX96, Bio-rad). Finally, the expressions of miR and mRNA were calculated according to the 2 −ΔΔCt method, with U6 as the internal reference for miR-182 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for YAP, HIF-1α, and EGR2. The primer sequences designed by Shanghai Sangon Biotech (Shanghai, China) are presented in Table 2. The experiment was repeated 3 times. Table 2 Primer sequences for reverse transcription quantitative polymerase chain reaction

Dual Luciferase Reporter Assay
The

HDL-C Determination
The HDL-C content was determined according to the manual of the HDL-C detection kit (ab65390, Abcam). The experiment was repeated 3 times.

Enzyme-linked Immunosorbent Assay (ELISA)
The levels of IL-17A in Th17 cells of mice were measured using an IL-17A ELISA kit (M17AF0, R&D Systems) according to the manufacturer's instructions. The serum levels of IL-17A in human were measured according to the instructions provided by the ELISA kit (D1700, R&D Systems).

Chromatin Immunoprecipitation (ChIP) Assay
The ChIP assay was conducted according to the instructions of the EZ-Magna ChIP kit (EMD Millipore, USA). In brief, the Th17 cells were xed with 1% paraformaldehyde and cross-linked with glycine for 10 min to produce DNA-protein cross-linking. The cells were then subjected to sonication to shear the DNA into 200-300 bp fragments. The supernatant was then collected and incubated with protein-A coated magnetic beads, followed by the addition of IgG (ab172730, dilution ratio of 1: 100, Abcam) or antibody against HIF-1α (ab2185, dilution ratio of 1: 20, Abcam). The protein-DNA complexes immobilized by magnetic beads were washed and de-crosslinked. Finally, the miR-182 promoter region in the complexes was determined by RT-qPCR (miR-182 ChIP primer: F: 5'-GAGTGTCCAGGGTTCGTCTG-3', R: 5'-GGTACACTTCTTTGCCCCCA-3').  Figure 1 Increased YAP, HIF-1α and miR-182 and decreased HDL-C level in asthma. A, the volcano plot of DEGs related to asthma in peripheral blood cells of asthma patients obtained from the GSE97049 dataset. The red points indicate the signi cantly upregulated genes, and the green points indicate the signi cantly downregulated genes; B, the Venn diagram of the DEGs in peripheral blood cells of asthma patients from GSE97049 dataset and the human transcription factors obtained from the Cistrome database; C, the PPI network of the 5 intersected transcription factors in panel B and the related genes; the larger circle at which genes are located re ects higher core degree of the gene and the smaller circle re ects lower core degree. D, the co-expression of YAP1 and HIF1A predicted by the MEM website (p = 3.76e-18); E, the target relationship between YAP1 and  miR-182 inhibited EGR2 expression and then promoted Th17 cell differentiation. A, the heatmap of DEGs related to asthma in respiratory epithelial cells obtained from the GSE64913 dataset; the right upper histogram represents color gradation; B, the Venn diagram of the DEGs in respiratory epithelial cells from the GSE64913 dataset, downstream genes of miR-182 from the miRDIP database and human transcription factors from the Cistrome database; C, EGR2 mRNA expression in human PBMCs detected by RT-qPCR; D, Western blot analysis of EGR protein in human PBMCs; E, EGR2 mRNA expression in mouse spleen cells detected by RT-qPCR; F, Western blot analysis of EGR2 protein in mouse spleen cells; G, the binding site between miR-182 and EGR2 3'UTR in mice predicted by the starbase website; H, miR-182 expression detected by RT-qPCR and the binding of miR-182 to EGR2 con rmed by dual luciferase reporter assay; I, miR-182 expression and EGR2 mRNA expression detected by RT-qPCR; J, Western blot analysis of the EGR2 protein in cells after different treatments; K, the Th17 cell proportion after different treatments determined by ow cytometry; L, the level of IL-17A after different treatments by ELISA; M, RORγt mRNA expression in cells after different treatments detected by RT-qPCR; N, Western blot analysis of the RORγt protein in cells after different treatments. Comparisons between two groups were conducted by unpaired t test, and those among multiple groups were conducted by one-way ANOVA with Tukey's post hoc test; the experiments were repeated 3 times; * p < 0.05, compared with the normal individuals (normal), normal mice (normal-M), cells treated with mimic-NC, inhibitor-NC, oe-NC + mimic-NC, or sh-NC + inhibitor-NC; # p < 0.05, compared with the cells treated with miR-182 mimic + oe-NC or miR-182 inhibitor + sh-NC; n = 22 in normal individuals; n = 29 in asthma patients; n = 12 in normal mice; n = 12 in asthma mice.  EGR2 overexpression alleviated the asthma and lipid metabolism dysfunction induced by the YAP/HIF-1α/miR-182 axis in vivo. A, the Th17 cell proportion in mouse spleen cells determined by ow cytometry; B, the level of IL-17A in mouse serum measured by ELISA; C, RORγt mRNA expression in mouse spleen cells detected by RT-qPCR; D, Western blot analysis of RORγt protein in mouse spleen cells; E, Th17 cell proportion in mouse lung tissues detected by ow cytometry. F, hematoxylin-eosin staining of mouse lung tissues (400 ×); G, the thickness of airway smooth muscle, airway wall, and airway epithelium mucosa of mice; H, the level of HDL-C in mouse serum. Comparisons among multiple groups were conducted by one-way ANOVA with Tukey's post hoc test, n = 12 for mice following each treatment; * p < 0.05, compared with the mice treated with sh-NC, oe-NC, or mimic-NC + oe-NC; # p < 0.05, compared with the mice with oe-YAP, oe-HIF-1α, or miR-182 mimic + oe-NC.

Figure 7
The mechanistic diagram illustrating the role of the YAP/HIF-1α/miR-182/EGR2 axis in asthma. The overexpression of YAP and HIF-1α promoted the expression of miR-182, thereby inhibiting EGR2 expression, increasing the expression of RORγt and IL-17A, and prompting the differentiation of CD4+T cells into Th17 cells, which ultimately aggravated asthma and lipid metabolism dysfunction.

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