miRNA‐34b/c regulates mucus secretion in RSV‐infected airway epithelial cells by targeting FGFR1

Abstract Respiratory syncytial virus (RSV) infection in airway epithelial cells is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV‐related LRTI). However, the pathological processes of mucus hypersecretion in RSV‐infected airway epithelial cells remains unclear. The current study explores the involvement of miR‐34b/miR‐34c in mucus hypersecretion in RSV‐infected airway epithelial cells by targeting FGFR1. First, miR‐34b/miR‐34c and FGFR1 mRNA were quantified by qPCR in throat swab samples and cell lines, respectively. Then, the luciferase reporters’ assay was designed to verify the direct binding between FGFR1 and miR‐34b/miR‐34c. Finally, the involvement of AP‐1 signalling was assessed by western blot. This study identified that miR‐34b/miR‐34c was involved in c‐Jun‐regulated MUC5AC production by targeting FGFR1 in RSV‐infected airway epithelial cells. These results provide some useful insights into the molecular mechanisms of mucus hypersecretion which may also bring new potential strategies to improve mucus hypersecretion in RSV disease.


| INTRODUC TI ON
Severe bronchiolitis is always caused by respiratory viruses during infancy, and respiratory syncytial virus (RSV) infection is the predominant reason. In hospitalized cases of infant bronchiolitis, the RSV infection rate is up to 50%-80%. 1,2 Compared with bronchiolitis induced by other common pathogens, such as rhinovirus (RV) and metapneumovirus, bronchiolitis caused by RSV is usually associated with prolonged hospital stay and intensive care, 3 which is also characterized by young age and severe airway obstruction. Excessive mucus secretion is the critical reason for airway obstruction and aggravation of RSV infection. 4 Moreover, severe cases of bronchiolitis in infancy are associated with a high risk of subsequent childhood asthma. 5 Thus, infant bronchiolitis is assumed to be a key event in the secondary prevention strategy of asthma, as intervention subjects are infants and young children who have not developed asthma but have high-risk signs of asthma (such as RSV infection). Add all this together, exploring the mechanism of abnormal mucus secretion after RSV infection has important implications for preventing the occurrence and development of severe bronchitis. Although the existing treatment is unable to satisfy the requirements of the secondary strategy, accumulating researches have verified that reducing the medical burden of bronchiolitis by cause-based diagnosis and treatment has a deep and profound significance. 6,7 Therefore, deeper studies are needed to understand the specific pathological process of mucus secretion in early RSV infection. miRNAs participate in gene expression which is involved in many biological processes, including cellular metabolism and immune responses. 8 Of note, miRNAs in lungs have been verified to be an important regulatory factor of mucus secretion in airway epithelial cells. [9][10][11] Our previous study has found that downregulated miRNA-34b/c induces MUC5AC overexpression in severe RSV-infected airway epithelia. 12 However, in the process of mucus hypersecretion, the specific target and signalling pathway regulated by miR-34b/ miR-34c is still obscure.
Bioinformatics analysis and miRNA binding site prediction revealed that Fibroblast growth factor receptor 1 (FGFR1) is the potential direct target in the process of mucin production regulated by miR-34b/miR-34c. 12 FGFR1 belongs to the FGFR family, acts as a tyrosine kinase receptor to activate intracellular signalling pathways, including P13K, AKT, MAPK, etc. 13 In airway epithelia, FGFR1 has been studied extensively which is involved in cell proliferation, stress response and epithelialmesenchymal transition (EMT), etc. 14,15 However, little is known about the influence of FGFR in epithelial mucin production. In this study, we first tested the expression of FGFR1 in airway epithelia after RSV infection in vitro and in vivo, respectively. Then, dual-luciferase was used to confirm the direct binding between FGFR1and miR-34b/miR-34c.
Finally, the FGFR1/AP-1/MUC5AC pathway was detected to verify the involvement of FGFR1 in epithelial mucin production.

| Cell culture, treatment and RSV infection
Normal primary human bronchial epithelial cells (HBECs) and A549 cells were purchased and cultured following the previous literature. 12,17 Purified RSV-A2 (MOI = 3) was incubated with HBECs or A549 cells for 24 h.

| Screening and analysis of public RNA profiles from RSV infected patients
Transcriptional profiles of RSV-infected patients and controls were screened and selected from the GEO database.
The screened target datasets were included according to the following criteria: (1) whole blood sample, nasal/throat swab sample, bronchial lavage fluid or induced sputum from human; (2) RNA-seq; (3) the presence of RSV was confirmed by quantitative real-time PCR. Profile graph function from the GEO2R program was used to obtain FGFR1 expression value and expression profile graph in different groups. The information of these datasets is described in Table 1.

| RNA extraction, qRT-PCR, miRNA qRT-PCR
Total RNA was extracted from HBECs with RNAiso Plus (Takara). The primers are listed in Table S1. β-actin and U6 serve as internal controls, respectively.

| Western blot
The steps of western blot were described in previous publications. 20 Briefly, cells are lysed by RIPA lysis buffer and protein were collected.
Total protein was separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. Then, 5% bovine serum albumin (BSA) was used to block non-specific sites. After that, membranes were incubated with primary antibodies overnight at 4℃ and incubated with secondary antibody the next day. The primary antibodies are listed as follows: c-Jun (Santa Cruz, sc-74543), FGFR1 (Abcam, ab824), β-actin(Santa Cruz, sc-84322) and phosphorylated c-Jun (Santa Cruz, sc-822).

| Cell Counting Kit-8 assays
To accessed cell viability, HBECs were cultured and grown to a density of 70% in a 96-wells plate. After being treated by miR mimic or miR NC, cell viability assays were operated following the CCK-8 kit protocol (Dojindo). The cell density at 450 nm was determined using a microplate reader (Bio-rad).

| Statistical analysis
The differential expression between the RSV infection group and the control group in each database was presented as mean ± SD, pvalue < 0.05, analysed by unpaired t-test. Other data were analysed with GraphPad Version 7 from minimum of three independent experiments, presented as mean ± SEM, analysed by one-way analysis of variance (ANOVA) or unpaired t-test. p-value < 0.05 were considered differences significant.

| The protein level of FGFR1 was upregulated in RSV-infected airway epithelial cells
To verify the involvement of FGFR1 in airway epithelia after RSV infection, the mRNA and protein level of FGFR1 were analysed in RSV-infected HBECs. As shown in Figure 1A, there is no significant change of FGFR1 mRNA expression between RSV-infected HBECs and controls. However, FGFR1 protein expression was significantly higher in RSV-infected HBECs than controls ( Figure 1B). Moreover, the mRNA level of FGFR1 was also analysed in throat swab samples from 11 healthy controls and 11 RSV patients. Consistent with the in vitro results, no significant difference has been found between the two enrolled clusters ( Figure 1C). These results demonstrated that the protein level of FGFR1 in airway epithelia was upregulated after RSV infection, but no significant difference was detected in the mRNA level of FGFR1.

| FGFR1 expression was analysed by RNA profiling in RSV-infected patient datasets
To further confirm the expression mode of FGFR1 after RSV infection, we screened RNA profiling subjecting of RSV-infected patients and healthy controls from public databases. Three datasets (GSE10 5450, GSE97742, GSE11 7827) were finally included in this study according to previous criteria. Characteristics of these three datasets was shown in Table 1. We analysed the differential expressed genes and the RNA-seq value of FGFR1 in each dataset which are shown in Figure 2. Consistent with expectations, there is no significant difference in FGFR1 mRNA expression between the RSV infection group and the control group.
Thus, RSV infection does not influence the mRNA expression of FGFR1 in vivo.

| miR-34b/miR-34c inhibited MUC5AC overexpression in RSV-infected airway epithelial cells
Next, we investigated the expression changes and the influence of miR-34b/miR-34c in airway epithelia during RSV infection. Our previous research found the RSV infection decreased the expression of miR-34b/miR-34c in airway epithelia which could further induce MUC5AC overexpression. 12 Here, the level of miR-34b/ miR-34c after RSV infection was further analysed in HBECs and throat swab samples, respectively. As is shown in Figure 3A,B, not only in HBECs but also in throat samples, the expression of miR-34b/miR-34c from RSV-infected HBECs were at a significantly lower level than the corresponding control HBECs. The transient transfection of miR-34b/34c mimics obviously elevated the level of miR-34b/miR-34c in HBECs ( Figure 3C). In addition, cell viability assay showed that miR-34b/ miR-34c mimics have no effect on cell viability ( Figure 3D). Moreover, miR-34b/miR-34c overexpression blocked MUC5AC expression significantly in HBECs after RSV infection ( Figure 3E).
Then, HBECs and A549, which represented the major target cells of RSV infection, were used as in vitro models. 22,23 The interaction of this FGFR1 with miR-34b/miR-34c was examined by the luciferase reporter gene containing the binding site of WT or MUT FGFR1. Compared with FGFR1 MUT, the luciferase density driven by FGFR1 WT and miR-34b/miR-34c mimics was decreased conspicuously both in A549 cells and HBECs ( Figure 4B-E). Then, the effects of miR-34b/miR-34c on the expression of FGFR1 were further verified, which revealed that miR-34b/miR-34c mimics induced the suppression of FGFR1 ( Figure 4G). These results suggest that the binding of FGFR1 3′-UTR -miR-34b/c inhibited the expression of FGFR1. In addition, the overexpression of miR-34b/miR-34c significantly reduced the protein expression of FGFR1, but not the mRNA expression ( Figure 4F). Thus, RSV infection in airway epithelia only upregulated the protein expression of FGFR1. The above results suggested that the regulation of FGFR1 by miR-34b/miR-34c may inhibit its post-transcriptional translation by loosely binding to its 3'UTR region.

| miR-34b/ miR-34c induced MUC5AC overexpression through AP-1 signalling in RSVinfected HBECs by targeting FGFR1
It had been proved that through activating c-Jun, a part of transcription factor AP-1, the decreased miR-34b/miR-34c contribute to RSV-induced abnormal secretion of mucin. 12 In this study, we further explored whether miR-34b/miR-34c could exert its effects on In recent years, the regulation of respiratory virus infections by miRNAs has been extensively studied. Our previous study also found that miRNA-34b/c contributed to the RSV-induced mucin production in airway epithelial cells through the functional modification F I G U R E 2 Fibroblast growth factor receptor 1 expression in RSV-infected patients and healthy controls in public RNA profiling datasets.
(A) FGFR1 expression profile graph in GSE10 5450 and differential expression analysis between RSV patients and healthy controls. (B) FGFR1 expression profile graph in GSE97742 and differential expression analysis between acute RSV patients and discharge RSV patients.
(C) FGFR1 expression profile graph in GSE11 7827 and differential expression analysis between RSV infection patients and asymptomatic controls of c-Jun, the AP-1 subunit. 12 However, c-Jun was not the directtargeted gene of miRNA-34b/c. In this study, we confirmed the decreased expression of miR-34b/miR-34c after RSV infection from throat swab samples and airway epithelial cells, respectively. Then, the direct binding between FGFR1 and miR-34b/miR-34c was verified through dual-luciferase experiment verification in this study.
Also, the involvement of further influences the signal mechanism of AP-1 activation. 36,37 Consistent with the previous findings, our results further verified the regulation of FGFR1 on AP-1 signalling in airway epithelial cells following RSV infection.
It is known that the downregulation of miR-34b/miR-34c was engaged in MUC5AC overexpression in RSV-infected airway epithelial cells. 12,38 Here, we further verified that miR-34b/miR-34c inhibited These results suggested that the post-transcriptional regulation of FGFR1 by miR-34b/miR-34c is achieved by regulating the process of protein translation. This is partly due to the incomplete complementarity and loose combination of FGFR1 3'UTR with miR-34b/miR-34c.
Although our research confirmed the key role of FGFR1 in epithelial mucus hypersecretion after RSV infection which is modulated by miR-34b/miR-34c, there are still some limitations. The first one is that the inhibitory level of miR-34b/miR-34c on c-Jun/MUC5AC activation should be detected through the FGFR1 overexpression plasmid. Besides, xenograft mouse models or organoids should be applied to study the function of FGFR1 and miRNA-34b/c after RSV infection.
In conclusion, our study validated that miR-34b and miR-34c regulate the overexpression of MUC5AC in RSV-infected airway epithelial cells by targeting FGFR1, which further induce mucus hypersecretion. This study offers some novel perceptions of the mechanisms of RSV-induced mucus secretion which may also bring novel strategies to treat mucus hypersecretion and RSV infection effectively.

ACK N OWLED G EM ENTS
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Funding acquisition (equal); Writing-original draft (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the National Center for Biotechnology Information (NCBI) Gene