Role of miR‐223‐3p in pulmonary arterial hypertension via targeting ITGB3 in the ECM pathway

Objectives To investigate the functions of miR‐223‐3p and ITGB3 in pulmonary arterial hypertension (PAH). Materials and Methods Microarray analysis was used to detect differentially expressed genes and microRNAs. In in vitro models, the expressions of miR‐223‐3p and ITGB3 were detected by qRT‐PCR and Western blot. α‐SMA expression and cell proliferation were analysed by immunofluorescence and MTT assay, respectively. In in vivo models, PAH progressions were determined by measuring the levels of mPAP and RVSP. Lung and myocardial tissues were subjected to HE staining and Masson and Sirius red‐saturated carbazotic acid staining to investigate the pathological features. Results The microarray analysis revealed that ITGB3 was upregulated, while hsa‐miR‐223‐3p was downregulated in PAH. After the induction of hypoxia, miR‐223‐3p was downregulated and ITGB3 was upregulated in PASMCs. Hypoxia induction promoted cell proliferation and inhibited α‐SMA expression in PASMCs. Both the upregulation of miR‐223‐3p and the downregulation of ITGB3 attenuated the aberrant proliferation induced by hypoxia conditions. After approximately 4 weeks, the mPAP and RVSP levels of rats injected with MCT were decreased by the overexpression of miR‐223‐3p or the silencing of ITGB3. The staining results revealed that both miR‐223‐3p overexpression and ITGB3 knockdown alleviated the pulmonary vascular remodelling and improved the PAH pathological features of rats. Conclusions MiR‐223‐3p alleviated the progression of PAH by suppressing the expression of ITGB3, a finding which provides novel targets for clinical treatment.


| INTRODUC TI ON
Pulmonary arterial hypertension (PAH) refers to a high resting mean pulmonary artery pressure (≥25 mm Hg). 1 PAH is a progressive pulmonary vascular disease related to the dysfunction of pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs). 2 PAH often leads to the remodelling and dysfunction of the pulmonary vasculature, right ventricular hypertrophy and even death. 3 Optimal combination therapies are required to improve the quality of life of patients. 4 Thus, developing novel therapeutic strategies for treating PAH is essential and urgent.
MicroRNAs (miRNAs), which are 20-22 nucleotides in length, are endogenously expressed non-coding RNAs that inhibit or degrade their target RNAs. MiRNAs play important roles in cell differentiation, proliferation, migration, apoptosis and stress responses. 5 Recent studies have indicated that miRNAs can serve as biomarkers for different vascular pathologies, including early myocardial infarction and heart failure in humans. 6 Several miRNAs have been implicated in the pathophysiological mechanisms of PAH. Sarrion et al 7 reported that miR-23a is correlated with pulmonary function parameters. Courboulin et al 8 indicated that the reduction in miR-204 levels promotes PASMC proliferation and induces resistance to apoptosis. Meloche et al 9 reported that the downregulation of miR-223 plays an important role in PAH, and restoring the expression of this miRNA can reverse PAH.
Integrin-β 3 subunit gene (ITGB3) is known as platelet glycoprotein IIIa and antigen integrin β3 (CD61). ITGB3 has been demonstrated to be modulated by miR-95 to regulate the proliferation, migration and invasion of non-small-cell lung cancer. 10 In addition, abnormal PASMC proliferation is a pathological feature of PAH, 11 although there has been no specific research investigating the role of ITGB3 in PAH. Moreover, the functions of miR-223-3p and ITGB3 in PAH are still not fully understood.
In this study, we investigated the expression of miR-223-3p and ITGB3 in PASMCs subjected to hypoxia in vitro and in the pulmonary artery tissues of a PAH rat model. miR-223-3p was found to hinder the deterioration caused by PAH, whereas ITGB3 contributed to the progression of PAH. These discoveries suggest the great potential of miR-223-3p and ITGB3 as novel PAH therapeutic targets and biomarkers for PAH.

| Bioinformatics analysis
The microarrays GSE33463 and GSE67597 were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm. nih.gov/geo/). Differentially expressed genes and dysregulated pathways were uncovered through the R language and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. The Linear Models for Microarray and RNA-seq Data (Limma) package was utilized to investigate gene expression between the IPAH group and healthy controls. The threshold used to screen for upregulated and downregulated mRNA was P < 0.05 and |log (FC)| > 1. The differentially expressed genes were clustered using the heatmap package.

| Cell isolation and cultivation
Healthy male Sprague Dawley (SD) rats weighing 200 g were anesthetized with 200 g/L urethane at a dose of 5 mL/kg. Pulmonary arteries were isolated in aseptic operations, and PASMCs were isolated from sections of pulmonary arteries as previously described. 12 In the subsequent experiments, the cells were cultured at 37°C in an atmosphere with 5% CO 2 .

| Hypoxia induction
When the cells were reached 70% confluence, a serum-free medium was utilized to replace the initial medium that contained 10% FBS.
Positive control PASMC lines were purchased from Cell Applications (San Diego, CA, USA). After being cultured for 12 hour, the cells were divided into two groups, namely the control group and the hypoxia induction group. Cells in the control group were incubated under 21% O 2 , 5% CO 2 and balanced N 2 ; cells in the hypoxia induction group were incubated under 2% O 2 , 5% CO 2 and balanced N 2, as described previously. 13

| Western blot
Approximately 1 × 10 7 cells were solubilized in lysis buffer purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to separate the proteins. Afterwards, approximately 60 µg of the purified proteins was transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membrane with the adsorbed proteins was incubated with Tris-buffered saline with Tween 20 (TBST) buffer obtained from Fanke Biotech Co., Ltd. (Shanghai, China) at room temperature, supplemented with 5% non-fat milk. After 1 hour, the membrane was incubated with the TA B L E 1 Sequences of primers utilized in qRT-PCR

TA B L E 2 Sequences of oligonucleotides utilized for transfection
Oligonucleotides Sequence Control inhibitor 5′-CAGUAGCATTGAGTUGCCAGAGCG-3′ primary antibodies overnight at room temperature, followed by incubation with the corresponding secondary antibody for 4 hour. In the present study, the primary antibodies used were as follows: rabbit anti-α-SMA (ab124964, 1:10 000 dilution; Abcam), rabbit anti-

| Haemodynamics
The pulmonary pressure changes were measured. In brief, a 13cm-long, heparin-priming polyethylene catheter (outer diameter, 0.9 mm) connected to PowerLab 16/30 (ADInstruments, Dunedin, New Zealand) through a pressure transducer was introduced into the right external jugular vein and advanced into the right ventricle and the main pulmonary artery. The right ventricular systolic pressure (RVSP), pulmonary arterial systolic pressure (PASP), total pulmonary resistance (TPR) and mean pulmonary arterial pressure (mPAP) were recorded. Meanwhile, the cardiac output (CO) and enddiastolic pressure were assessed, as previously described. 19-22

| Haematoxylin-eosin (HE) staining
Paraffin-embedded tissue sections were roasted and dried for 20 minute in the oven at 75°C, followed by immersion in xylene for 15 minute, anhydrous ethanol for 5 minute, 95% ethanol for 5 minute, 80% ethanol for 5 minute and 70% ethanol for 5 minute. All operations were conducted twice. Briefly, after the sections were dried, haematoxylin was used to stain the nuclei, and eosin was used to stain the cytoplasm after dewaxing and hydration, as previously described. 23,24 To examine the degree of fibrosis, microscopic images of the HEstained tissues were graded according to the Ashcroft method. 25

| Masson staining
For Masson staining, paraffin-embedded tissue sections were stained with haematoxylin for 1 minute and then Masson Li Chunhong acid fuchsine solution for 5 minute. The detailed histological protocols were previously described. 26 F I G U R E 2 Gene oncology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of differentially expressed genes in IPAH. A, The seven most distinctively activated GO pathways between healthy and IPAH tissues. B, The seven most distinctively activated KEGG pathways in healthy and IPAH tissues

| Sirius red-saturated carbazotic acid staining
Paraffin-embedded tissue sections were washed with distilled water and then stained with celestine blue for 10 minute followed by three washes with distilled water. Then, Sirius red-saturated carbazotic acid was applied for 20 minute. Afterwards, the sections were treated with 0.2% ethylic acid for 1 minute, twice. After dehydration, transparency and sealing, the tissues were observed using ordinary optical or polarized light microscopy. 27,28

| Immunohistochemistry
Immunohistochemistry (IHC) analysis was performed on 5-μm paraffin-embedded tissue sections that were rehydrated in a graded series of ethanol (99%-70%) and finally in distilled water.

| Data analysis
Data were presented as the mean ± standard deviation (SD) of at least three independent replicates. Data were analysed with F I G U R E 3 Results of gene oncology (GO) analysis. A and B, The GO analysis revealed differentially modulated pathways in the IPAH group compared to the normal group. In the dotplot, the graph size represents the number of genes. The colour represents the P value.
In the joyplot, the enrichment significance (the adjusted P value) is reflected by the colour intensity of the peaks. Pathways with ridges on the left side of 0 were downregulated, while those with ridges on the right side of 9 were upregulated. The gene ratio in the horizontal axis represents the proportion of differential genes in the gene set GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA), and comparisons between two groups were analysed by the Student's t test. Multiple groups were compared with one-way ANOVA.
Differences were considered significant at values of P < 0.05.

| Downregulation of miR-223-3p in IPAH and related dysregulated pathways
Differentially expressed genes and dysregulated pathways were determined using the R language and KEGG database, respectively. Figure 1A shows the top 10 differentially expressed genes with aberrantly high and low expression. ITGB3 was shown to be upregulated in IPAH tissues. As shown in Figure 1B, hsa-miR-223-3p was downregulated in patients with IPAH. The top 14 significantly activated and inactivated pathways in IPAH are shown in Figure 2A.
The Gene Oncology (GO) analysis revealed that these co-expressed genes in IPAH were associated with several biological processes, including autophagy and phagocytic vesicle membrane (Figure 2A).
The KEGG analysis results revealed upregulated pathways in IPAH, such as olfactory transduction, pantothenate and CoA biosynthesis, regulation of actin cytoskeleton and ubiquitin-like protein ligase binding. Compared to the normal group, the extracellular matrix (ECM) receptor interaction pathway was significantly upregulated in the IPAH group ( Figure 2B). The joyplot ( Figure 3A) and the dotplot F I G U R E 4 Results of Kyoto encyclopedia of genes and genomes (KEGG) analysis. A and B, The KEGG analysis revealed differentially modulated pathways in the IPAH group compared to the normal group, including the extracellular matrix (ECM) receptor interaction pathway. In the dotplot, the graph size represents the number of genes. The colour represents the P value.
In the joyplot, the enrichment significance (the adjusted P value) is reflected by the colour intensity of the peaks. Pathways with ridges on the left side of 0 were downregulated, while those with ridges on the right side of 9 were upregulated. The gene ratio in the horizontal axis represents the proportion of differential genes in the gene set ( Figure 3B) illustrate the activated and suppressed GO pathways in IPAH tissues. Figure 4A,B illustrates the activated and suppressed KEGG pathways in IPAH tissues, including the ECM receptor interaction pathway.

| Visual characterization of PASMC and analysis of the expressions of miR-223-3p and ITGB3 after hypoxia induction
PASMCs were observed under the microscope and "peak and valley" characteristics were exhibited ( Figure 5A). α-SMA was strongly expressed in PASMCs ( Figure 5B). After the induction of hypoxia, the expression of miR-223-3p was suppressed in the hypoxia group ( Figure 5C, P < 0.01), whereas ITGB3 expression was elevated in the hypoxia group compared with normal group (Figure 5D,E, P < 0.01).

| The expression of α-SMA in PASMCs cultured under normal or hypoxic condition after transfection
Transfected cells were incubated under normal or hypoxic conditions, and then, the total proteins were extracted. Because the phenotypic modulation of pulmonary artery smooth muscle cells (PASMCs) can be triggered by hypoxia, 32,33 and α smooth muscle actin (α-SMA), a phenotype marker of PASMCs, is the first F I G U R E 6 MiR-223-3p and ITGB3 expression level after transfection. A, ITGB3 expression after transfection with three specific siRNAs was determined. B, MiR-223-3p expression level after transfection with miR-223-3p mimics or inhibitor was confirmed via qRT-PCR. C, The ITGB3 mRNA level after transfection with pcDNA-ITGB3 or si-ITGB3 transfection was compared with matched control groups via qRT-PCR. D, The protein expression of ITGB3 was measured and compared with the matched control group after transfection with pcDNA-ITGB3 and si-ITGB3 or co-transfection with pcDNA-ITGB3 and miR-223-3p mimics. E, Design of ITGB3 3′UTR (wild type and mutant) was the target sequence. F, The dual luciferase reporter assay was performed, and decreased luciferase activity was observed in miR-223-3p mimics + pGL3-ITGB3 wt group. All experiments were performed in triplicate. **P < 0.01 compared with control siRNA, control mimics, pcDNA Vector or pGL3-NC group; ## P < 0.01 compared with control inhibitor or control siRNA known protein to be expressed during the differentiation of the smooth muscle cell (SMC) in development, 34 Western blot was employed to evaluate the protein expression of α-SMA. As shown in Figure 12A

| Rat model building
The lung tissues of the control rats presented were pink with smooth and full surfaces. The rats in the MCT group exhibited clearly reduced activity levels, reduced consumption of food, disordered hair, and cyanotic noses and lips after 2 weeks. The chests were analysed after 4 weeks. The lung tissue was grey with poor elasticity. The right ventricular wall was found to be thickened after the heart was cut. Notably, the agomiR-223-3p and sh-ITGB3 adenovirus transfection groups exhibited higher activity levels and a few bruises at lung tissue surface. However, a situation similar to the control group was observed for the agomiR-223-3p and sh-ITGB3 co-transfection groups. The observed phenotypes of each group are presented in Table 3. After 4 weeks, weight loss was observed in the MCT group compared with the other group, and the co-transfection group was healthier overall compared with the sh-ITGB3 adenovirus transfection group. Moreover, the weight of the rats in the co-transfection group was close to that of the rats in the control group. The weight of each group is presented in Table 4.

| Hemodynamic and the index of right ventricular hypertrophy
The levels of mPAP, TRP, EDP and RVSP in the MCT induced rats were elevated compared with the normal rats at 4 weeks, and the right ventricular mass ratio of the rats in the MCT group was also increased (P < 0.01) with obvious pulmonary hypertension. In addition, the CO of the experimental rats was attenuated compared with the normal rats, but the overexpression of miR-223-3p and the knockdown of ITGB3 increased the CO  Given that Shi et al reported that miR-223-3p was downregulated in the lung and right ventricle (RV) due to hypoxia, and the right ventricle is affected by PAH, 35 we investigated the expression of miR-223-3p in the right ventricle of the PAH models. The results were consistent with the results in the pulmonary artery ( Figure 14D, P < 0.01). Furthermore, immunohistochemical (IHC) analysis demonstrated that ITGB3 expression in the MCT group was higher compared with the normal group, and moreover, lower ITGB3 expression was observed in the agomiR-223-3p and sh-ITGB3 transfection groups. There was an even more obvious decrease in ITGB3 expression in the group that was co-treated with agomiR-223-3p and sh-ITGB3 ( Figure 14E, P < 0.01).

| Pathological features of rat lung tissues and the right ventricular myocardium
Pathological staining of rat lung tissues was performed to inves-

E TH I C A L A PPROVA L
All procedures performed in studies involving animals were in accordance with the ethical standards Beijing Anzhen Hospital.

CO N FLI C T O F I NTE R E S T
The authors confirm that there is no conflict of interest.