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Original Article Potential Biomarkers of Idiopathic Pulmonary Fibrosis Discovered in Serum by Proteomic Array Analysis

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Original Article Potential Biomarkers of Idiopathic Pulmonary Fibrosis Discovered in Serum by Proteomic Array Analysis Int J Clin Exp Pathol 2016;9(9):8922-8932 www.ijcep.com /ISSN:1936-2625/IJCEP0036540 Original Article Potential biomarkers of idiopathic pulmonary fibrosis discovered in serum by proteomic array analysis Rui Niu1, Xiaohui Li2, Yuan Zhang3, Hui Wang1, Yongbin Wang1, Ying Zhang1, Wei Wang1 Departments of 1Respiratory Medicine, 2Nursing, 3Evidence-Based Medicine, Second Hospital of Shandong University, Shandong, China Received July 24, 2016; Accepted July 25, 2016; Epub September 1, 2016; Published September 15, 2016 Abstract: Idiopathic pulmonary fibrosis (IPF) is a serious interstitial pneumonia leading to considerable morbidity and mortality. The unavailability of prompt biomarkers for IPF has hampered our ability to uncover preventive and therapeutic measures for this disease in a well-timed manner. The objective of this study was to identify valuable IPF associated blood biomarkers for stratifying patients and predicting outcome. By analyzing the expression of proteins in sera of 50 IPF patients and 10 healthy individuals using Biotin Label-based Antibody Array, we identified a signature of 46 differentially expressed proteins including 15 up-regulated and 31 down-regulated proteins as potential IPF biomarkers. The PPI network showed strong and complex interactions between identified biomarkers while functional enrichment analysis revealed their implications in 589 biological processes and 40 KEGG meta- bolic pathways. Western blotting and RT-PCR validation results corroborated with the microarray data. Our research unearthed candidate biomarkers with great potential for diagnosis of IPF and suggested that recombinant throm- bopoietin and anti-CCL18 antibodies, as well as other identified biomarkers may represent a novel advance in the medical treatment of patients with IPF. Keywords: Idiopathic pulmonary fibrosis, biomarkers, proteomic array Introduction that are responsible for the development of specific diseases [4]. Compared to RNA-seq or Diverse responses and reactions that occur in microarray-based profiling of gene expression, the lung, including granulomatous disorders, which have been previously applied to IPF, pro- exposure to environmental dusts and toxins, teomics analysis offers various advantages [5, autoimmune diseases and drugs, generally 6]. In particular, proteomics indicates the effec- lead to pulmonary fibrosis. The most common tive existence of functional proteins in studied and severe form of pulmonary fibrosis is idio- samples. In addition, high transcriptional level pathic pulmonary fibrosis (IPF) which is charac- producing abundant quantity of mRNA does not terized by a progressive distortion of the alveo- imply the high amount of the corresponding pro- lar architecture and the replacement by fibrotic tein or its effectual activity [7]. Therefore, pro- tissues, resulting in a progressive dyspnea and teomics is significantly advantageous in facili- a loss of the respiratory function [1]. On aver- tating the discovery of specific diagnostic bio- age, half of patients with IPF decease within 3 markers and new therapeutic targets. Proteome years after diagnosis as a result of respiratory profiler antibody arrays are suitable for screen- or heart failure [2, 3]. The high incidence of IPF ing biomarkers involved in a specific disease has conducted to the search for biomarkers as because they allow high-throughput and rapid an essential asset for diagnosis and prognosis of IPF patients and, more importantly, for the detection of multiple proteins with low sample assessment of therapeutic interventions. requirement [8-10]. In laboratories and clinics, antibody arrays have been successfully applied Proteomics tools allow the large-scale study of to uncover biomarkers of a variety of diseases gene expression at the protein level, and there- such as cancers [11-13]. Nevertheless, to the by enables the identification of gene regulation best of our knowledge, up to date, the applica- IPF biomarkers tion of antibody arrays to the identification of Collection and storage of blood biological markers in sera of IPF patients has not been deeply assessed. From each fasting subjects, 5 mL of peripheral blood were collected into a BD Vacutainer tube In this study, we used a RayBio® Human Biotin- without anticoagulant using standardized phle- labeled Antibody Array I to concurrently identify botomy procedures. After centrifugation at and determine concentrations of 507 serum 10,000 rpm for 10 minutes at 4°C, all samples proteins in a cohort of IPF patients and healthy were rapidly aliquoted and stored at -80°C until individuals. The study allowed the discovery of analysis. Only samples obtained before treat- a panel of 46 serum proteins as IPF biomarkers ment of IPF patients were employed for pro- and indicated their promising application in teomic analysis. All serum samples from IPF personalized medicine for discriminating pati- patients and healthy individual groups were ents with IPF and healthy subjects. randomized, and the researcher was blinded to their characteristics. Materials and methods Protein expression profiling using antibody Study population chip technology This study included 50 IPF patients recruited at We employed the RayBio® Human Biotin-la- Second Hospital (20 cases) and Qilu Hospital beled Antibody Array I (AAH-BLG-1, RayBiotech, (10 cases) of Shandong University, Shandong Norcross, GA, USA), constituted of 507 differ- Province Hospital (10 cases) and Qianfoshan ent human proteins, to determine proteins dif- Hospital of Shandong (10 cases). The diagnosis ferentially expressed between IPF patients and of IPF was based on published consensus control subjects. The first step consisted in the guidelines [14] and the “Guidelines for biotinylation of the primary amine of the pro- Diagnosis and Management of Idiopathic teins in serum samples. Thereafter, the glass Pulmonary Fibrosis” established by the Chinese chip arrays were blocked and 400 μl biotin- Medicine Association and determined on the labeled samples were added onto the glass basis of high-resolution computed tomography chip which was preprinted with capture anti- (HRCT) or surgical lung biopsy showing a bodies, and incubated at room temperature for definite usual interstitial pneumonia pattern. 2 h. Subsequently, the chips were washed to The most common symptoms at the onset of remove free components. Protein chips were the disease were irritating cough, shortness of subsequently incubated at room temperature breath, dyspnea, loss of endurance activities for 1 h with streptavidin-conjugated fluorescent TM and other symptoms of dyspnea. The anoma- dye, HiLytePlus 532 (Cy3 equivalent) that was lies of pulmonary function included restriction purchased from AnaSpec (Freemont, CA, USA). of ventilation function and impairment of lung After that, the excess of streptavidin was diffusion. The X film of the chest showed a retic- removed and the glass chip dried. Finally, the TM ular shadow in the surrounding lung area, which signals were scanned using a GenePix 4000B laser scanner (Axon Instruments, Sunnyvale, occurred mainly in the basal part of the lung. ® The main HRCT results showed double lung CA 94089, USA) coupled with GenePix bottom patch or mesh like changes, with vary- ProMicroarray Image Analysis Software which was used for image analysis. ing degrees of grinding glass-like shadow. The healthy controls (10 cases) were selected after Protein array data analysis a rigorous physical examination at the Physical Examination Center of Second Hospital of The data resulted from RayBio® Human Biotin- Shandong University. Written informed consent labeled Antibody Array I assay of serum sam- was obtained from all subjects according to the ples stemming from IPF patients and healthy declaration of Helsinki and the study was controls were normalized based on the positive approved by the institutional review board of control signal, consisting of biotin-labeled anti- Second Hospital and Qilu Hospital of Shandong bodies printed on each array, compared to a University, Shandong Province Hospital, and common reference array. Signals of adjacent Qianfoshan Hospital of Shandong prior to spots were measured as background signals. patients’ enrollment. After withdrawing background signals and nor- 8923 Int J Clin Exp Pathol 2016;9(9):8922-8932 IPF biomarkers Table 1. Clinical and demographic IPF variables catego- Retrieval of Interacting Genes (STRING) rized by forced vital capacity (FVC, (%)) and diffusing database (http://string-db.org) to dis- close possible connections among pro- capacity for carbon monoxide (DLCO (%)) teins and visualize the PPI (protein-pro- Healthy Variable Characteristics IPF (N=50) control tein interaction) network. The PPI net- (N=10) work was constructed using the cut-off Predicted FVC (%) 80±10% 100% value of a confidence score >0.15. The GO and KEGG pathway enrichment anal- Predicted DLCO (%) 72±8% 100% yses of genes in the PPI network were Age (years) Mean ± SD 64.3±10.56 66.98±8.48 realized to pinpoint their biological func- Range 41-71 49-80 tions and pathways, based on the hyper- Sex Male 34 6 geometric distribution algorithm. P<0.05 Female 16 4 was chosen as the cut-off value for sig- Smoking status Current smokers 0 0 nificantly enriched functions and path- Former smokers 11 0 ways. Never smoked 30 0 Validation of the proteomic array data Not reported 9 0 Protein isolation and western blot analy- Table 2. Primers used in this study sis Gene name Primer Prior to western blotting analysis, total THPO F: 5’-ATGGAGCTGACTGAATTGCTCCTCG-3’
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