Idiopathic Pulmonary Fibrosis (IPF): tissue identification of crucial biomarkers by RNA-Sequencing approach.

Background. Idiopathic pulmonary fibrosis (IPF) is one of the most common idiopathic interstitial lung diseases characterized by progressive lung scarring and a very poor overall prognosis with a median survival of 2-3 years. Despite extensive research efforts, the etiopathogenesis and pathophysiology of IPF are still little understood and consequently only slight improvement has been made for appropriate management and effective therapies. Some advances have been made in understanding the multiple interrelated pathogenic pathways underlying IPF and the disease is now considered to result from complex interactions among genetic, epigenetic, transcriptional, post-transcriptional, metabolic and environmental factors. Thus, the discovery and validation of theranostic biomarkers are necessary to enable a more precise and earlier diagnosis of IPF and to improve the prediction of future disease behavior. Usual interstitial pneumonia (UIP) is the histopathological pattern of IPF characterized by spatial/temporal heterogeneous histological lesions. Fibroblastic focus (FF) areas include fibroblasts and myofibroblasts arranged in a linear fashion with a pale staining matrix, with metaplastic epithelial cells lining on top of them. They are usually detected in the transitional area from dense scarring to normal lung and are considered an “active” component in IPF pathogenesis. Aim of the research. The main goal of the present PhD research project was the identification of crucial biomarkers in IPF pathogenesis by extracting RNA from FF areas (FF plus metaplastic epithelial cells lining FF). The main steps of the study were the following: 1) validation of a protocol for the isolation of FF areas by laser microdissection of formalin-fixed and paraffin-embedded (FFPE) tissues of IPF patients and controls; 2) total RNA extraction, quality and quantity evaluation; 3) creation of cDNA libraries starting from the extracted RNAs; 4) transcriptomic analysis by a Next-Generation Sequencing (NGS) approach (RNA-sequencing; RNA-Seq); 5) validation of the biomarker emerged from the transcriptomic analysis in a more extensive (retrospective) cases series using immunohistochemistry. Material and Methods. Total RNA was extracted from fibroblastic focus areas isolated with laser microdissection in 10 FFPE IPF lung tissues: 8 from lung transplanted patients and 2 from surgical biopsy. Microdissected fibroblastic focus areas from 2 patients with recurrent pneumothorax were also analyzed and considered as controls. The RNA was extracted using a modified protocol which provides an overnight tissue incubation at 43°C with 10 μl proteinase K. RNA was then preserved by adding RNase inhibitors at the end of the extraction procedure. This was a custom-made protocol (RNeasy® FFPE kit; Qiagen, Hilden, Germany) with additional procedures optimized during my PhD research study. The final RNAs quality and quantity were valuated with an Agilent RNA 6000 Pico Kit using a 2100 Bioanalyzer instrument. Quality was expressed as DV200 (percentage of RNA fragments > 200 nucleotides). Libraries were obtained with the SMARTer Stranded Total RNA-Seq Kit pico input mammalian of Takara Bio. DNA library was sequenced with a paired-end sequencing 2x150 bp on a HiSeq 4000 System sequencer of Illumina. MUC5B immunohistochemistry (clone 4A10-H2; 1:200, Novus Biologicals, Centennial, Colorado, USA) was performed in 44 interstitial lung disease (ILD) cases (39 UIP/IPF and 5 ILD with no UIP pattern) and 6 controls (5 spontaneous pneumothorax and 1 emphysema) following the antibody manufacturer's protocol using a Leica Bond-III Autostainer. Results. Considering the whole population the mean quantity of extracted RNA was 2992.8 pg/µl±2473 (mean ± SD), ranging from 840 pg/µl to 7530 pg/µl. Quality evaluation showed 42% of total cases with a medium/high quality (DV200>50%). In all cases molecular analyses were performed and final libraries were obtained with a concentration ranging from 3.4 to 22.6 ng/ul and a mean cDNA fragment length of 289 nucleotides. RNA-Seq analysis showed that 323 genes were differentially expressed in UIP/IPF cases than controls: 14 of them were up-regulated and 309 down-regulated. The most significant up-regulated gene was MUC5B, the other up-regulated genes were those involved in epithelial-to-mesenchymal transition (EMT) and epithelial carcinogenesis process. Gene Ontology Enrichment Analysis (GOEA) was performed to identify the most enriched Gene Ontology (GO) categories for the down-regulated genes. We found that extracellular matrix structure and organization were the principally down-regulated pathways. The overexpressed gene MUC5B was validated by immunohistochemistry. MUC5B was expressed only in IPF/UIP and ILDs, never in control group. The MUC5B expression was mainly detected in metaplastic epithelial cells lining: a) honeycomb areas, b) other alveolar spaces and c) in the metaplastic epithelial cells lining FF. Interestingly, a gradient of MUC5B expression was detected both in IPF/UIP and ILDs samples where MUC5B was overexpressed in lower lobes. Interestingly, MUC5B was overexpressed in upper and middle lobes of IPF/UIP compared with the same lobes of other ILDs. Conclusion. The principal results obtained from the present research study offer interesting insights into the complex molecular system signature of IPF: 1) adequate quantity and quality of RNA was extracted from microdissected FF areas of FFPE IPF lung tissues. The quantity/quality of RNA was suitable to create cDNA libraries for transcriptomic analysis by RNA-seq: this represents an important step forward in tissue molecular investigation of this disease characterized by high tissue heterogeneity. Only a very few papers in the literature have used lung FFPE tissue for molecular analysis, in particular, this molecular approach on specific affected IPF tissues has not previously used. 2) Comparative analysis performed on selected areas found an overexpression of epithelial proliferation/cancer progression and EMT transcripts: this highlights the crucial role of metaplastic epithelial cells that are the key actors also in the FF areas, considered the active injured lesion in IPF. 3) The up-regulated transcript MUC5B, validated also by immunohistochemistry, confirms the crucial role of this mucin in the disease. Indeed previous works, mainly performed in blood samples, had highlighted the importance of this gene in the disease. Selective regulation of MUC5B in experimental models could open up an entire line of investigation that could bring us closer to understanding regulation of MUC5B and providing novel therapeutic options.