Introduction

Although rheumatoid arthritis (RA) is recognized as a chronic autoimmune disease that affects multiple joint structures and synovial membranes [1], the pathogenesis of ‘RA’ is still far from clear. Recently, epigenomic control involving microRNA (miRNA) regulation in ‘RA’ has received considerable attention and importance [1]. Most of studies carried so far revealed up- or down-regulation of different miRNAs according to the cell type analyzed from tissue sample of ‘RA’ subjects [1]. Little heed has been paid to unfold the possible correlation between miRNA expression, within blood mononuclear cells, and disease activity. Although miR-146a expression in peripheral blood mononuclear cells (PBMCs) has been shown to mimic that of ‘RA’ synovial tissue and fibroblasts [1], the elevated expression of miR-146a has found no specificity to ‘RA’ [1]. A new dimension was added to the emerging field of miRNA-based epigenomic control, by the discovery of a novel immunomudulatory microRNA (designated ARCHIVES OF MEDICINE 2015 Vol. 7 No. 1:1 2 This article is available from: www.archivesofmedicine.com as miR-2909) encoded by human cellular AATF genome [2-4]. In this context, the present study was addressed to explore the nature of miR-2909 RNomics and its impact on immune response exhibited by PBMCs derived from human ‘RA’ subjects.

Material and Methods

Human subjects: Selection criteria

Freshly diagnosed and untreated rheumatoid arthritis (RA) patients (n=50) were selected from the outpatient “Rheumatology Clinic” of Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India with prior informed consent and approval by our Institutional Ethics (IEC) as well as control human volunteers (n=25; with their prior informed consent), who had abstained from any medication for 2 weeks before blood donation, were employed in the present study.

Cellular model employed

Peripheral blood mononuclear cells (PBMCs) were employed as a cellular model for the present study. Blood was drawn through venipuncture into heparinized tubes and PBMCs were isolated using density gradient centrifugation method. Briefly, 5 ml of heparinized blood (from either ‘RA’ or control subjects) was gently layered onto 4 ml of Histopaque (Sigma solution containing polysucrose and sodium diatrizoate, adjusted to a density of 1.077 ± 0.001 gm/ml) and centrifuged at 400xg in the swinging bucket rotor for 30 minutes at room temperature. After centrifugation steps, the layer of PBMCs were recovered and subsequently used for gene expression analysis and sequencing study.

Gene expression analysis, DNA sequencing

Human PBMCs (from ‘RA’ and control subjects) were processed for total as well as small non-coding RNA extraction using miReasy mini kit (Qiagen) in accordance to the manufacturer’s protocol as reported by us earlier [2]. The extracted RNA was reversetranscribed using miScript reverse transcription kit (Qiagen) and subjected to quantitative real-time PCR analysis [2] using gene-specific primers. Genes coding β-Actin and U6 were used as invariant controls for studying the differential expression of cellular mRNAs (IL-6, IFN-γ. IL-17, RIG-1, CCL5, Sp1) and miR-2909 respectively as reported earlier [2,3]. For studying differential gene expression at the translational level, total cellular protein was extracted and subsequently subjected to SDS-PAGE followed by western blotting and immuno-detection using specific antibodies for genes coding for KLF4, IL-6, CCL-5, IL-17, Sp1 and β-Actin (used as invariant control).