Background/Purpose:  Osteoarthritis (OA) is a chronic and debilitating disease of articulating joints. Mechanical stress, genetic and environmental factors play critical role in the pathogenesis of OA. Micro-RNAs (miRNA) are endogenous, short ~22-nucleotides non-coding single stranded RNAs, that act as a regulator of gene expression. In order to understand the role of miRNAs in OA pathogenesis we employed deep sequencing technology to comprehensively profile the miRNA population in OA chondrocytes.

Methods:   Chondrocytes were prepared from human cartilage samples obtained from OA patients at the time of knee arthroplasty and were treated with IL-1β (2ng/ml) for 1, 12, 24 h. Total RNA was isolated using miRNeasy Mini kit (Qiagen) and a small RNA library was prepared using TrueSeq Small RNA Library Preparation Kit (Illumina). RNA purity, integrity and library integrity and purity was verified using Agilent 2100 Bioanalyzer. Cluster generation and sequencing was performed on MiSeq (Illumina). The small RNA-Seq reads were aligned to genomic reference (hg19) and subsequently miRNAs were annotated to generate the miRNA abundance profile and novel miRNA sequences from NGS data using Strand NGS software. Data was normalized followed by differential expression analyses in control and treated samples using Strand NGS software. Expression of selected miRNAs was verified by TaqMan assays. Differentially regulated miRNAs were used to construct the signaling pathways that were enriched by the treatment using DIANA-mirPath open web tool.

Results: We obtained 2.0-2.5 million reads per sample and more than 95% reads pass the quality filter. We identified 1548 miRNAs in our sequencing reads that were unique. miRNAs with less than 5 sequencing reads were excluded from the analyses which left 511 differentially expressed miRNAs. In chondrocytes, expression of 10 miRNAs (miR-100, 26a, 22, 148a, 27b,125b, 99b, 21, let-7a and 7b) on average constitute ~70% of all miRNAs expression across the samples. Differentially expressed miRNAs were identified across different time points by using 1.5 fold as cut-off criteria. We identified 46 miRNA that were upregulated and 41 miRNAs that were downregulated compared to untreated control sample at all the time points analyzed. Highest upregulated miRNA was miR-146a (30 fold) which has been implicated in OA pathogenesis and most downregulated miRNA was miR-452 (5 fold). Some miRNAs exhibited dynamic expression pattern. Fifteen miRNAs were identified as potential novel miRNAs by the Strand NGS software. One sequence (newgene 105) annotated in bovine as bta-miR-2904 was also found to be expressed in human chondrocytes. Several of the differentially expressed miRNAs were predicted to target mRNAs associated with MAPK signaling, PI3K-AKT signaling, focal adhesion and regulation of actin cytoskeleton.

Conclusion: Deep sequencing revealed several novel miRNAs in OA chondrocytes. Expression of Newgene 105 (miR-2904 in Bos taurus) has been documented for the first time in human chondrocytes. These newly identified miRNAs could have novel functions in OA pathogenesis. Our work deliver a clear picture of miRNA profile and associated regulatory network in OA chondrocytes.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-novel-micro-rnas-in-il-1-stimulated-oa-chondrocytes-by-next-generation-sequencing/