Background/Purpose:  Osteoarthritis (OA) is a chronic and debilitating disease of the articulating joints in every population. Micro-RNAs are ~22 nucleotides long non-coding single stranded RNAs that mostly regulate post-transcriptional gene expression. In order to dissect the role of novel miRNAs in OA pathogenesis we exploited the next generation sequencing technology and comprehensively profiled the miRNAs expression in OA chondrocytes.

Methods:  Chondrocytes were prepared by the enzymatic digestion of discarded human cartilage from donors who underwent total knee arthroplasty due to OA. Total RNA directly from the cartilage or from chondrocytes was prepared using Trizol and miRNeasy Mini kit respectively. RNA purity, integrity and NGS library integrity was verified using Agilent 2100 Bioanalyzer. The small RNA library was prepared using a kit (Illumina). Cluster generation and sequencing was performed on MiSeq system. The small RNA sequencing reads were aligned to genomic reference (hg19) and novel miRNA sequences were extracted from NGS data using Strand NGS software. miRNA targets were identified using open web bioinformatics program Diana Tools. Expression of mature miRNAs was quantified using individual miScript Primer Assay. Expression of KiSS1 and other OA related catabolic and anabolic genes was quantified using TaqMan Assays and of proteins by Western blotting. Chondrocytes were transfected with siRNAs, control miRNA, miR-N105 mimic or inhibitor using Amaxa Nucleofactor System.

Results: We analyzed the 95% of the ~20 million reads from the miRNA-Seq which passed the quality filter using the Strand NGS Software and identified 15 miRNAs as potential novel miRNAs. Based on differential expression and abundance novel miRNA miR-N105 was selected for further analysis. Expression of miR-N105 was downregulated upon IL-1β treatment in chondrocytes in a time dependent manner. Interestingly expression of miR-N105 was significantly low in damaged cartilage compared to the expression levels detected in the smooth cartilage of the same patients (n=8). Diana Tool predicted KiSS1 (KiSS-1 metastasis-suppressor) as a potential target of miR-N105. Expression of KiSS1 was 4 fold induced in OA chondrocytes upon IL-1β treatment. In the damaged cartilage expression of KiSS1 was significantly higher compared to the smooth cartilage. Overexpression of miR-N105 inhibited the IL-1β induced expression of KiSS1 which was rescued upon transfection of miR-N105 inhibitor suggesting KiSS1 is a bona fide target of miR-N105. siRNA-mediated knockdown of KiSS1 or its inhibition by overexpression of miR-N105 enhanced the expression of ACAN and reduced the expression levels of ADAMTS4 and ADAMTS5 in IL-1β-stimulated OA chondrocytes. Importantly, overexpression of KiSS1 inhibited the ACAN expression but significantly enhanced the expression of ADAMTS4 and ADAMTS5 in IL-1β-stimulated OA chondrocytes.

Conclusion:  We identified a novel miRNA-N105 that was differentially expressed in smooth and damaged OA cartilage, and validated KiSS1 mRNA as its target in OA chondrocytes. Our data also identifies for the first time KiSS1 as an important regulator of genes associated with the pathogenesis of OA.   

« Back to 2016 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/kiss1-is-a-regulator-of-adamts4-and-adamts5-expression-and-is-post-transcriptionally-regulated-by-micro-rna-n105-in-human-oa-chondrocytes/