Background Piwi-interacting RNAs (piRNAs, 26-31 nucleotides) are recently described short regulatory RNAs, which build functional complexes with the four argonaute-subfamily members PIWI-like proteins to repress gene expression of retrotransposons. We recently presented that PIWIL4 is highly expressed in RA synovial fibroblasts.
Objectives The aim of our study here was to analyze the expression of piRNAs (piR-651, piR-823 and piR-4987) which are associated with invasion and proliferation of cancer cells.
Methods Expression of piRNAs (piR-651, piR-823 and piR-4987) was analyzed by TaqMan RealTime-PCR and normalized to RNU6B and RNU48 in synovial fibroblasts (SF) from rheumatoid arthritis (RA) and osteoarthritis (OA) patients (n=8 each) and in peripheral blood mononuclear cells (PBMC) isolated from patients with RA and from healthy controls (HC, n=8 each). HeLa and HEK293 cell lines served as positive controls.
RASF (n=4-8) were stimulated or not for 24 hours with Poly(I:C) (10ug/ml), LPS (100ng/ml) or TNFα (10ng/ml) in combination with IL1β (1ng/ml). PIWIL4 was silenced in RASF (n=5) with siRNA and the expression of piRNAs was analyzed 48h later.
Results High expression of piR-651, piR-823 and piR-4987 could be detected in both RA and OASF (RA/OA mean dCt= -9.0/-8.9, -4.7/-4.2, 0.8/0.9). A strong expression of piR-651, piR-823 and piR-4987 was also found in PBMC of patients with RA and HC (mean dCt= - 3.4/-3.7, 7.2/7.1, 11.7/12.7). In the cancer cell lines, piR-651, piR-823 and piR-4987 were expressed as follows: HeLa (dCt= -10.2/-3.6/-1.1), HEK293 (dCt= -9.9/-4.9/-1.3).
Levels of piR-4987 in RASF were further enhanced by Poly(I:C) (to 3.0-fold, p=0.002) and by LPS (to 3.5-fold, p=0.071), but not by TNFα in combination with IL1β. Expression of piR-651 and piR-823 was not changed neither by Poly(I:C) nor by LPS or TNFα in combination with IL1β.
PIWIL4 silencing (to 0.3-fold, p<0.0001) in RASF did not change expression of the piR-651 (to 1.0-fold, p=0.968), piR-823 (to 0.8-fold, p=0.107) or piR-4987 (to 0.8-fold, p=0.075).
Conclusions We have demonstrated high expression of piR-651, piR-823 and piR-4987 in synovial fibroblasts and PBMC in RA. Levels of those piRNAs in RA are similar to those in OA. The up regulation of piR-4987 after innate immune stimulation suggests a functional role in synovial fibroblasts in RA.
Acknowledgements OSTEOIMMUNE, IMI-BTCure, EuroTEAM, IAR, MHCR project 023728
Disclosure of Interest None declared