TLR2 expression is regulated by microRNA miR-19 in rheumatoid fibroblast-like synoviocytes.

Resident cells, such as fibroblast-like synoviocytes (FLS), play a crucial role in rheumatoid arthritis (RA). They are implicated in the inflammatory response and play a key role in osteoarticular destruction. Moreover, RA FLS spread RA to unaffected joints. Pathogen-associated molecular patterns and damage-associated molecular patterns have been found to activate RA FLS by interacting with pattern recognition receptors, such as TLR. RA FLS express a large number of TLR, and TLR2 was demonstrated to be involved in RA inflammation. Because microRNA have emerged as important controllers of TLR expression and signaling, the aim of this study was to evaluate their potential involvement in the control of TLR2 expression by RA FLS. We first showed that Tlr2 expression is strongly upregulated in RA FLS in response to TLR2 ligands. Using a microRNA microarray analysis, we identified one miRNA in activated RA FLS, miR-19b, which was downregulated and predicted to target Tlr2 mRNA. Downregulation of miR-19b and miR-19a, which belongs to the same cluster, was confirmed by real-time quantitative PCR. Transfection of RA FLS with miR-19a/b mimics decreased TLR2 protein expression. In parallel, we found that both IL-6 and matrix metalloproteinase 3 secretion was significantly downregulated in activated FLS transfected with either mimic. Moreover, using a luciferase assay, we showed that miR-19a/b directly target Tlr2 mRNA. Taken together, our data point toward an important role for miR-19a/b in the regulation of IL-6 and matrix metalloproteinase 3 release by controlling TLR2 expression, as well as provide evidence that miR-19a/b can act as negative regulators of inflammation in humans.

Kaposi's sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis.

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.

Bruton's tyrosine kinase is involved in miR-346-related regulation of IL-18 release by lipopolysaccharide-activated rheumatoid fibroblast-like synoviocytes.

MicroRNAs (miRNAs) have emerged as key players in the regulation of expression of target mRNAs expression. They have been associated with diverse biological processes, and recent studies have demonstrated that miRNAs play a role in inflammatory responses. We reported previously that LPS-activated fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients express IL-18 mRNA but they do not release IL-18. Based on the observation that this inhibition was due to a rapid degradation of IL-18 mRNA, our group has conducted a study to identify miRNAs that could play a role in the "antiinflammatory" response of LPS-activated RA FLS. LPS challenge modulated the expression of 63 miRNAs as assessed by microarray analysis. Fifteen miRNAs were up-regulated, including miR-346, for which overexpression upon LPS treatment was validated by quantitative RT-PCR. We then transfected FLS with an antisense oligonucleotide targeting miR-346 and found that, in these conditions, IL-18 release could be measured upon LPS activation of FLS. Moreover, we also demonstrated that miR-346 indirectly regulated IL-18 release by indirectly inhibiting LPS-induced Bruton's tyrosine kinase expression in LPS-activated RA FLS. These findings suggest that miRNAs function as regulators that help to fine-tune the inflammatory response in RA.

miR-346 controls release of TNF-α protein and stability of its mRNA in rheumatoid arthritis via tristetraprolin stabilization.

TNF-α is a major cytokine implicated in rheumatoid arthritis. Its expression is regulated both at the transcriptional and posttranscriptional levels and recent data demonstrated that miRNAs are implicated in TNF-α response in macrophages. LPS-activated FLS isolated from RA patients express TNF-α mRNA but not the mature protein. This prompted us to look for miRNAs which could be implicated in this anti-inflammatory effect. Using a microarray, we found two miRNAs, miR-125b and miR-939 predicted to target the 3'-UTR of TNF-α mRNA, to be up-regulated in RA FLS in response to LPS, but their repression did not restore mature TNF-α expression in FLS. We showed previously that miR-346, which is upregulated in LPS-activated FLS, inhibited Btk expression that stabilized TNF-α mRNA. Blocking miR-346 reestablished TNF-α expression in activated FLS. Interestingly, transfection of miR-346 in LPS-activated THP-1 cells inhibited TNF-α secretion. We also demonstrated that TTP, a RNA binding protein which inhibited TNF-α synthesis, is overexpressed in activated FLS and that inhibition of miR-346 decreases its expression. Conversely, transfection of miR-346 in LPS-activated THP-1 cells increased TTP mRNA expression and inhibited TNF-α release. These results indicate that miR-346 controls TNF-α synthesis by regulating TTP expression.

Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay.

The mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysis (RIP-Chip). Here we used Ago2-based RIP-Chip to identify transcripts targeted by Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (n = 114), Epstein-Barr virus (EBV) miRNAs (n = 44), and cellular miRNAs (n = 2337) in six latently infected or stably transduced human B cell lines. Of the six KSHV miRNA targets chosen for validation, four showed regulation via their 3'UTR, while two showed regulation via binding sites within coding sequences. Two genes governing cellular transport processes (TOMM22 and IPO7) were confirmed to be targeted by EBV miRNAs. A significant number of viral miRNA targets were upregulated in infected cells, suggesting that viral miRNAs preferentially target cellular genes induced upon infection. Transcript half-life both of cellular and viral miRNA targets negatively correlated with recruitment to RISC complexes, indicating that RIP-Chip offers a quantitative estimate of miRNA function.