BACKGROUND. I micro-RNA (miRNA) sono piccoli RNA, a singola elica, non codificanti che agiscono legandosi a specifiche regioni non tradotte 3’ (UTRs) di mRNA target inibendo l’espressione proteica e promuovendone la degradazione. Essi influenzano inoltre i pathways molecolari che controllano lo sviluppo e la funzione delle risposte del sistema immune, sia innato che adattativo. E’ stato dunque studiato il ruolo di molteplici miRNAs nelle malattie autoimmunitarie come l’Artrite Reumatoide (AR). Recentemente è stata analizzata l’espressione di miRNAs in diverse tipologie cellulari come i fibroblasti sinoviali di pazienti affetti da AR, i quali dopo stimolazione con TNF-α mostrano una up-regolazione del miR-146a e del miR-155. Scopo del presente studio è quello di analizzare il ruolo dei miR-34a/c e miR-22, importanti nella regolazione del ciclo e della differenziazione cellulare nei monociti e nelle cellule dendritiche di pazienti affetti da AR. METODI. Tessuto sinoviale di pazienti affetti da AR e osteoartrite (OA) sono stati sottoposti a ibridizzazione in situ per miR-34a e miR-22 e a ibridizzazione in situ con fluorescenza per identificare la natura delle cellule positive. Inoltre, dopo isolamento da sangue periferico (pb) di pazienti affetti da AR [attraverso centrifugazione a gradiente (Histopaque) e separazione automatizzata (AutoMACS)] le cellule CD14+ sono state sottoposte a lipofezione con miR-34a/c e miR-22 mimic (SIGMA). Le cellule sono state quindi sottoposte a stimolazione con LPS, CL097 o con solo terreno di coltura dopo 48 h di lipofezione. Dopo 14h dalla stimolazione il sovranatante è stato raccolto e sottoposto ad analisi (Luminex assay) di panel di citochine. Le cellule sono state analizzate per la presenza di markers di attivazione cellulare (CD16, MHC II, CD80, CD86) attraverso metodica citofluorimetrica e per il possibile effetto del miR-34a/c e miR-22 sul ciclo cellulare (CFSE staining, annexin-V staining). Le cellule CD14+ isolate da PB di soggetti sani [attraverso Histopaque e separazione automatizzata (AutoMACS)] sono state isolate e transfettate con miR-34a, miR-34c e miR-22 (SIGMA). Esperimenti simili sono stati effettuati su cellule dendritiche ottenute dopo incubazione di cellule CD14+ con GM-CSF e IL-4 per 5 giorni. Dopo 48h dalla transfezione e 14h dalla stimolazione con LPS o CL097 il sovranatante di cellule CD14+ e DC è stato raccolto per analisi di un panel di fattori solubili coinvolti nell’attivazione cellulare (Luminex assay). Il pellet cellulare è stato raccolto in Qiazol per analisi di gene expression (Agilent). Attraverso l’utilizzo di un algoritmo predittivo computerizzato e il profilo trascriptomico sono stati identificate mRNA target per miR-34a/c e miR-22. Inoltre i target selezionati verranno confermati sperimentalmente attraverso QPCR e Luciferase assay. RISULTATI. Dati preliminari hanno mostrato un’over-espressione di miR-34a/c e miR-22 nelle cellule CD14+ isolate da liquido sinoviale (LS) rispetto a cellule CD14+ isolate da PB. Tale reperto è stato confermato attraverso l’analisi in Real-time PCR (RT-PCR) in cellule CD14+ isolate da pazienti affetti da RA. Inoltre è stata dimostrata una over-espressione di miR-34b/c nelle cellule CD1c+ isolate da LS rispetto a cellule CD1c+ isolate da PB in pazienti affetti da AR. Inoltre è stato dimostrato che cellule CD14+ isolate da PB over-esprimono miR-34a quando incubate con LS suggerendo che componenti presenti nel LS possano essere responsabili dell’over-espressione di miR-34a. Il tessuto sinoviale di pazienti affetti da AR mostra un’over-espressione del miR-34a e nel miR-22 rispetto alla membrana sinoviale dei pazienti affetti da OA. Inoltre cellule CD68+ sono risultate esprimenti il miR-34a a livello del tessuto sinoviale in corso di AR. E’ stata confermata l’efficienza e la stabilità del protocollo di transfezione di monociti e cellule dendritiche utilizzando miR-34a/c e miR-22 mimic. I dati preliminari ottenuti hanno mostrato che inducendo l’over-espressione di miR-34a/c attraverso transfezione con miR-34a/c mimic si riscontrano cambiamenti significativi nel fenotipo cellulare e produzione di TNF-α, IL-6 e chemochine. Attraverso un’analisi integrata (combinando la signature CD14 trascriptomica e software automatizzati- Targetscan) sono stati identificati AXL e Tyro3 come possibili mRNA target del miR-34a/c e miR-22 rispettivamente. Tale relazione è stata confermata sperimentalmente attraverso RT-PCR e Luciferase assay. Inoltre in vivo è stata confermata la tendente down-espressione di Axl nelle cellule CD14+ e CD1c+ isolate dal PB e dal LS di pazienti con AR. Considerati nell’insieme, questi dati indicano che i miR-34a/c e miR-22 possano avere un effetto importante sulle cellule mieloidi e che una sua futura analisi possa fornire importanti informazioni utili per comprendere l’attivazione cronica di tali cellule del tessuto sinoviale e nuovi targets terapeutici per l’AR.

Micro-RNA (miRNAs) are small, single-stranded non-coding RNAs, many of which have been highly conserved throughout evolution. They act by direct binding to the 3‘ untranslated regions (UTRs) of specific target mRNAs, leading to the repression of protein expression. Recently, the miRNAs profile in Rheumatoid Arthritis (RA) has been studied. In the host laboratory high expression of miR-34a/c and miR-22 was detected in synovial fluid CD14+ cells compared to matched PB CD14+ cells as well as miR-34a/c and miR-22 in SF CD1c+ cells compared to PB suggesting that those miRNAs may contribute in the induction of activated phenotype of these cells. A limited number of studies suggest that miR-34a play an important role in myeloid cell biology. Inhibition of miR-34a stopped DC differentiation showing a crucial role of this miRNA in DC fate. Moreover it was confirmed that the 3’UTR of JAG1 was its functional target since exogenous addition of Jagged-1 also stopped DC differentiation suggesting that miR-mediated inhibition of endogenous JAG1 expression is important for proper DC differentiation. These data indicated that miR-34a is a potent regulator of cell proliferation and differentiation. The purpose of my study was to investigate the consequences of miR-34/22 overexpression in RA synovial fluid DCs and monocytes. This could reveal information about the function of those miRNAs beyond cell cycle and most importantly could reveal the mechanism responsible for activation of DCs and monocytes in RA patients METHODS: Blood and synovial fluid from 10 RA patients and blood from age-matched healthy volunteers (n = 8) were used for miRNA profiling. The study protocols were approved by the local ethics committees, and all subjects provided signed informed consent. MiRNAs signature was obtained through TLDA plates and RT-PCR. TaqMan mRNA assays (Applied Biosystems) or miScript primer assay (Qiagen) were used for semiquantitative determination of the expression of human miR-34a, human miR-34c and human miR-22. This analysis provided miRNAs profile in RA patients compared to healthy subjects and the miRNAs expression in different compartments in RA patients. In Situ Hybridization on Paraffin-Embedded Tissues. Synovial tissue specimens were obtained during synovectomy from patients who had RA or OA. RA and OA tissues were evaluated using a numerical score based on the number of positive cells in the lining, sublining, and perivascular areas of the section (three different fields in each section), with a score of 0 indicating no positive cells; 1 indicating <10% positive cells; 2 indicating 10–50% positive cells; and 3 indicating >50% positive cells. Effect of dysregulated expression of miR-34 and miR-22 on the release of soluble mediators by PB monocytes and monocytes derived –DCs: CD14+ cells from SF or PB of RA patients and CD14+ cells from PB of healthy control subjects were isolated through Histopaque gradient and automated CD14+ cells isolation protocol (AutoMACS). Some CD14+ cells were differentiated into conventional myeloid DCs through incubation with IL-4 (20 ng/mL) and GM-CSF (100 ng/mL) for 6 days. CD14+ derived DCs were lately phenotyped through Flow-cytometry detecting conventional myeloid DCs cellular markers (MHC class II, CD1c and CD11c). Also, PB and SF CD14+ cells were transfected with either miR-34a, miR-34c and miR-22 mimics (each at 20 nM) or, as controls, with scrambled mimic (20 nM) (Thermo Scientific Dharmacon), using the N-TER nanoparticle siRNA transfection system (Sigma) for CD14+ cells and DhermaFect3 System for DCs. Cells and supernatant were collected after 48 h. LPS (10 ng/mL) and CL097 (1 μg/mL) (all from InvivoGen) were added to some cultures 24 h after transfection for a further 16 h of stimulation. To establish the transfection efficiency, CD14+ cells were transfected with miR mimic labelled with Dy547 using the N-TER nanoparticle siRNA transfection system (Sigma). After 48 h cells were collected and analyzed by flow cytometry (FL-2). Cytokine levels in serum samples or cell culture supernatants were measured using a 30-plex cytokine assay (Invitrogen). To identify mRNAs targeted by miR-34 and miR-22 we used prediction algorithms and transcriptomic signature of RA and PSA SF CD14+ cells. It should be underlined that transcriptomic signature of SF CD14+ has been already generated on the same RNA samples that were used for miRNA profiling. It allowed us to reduce the list of mRNA targets predicted by computational algorithm to mRNAs that are specific for SF cells. Among the possible miRNAs targets we selected Axl and Tyro-3 as possible miR-34 and miR-22 mRNA targets respectively, belonging to the TAM receptors family. We experimentally confirmed that Axl and Tyro-3 are mRNA targets of miR-34 and miR-22 respectively for degradation through quantitative PCR and Luciferase activity assay. To analyse the gene expression of TAM receptors (Axl and Tyro-3 respectively) in CD14+ as well as CD1c+ cells in RA patients compared to healthy subjects, CD14+ cells and CD1c+ cells were isolated from PB and SF of RA patients and PB of healthy controls as previously described. Axl and Tyro-3 genes expression was evaluated throught RT-PCR methodology. RESULTS: We have confirmed the overexpression of miR-34a, miR-34c and miR-22 in SF CD14+ cells compared to PB CD14+ cells in RA patients samples by QPCR. In addition, we were able to show that PB CD14+ upregulates miR-34a when incubated with synovial fluid suggesting that components of synovial fluid can be responsible for miR-34a overexpression. We were able to show the over-expression of miR-34a and miR-22 in the lining, sublining and perivascular areas in RA synovium compared to inflammatory and non inflammatory OA synovium through in-situ hybridization. Moreover, we were able to show that some of the miR-34a positive cells in RA synovium express CD68 marker after fluorescent in-situ hybridization, suggesting that not only monocytes but also RA synovial macrophages overexpressed mir-34a. Also, we have successfully established the transfection methodology for CD14+ cells and CD14+ derived DCs with miR-34a, miR-34c and miR-22 mimic. Our experiments showed that indeed overexpression of miR-34a in monocytes by miR-34a specific mimic changes their phenotype and induces TNF-α, IL-6 and MIP-1α production after 16h of TLR7-8 stimulus whereas no difference in the cytokine production was found after TLR-4 stimulus. Moreover, the overexpression of miR-22 in monocytes by miR-22 specific mimic induces IL-6 and MIP-1α production after 16h of TLR7-8 stimulus whereas no difference in the cytokine production was found after TLR-4 stimulus. No significant differences on the production of other soluble factors including IL-8, IL-10 and IL-12 was found in monocytes after TLR7-8 stimulus. Those data have been confirmed through gene expression analysis after transfection of CD14+ cells with miR-34a mimic showing a significant upregulation of TLR-7 gene after miR-34a mimic transfection compared to C-mimic transfected CD14+ cells. We have confirmed the overexpression of miR-34a, miR-34c in SF CD1c+ cells compared to PB CD1c+ cells in RA patients samples by RT-PCR. The functional part of the study showed that the overexpression of miR-34a and miR-22 enhances TNF-α and in a lesser extent IL-6 production from CD14+ derived DCs after TLR4 stimulus. Moreover, overexpression of miR-22 but not miR-34a induces production of IFN-a and chemokines (MIP-1α and MIP-1β) from CD14+ derived DCs spontaneously and after TLR4 stimulus. To identify the putative mechanism to explain the functional consequences of miR-34a/c and miR-22 overexpression a matched analysis combining Targetscan and the already known transcriptomic signature that includes all the differentially expressed genes of SF CD14 cells we selected Tyro3 and Axl as targets for miR-22 and miR-34a-c respectively for further analysis. We experimentally confirmed that Tyro3 and Axl are degraded after miR-22 and miR-34a-c overexpression respectively through Luciferase assay. We showed a quantitative reduction of Tyro3 and AXL mRNA after miR-22 and miR-34a-c overexpression respectively through RT-PCR on transfected CD14+ cells. Finally, at least a reduction in the Axl genes expression was found in CD14+ and CD1C+ cells isolated from SF and PB of RA patients compared to healthy controls suggesting the presence of functional mir-34a/Axl interaction in RA monocytes and DC that may contribute to chronicity of inflammation in RA. All together, these data indicate that miR-34a/c and miR-22 can have an important effect on myeloid cells. In particular, the miR-34a/c and miR-22 over-expression characterizes the activated status of myeloid cells. MiR-34a/c and miR-22 expression in CD14+ and CD1c+ cells is enhanced by TLRs ligands of which synovial fluid is enriched. This phenomenon leads to the degradation and down-expression of miR-34a/c and mir-22 target mRNAs (Axl and Tyro-3 respectively) inducing CD14+ and CD1c+ cells to develop an activated phenotype with an increased release of pro-inflammatory molecules. This biological phenomenon should be regulated in physiologic conditions leading those cells to restore the previous miRNAs expression level with the resolution of the inflammatory cascade. However, the prolonged miR-34a/c and miR-22 over-expression in RA myeloid cells could induce a pro-inflammatory phenotype contributing to the chronic inflammatory process.

THE ROLE OF MIR-34 AND MIR-22 IN THE ACTIVATION OF RA MONOCYTES AND DENDRITIC CELLS AND ITS CONTRIBUTION TO RHEUMATOID ARTHRITIS.

ALIVERNINI, Stefano
2013-01-01

Abstract

Micro-RNA (miRNAs) are small, single-stranded non-coding RNAs, many of which have been highly conserved throughout evolution. They act by direct binding to the 3‘ untranslated regions (UTRs) of specific target mRNAs, leading to the repression of protein expression. Recently, the miRNAs profile in Rheumatoid Arthritis (RA) has been studied. In the host laboratory high expression of miR-34a/c and miR-22 was detected in synovial fluid CD14+ cells compared to matched PB CD14+ cells as well as miR-34a/c and miR-22 in SF CD1c+ cells compared to PB suggesting that those miRNAs may contribute in the induction of activated phenotype of these cells. A limited number of studies suggest that miR-34a play an important role in myeloid cell biology. Inhibition of miR-34a stopped DC differentiation showing a crucial role of this miRNA in DC fate. Moreover it was confirmed that the 3’UTR of JAG1 was its functional target since exogenous addition of Jagged-1 also stopped DC differentiation suggesting that miR-mediated inhibition of endogenous JAG1 expression is important for proper DC differentiation. These data indicated that miR-34a is a potent regulator of cell proliferation and differentiation. The purpose of my study was to investigate the consequences of miR-34/22 overexpression in RA synovial fluid DCs and monocytes. This could reveal information about the function of those miRNAs beyond cell cycle and most importantly could reveal the mechanism responsible for activation of DCs and monocytes in RA patients METHODS: Blood and synovial fluid from 10 RA patients and blood from age-matched healthy volunteers (n = 8) were used for miRNA profiling. The study protocols were approved by the local ethics committees, and all subjects provided signed informed consent. MiRNAs signature was obtained through TLDA plates and RT-PCR. TaqMan mRNA assays (Applied Biosystems) or miScript primer assay (Qiagen) were used for semiquantitative determination of the expression of human miR-34a, human miR-34c and human miR-22. This analysis provided miRNAs profile in RA patients compared to healthy subjects and the miRNAs expression in different compartments in RA patients. In Situ Hybridization on Paraffin-Embedded Tissues. Synovial tissue specimens were obtained during synovectomy from patients who had RA or OA. RA and OA tissues were evaluated using a numerical score based on the number of positive cells in the lining, sublining, and perivascular areas of the section (three different fields in each section), with a score of 0 indicating no positive cells; 1 indicating <10% positive cells; 2 indicating 10–50% positive cells; and 3 indicating >50% positive cells. Effect of dysregulated expression of miR-34 and miR-22 on the release of soluble mediators by PB monocytes and monocytes derived –DCs: CD14+ cells from SF or PB of RA patients and CD14+ cells from PB of healthy control subjects were isolated through Histopaque gradient and automated CD14+ cells isolation protocol (AutoMACS). Some CD14+ cells were differentiated into conventional myeloid DCs through incubation with IL-4 (20 ng/mL) and GM-CSF (100 ng/mL) for 6 days. CD14+ derived DCs were lately phenotyped through Flow-cytometry detecting conventional myeloid DCs cellular markers (MHC class II, CD1c and CD11c). Also, PB and SF CD14+ cells were transfected with either miR-34a, miR-34c and miR-22 mimics (each at 20 nM) or, as controls, with scrambled mimic (20 nM) (Thermo Scientific Dharmacon), using the N-TER nanoparticle siRNA transfection system (Sigma) for CD14+ cells and DhermaFect3 System for DCs. Cells and supernatant were collected after 48 h. LPS (10 ng/mL) and CL097 (1 μg/mL) (all from InvivoGen) were added to some cultures 24 h after transfection for a further 16 h of stimulation. To establish the transfection efficiency, CD14+ cells were transfected with miR mimic labelled with Dy547 using the N-TER nanoparticle siRNA transfection system (Sigma). After 48 h cells were collected and analyzed by flow cytometry (FL-2). Cytokine levels in serum samples or cell culture supernatants were measured using a 30-plex cytokine assay (Invitrogen). To identify mRNAs targeted by miR-34 and miR-22 we used prediction algorithms and transcriptomic signature of RA and PSA SF CD14+ cells. It should be underlined that transcriptomic signature of SF CD14+ has been already generated on the same RNA samples that were used for miRNA profiling. It allowed us to reduce the list of mRNA targets predicted by computational algorithm to mRNAs that are specific for SF cells. Among the possible miRNAs targets we selected Axl and Tyro-3 as possible miR-34 and miR-22 mRNA targets respectively, belonging to the TAM receptors family. We experimentally confirmed that Axl and Tyro-3 are mRNA targets of miR-34 and miR-22 respectively for degradation through quantitative PCR and Luciferase activity assay. To analyse the gene expression of TAM receptors (Axl and Tyro-3 respectively) in CD14+ as well as CD1c+ cells in RA patients compared to healthy subjects, CD14+ cells and CD1c+ cells were isolated from PB and SF of RA patients and PB of healthy controls as previously described. Axl and Tyro-3 genes expression was evaluated throught RT-PCR methodology. RESULTS: We have confirmed the overexpression of miR-34a, miR-34c and miR-22 in SF CD14+ cells compared to PB CD14+ cells in RA patients samples by QPCR. In addition, we were able to show that PB CD14+ upregulates miR-34a when incubated with synovial fluid suggesting that components of synovial fluid can be responsible for miR-34a overexpression. We were able to show the over-expression of miR-34a and miR-22 in the lining, sublining and perivascular areas in RA synovium compared to inflammatory and non inflammatory OA synovium through in-situ hybridization. Moreover, we were able to show that some of the miR-34a positive cells in RA synovium express CD68 marker after fluorescent in-situ hybridization, suggesting that not only monocytes but also RA synovial macrophages overexpressed mir-34a. Also, we have successfully established the transfection methodology for CD14+ cells and CD14+ derived DCs with miR-34a, miR-34c and miR-22 mimic. Our experiments showed that indeed overexpression of miR-34a in monocytes by miR-34a specific mimic changes their phenotype and induces TNF-α, IL-6 and MIP-1α production after 16h of TLR7-8 stimulus whereas no difference in the cytokine production was found after TLR-4 stimulus. Moreover, the overexpression of miR-22 in monocytes by miR-22 specific mimic induces IL-6 and MIP-1α production after 16h of TLR7-8 stimulus whereas no difference in the cytokine production was found after TLR-4 stimulus. No significant differences on the production of other soluble factors including IL-8, IL-10 and IL-12 was found in monocytes after TLR7-8 stimulus. Those data have been confirmed through gene expression analysis after transfection of CD14+ cells with miR-34a mimic showing a significant upregulation of TLR-7 gene after miR-34a mimic transfection compared to C-mimic transfected CD14+ cells. We have confirmed the overexpression of miR-34a, miR-34c in SF CD1c+ cells compared to PB CD1c+ cells in RA patients samples by RT-PCR. The functional part of the study showed that the overexpression of miR-34a and miR-22 enhances TNF-α and in a lesser extent IL-6 production from CD14+ derived DCs after TLR4 stimulus. Moreover, overexpression of miR-22 but not miR-34a induces production of IFN-a and chemokines (MIP-1α and MIP-1β) from CD14+ derived DCs spontaneously and after TLR4 stimulus. To identify the putative mechanism to explain the functional consequences of miR-34a/c and miR-22 overexpression a matched analysis combining Targetscan and the already known transcriptomic signature that includes all the differentially expressed genes of SF CD14 cells we selected Tyro3 and Axl as targets for miR-22 and miR-34a-c respectively for further analysis. We experimentally confirmed that Tyro3 and Axl are degraded after miR-22 and miR-34a-c overexpression respectively through Luciferase assay. We showed a quantitative reduction of Tyro3 and AXL mRNA after miR-22 and miR-34a-c overexpression respectively through RT-PCR on transfected CD14+ cells. Finally, at least a reduction in the Axl genes expression was found in CD14+ and CD1C+ cells isolated from SF and PB of RA patients compared to healthy controls suggesting the presence of functional mir-34a/Axl interaction in RA monocytes and DC that may contribute to chronicity of inflammation in RA. All together, these data indicate that miR-34a/c and miR-22 can have an important effect on myeloid cells. In particular, the miR-34a/c and miR-22 over-expression characterizes the activated status of myeloid cells. MiR-34a/c and miR-22 expression in CD14+ and CD1c+ cells is enhanced by TLRs ligands of which synovial fluid is enriched. This phenomenon leads to the degradation and down-expression of miR-34a/c and mir-22 target mRNAs (Axl and Tyro-3 respectively) inducing CD14+ and CD1c+ cells to develop an activated phenotype with an increased release of pro-inflammatory molecules. This biological phenomenon should be regulated in physiologic conditions leading those cells to restore the previous miRNAs expression level with the resolution of the inflammatory cascade. However, the prolonged miR-34a/c and miR-22 over-expression in RA myeloid cells could induce a pro-inflammatory phenotype contributing to the chronic inflammatory process.
2013
microRNA; Rheumatoid Arthritis; monocytes
BACKGROUND. I micro-RNA (miRNA) sono piccoli RNA, a singola elica, non codificanti che agiscono legandosi a specifiche regioni non tradotte 3’ (UTRs) di mRNA target inibendo l’espressione proteica e promuovendone la degradazione. Essi influenzano inoltre i pathways molecolari che controllano lo sviluppo e la funzione delle risposte del sistema immune, sia innato che adattativo. E’ stato dunque studiato il ruolo di molteplici miRNAs nelle malattie autoimmunitarie come l’Artrite Reumatoide (AR). Recentemente è stata analizzata l’espressione di miRNAs in diverse tipologie cellulari come i fibroblasti sinoviali di pazienti affetti da AR, i quali dopo stimolazione con TNF-α mostrano una up-regolazione del miR-146a e del miR-155. Scopo del presente studio è quello di analizzare il ruolo dei miR-34a/c e miR-22, importanti nella regolazione del ciclo e della differenziazione cellulare nei monociti e nelle cellule dendritiche di pazienti affetti da AR. METODI. Tessuto sinoviale di pazienti affetti da AR e osteoartrite (OA) sono stati sottoposti a ibridizzazione in situ per miR-34a e miR-22 e a ibridizzazione in situ con fluorescenza per identificare la natura delle cellule positive. Inoltre, dopo isolamento da sangue periferico (pb) di pazienti affetti da AR [attraverso centrifugazione a gradiente (Histopaque) e separazione automatizzata (AutoMACS)] le cellule CD14+ sono state sottoposte a lipofezione con miR-34a/c e miR-22 mimic (SIGMA). Le cellule sono state quindi sottoposte a stimolazione con LPS, CL097 o con solo terreno di coltura dopo 48 h di lipofezione. Dopo 14h dalla stimolazione il sovranatante è stato raccolto e sottoposto ad analisi (Luminex assay) di panel di citochine. Le cellule sono state analizzate per la presenza di markers di attivazione cellulare (CD16, MHC II, CD80, CD86) attraverso metodica citofluorimetrica e per il possibile effetto del miR-34a/c e miR-22 sul ciclo cellulare (CFSE staining, annexin-V staining). Le cellule CD14+ isolate da PB di soggetti sani [attraverso Histopaque e separazione automatizzata (AutoMACS)] sono state isolate e transfettate con miR-34a, miR-34c e miR-22 (SIGMA). Esperimenti simili sono stati effettuati su cellule dendritiche ottenute dopo incubazione di cellule CD14+ con GM-CSF e IL-4 per 5 giorni. Dopo 48h dalla transfezione e 14h dalla stimolazione con LPS o CL097 il sovranatante di cellule CD14+ e DC è stato raccolto per analisi di un panel di fattori solubili coinvolti nell’attivazione cellulare (Luminex assay). Il pellet cellulare è stato raccolto in Qiazol per analisi di gene expression (Agilent). Attraverso l’utilizzo di un algoritmo predittivo computerizzato e il profilo trascriptomico sono stati identificate mRNA target per miR-34a/c e miR-22. Inoltre i target selezionati verranno confermati sperimentalmente attraverso QPCR e Luciferase assay. RISULTATI. Dati preliminari hanno mostrato un’over-espressione di miR-34a/c e miR-22 nelle cellule CD14+ isolate da liquido sinoviale (LS) rispetto a cellule CD14+ isolate da PB. Tale reperto è stato confermato attraverso l’analisi in Real-time PCR (RT-PCR) in cellule CD14+ isolate da pazienti affetti da RA. Inoltre è stata dimostrata una over-espressione di miR-34b/c nelle cellule CD1c+ isolate da LS rispetto a cellule CD1c+ isolate da PB in pazienti affetti da AR. Inoltre è stato dimostrato che cellule CD14+ isolate da PB over-esprimono miR-34a quando incubate con LS suggerendo che componenti presenti nel LS possano essere responsabili dell’over-espressione di miR-34a. Il tessuto sinoviale di pazienti affetti da AR mostra un’over-espressione del miR-34a e nel miR-22 rispetto alla membrana sinoviale dei pazienti affetti da OA. Inoltre cellule CD68+ sono risultate esprimenti il miR-34a a livello del tessuto sinoviale in corso di AR. E’ stata confermata l’efficienza e la stabilità del protocollo di transfezione di monociti e cellule dendritiche utilizzando miR-34a/c e miR-22 mimic. I dati preliminari ottenuti hanno mostrato che inducendo l’over-espressione di miR-34a/c attraverso transfezione con miR-34a/c mimic si riscontrano cambiamenti significativi nel fenotipo cellulare e produzione di TNF-α, IL-6 e chemochine. Attraverso un’analisi integrata (combinando la signature CD14 trascriptomica e software automatizzati- Targetscan) sono stati identificati AXL e Tyro3 come possibili mRNA target del miR-34a/c e miR-22 rispettivamente. Tale relazione è stata confermata sperimentalmente attraverso RT-PCR e Luciferase assay. Inoltre in vivo è stata confermata la tendente down-espressione di Axl nelle cellule CD14+ e CD1c+ isolate dal PB e dal LS di pazienti con AR. Considerati nell’insieme, questi dati indicano che i miR-34a/c e miR-22 possano avere un effetto importante sulle cellule mieloidi e che una sua futura analisi possa fornire importanti informazioni utili per comprendere l’attivazione cronica di tali cellule del tessuto sinoviale e nuovi targets terapeutici per l’AR.
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