RNAseq of mouse arthritic ankles with over-expression of miR-17-5p
ABSTRACT: DBA1 mice with collagen-induced arthritis were injected intra-articularly with miR-17-5p mimic lipoplex. At the peak of inflammation (day 7), ankles were processed for bulk RNAseq using Illumina NextSeq 500. Overall design: Ankle mRNA profiles of DBA1 mice with collagen-induced arthritis injected with miR-17-5p lipoplex (n=4) or control irrelevant miR lipoplex (n=3) were generated by deep sequencing using Illumina NextSeq 500.
Project description:The degradation of cruciate ligaments is frequently observed in degenerative joint diseases, such as osteo-arthritis (OA). The present study aimed to identify the differentially expressed microRNAs (miRNAs or miRs) in knee anterior cruciate ligament (ACL) tissues derived from patients with OA and in health subjects (non-OA). By using Affymetrix miRNA 4.0 microarrays, a total of 22 miRNAs (including let-7f-5p, miR-26b-5p and miR-146a-5p) were found to be upregulated, while 17 (including miR-18a-3p, miR-138-5p and miR-485-3p) were downregulated in the osteoarthritic ACL tissues (fold change ?2, P-value <0.05). The expression levels of 12 miRNAs were validated by quantitative PCR, and the corresponding results revealed an excellent correlation with the microarray data (R2=0.889). Genes (such as a disintegrin and metalloproteinase domain with thrombospondin type-1 motifs, bone morphogenetic protein-2, runt related transcription factor-2, collagen-1A1 and 2, interleukin-6 and transforming growth factor-?) involved in cartilage development and remodeling, collagen biosynthesis and degradation, inflammatory response and extracellular matrix homeostasis were predicted as potential targets of the dysregulated miRNAs. Moreover, a large set of putative genes were enriched in OA pathogenesis?associated pathways (such as mitogen-activated protein kinase and vascular endothelial growth factor signaling pathway). Collectively, the data from our study provides novel insight into the ligament injury-related miRNA dysregulation in patients with OA.
Project description:Periploca forrestii Schltr. (P. forrestii) is a species used in Traditional Chinese Medicine (TCM) known as "Miao medicine", and has a long history of use in the treatment of rheumatism, rheumatoid arthritis (RA), and joint pain. The present study aimed to evaluate the anti-arthritis effects of the cardenolide-rich and caffeoylquinic acid-rich fractions (CDLFs and CQAFs) of P. forrestii in collagen-induced arthritic (CIA) rats, and defined the mechanisms of therapeutic action in MH7A cells treated with TNF-?. Serum rheumatoid factor (RF), TNF-?, IL-6, IL-1?, PGE?, NO, SOD, and MDA were determined by ELISA or other commercially assay kits. Histopathological changes in ankle joint tissues were examined. The mRNA expressions of IL-1?, IL-6, COX-2, and iNOS in MH7A cells were measured by qRT-PCR assays. In addition, the expressions of iNOS, COX-2, and p65 proteins, and the phosphorylation of I?B?, p38, ERK1/2, and JNK proteins in MH7A cells were analyzed by Western blot. The results showed that CDLF and CQAF could suppress the paw swelling in CIA rats at different doses (125 mg/kg, 250 mg/kg, and 500 mg/kg). Histopathological examination suggests that the CDLF and CQAF significantly relieved the damage of the structure of the ankle joint in CIA rats. In addition, serum RF, TNF-?, IL-6, IL-1?, PGE?, NO, and MDA were decreased, along with increased activity of serum SOD. Furthermore, CDLF and CQAF downregulated the expressions of IL-1?, IL-6, COX-2, iNOS, and p65, and inhibited the phosphorylation of I?B?, p38, ERK1/2, and JNK in MH7A cells treated with TNF-?. These findings demonstrated that both CDLF and CQAF exhibited anti-arthritic activity, which might be associated with their inhibitory effects on the NF-?B and MAPK signaling pathways.
Project description:CONTEXT:Rheumatoid arthritis (RA) is a common systemic auto-immune disease, which is characterized by chronic and symmetry synovial inflammation. Crocin has been reported to exhibit anti-inflammatory effects in animal models. OBJECTIVE:This study investigates the anti-inflammatory and anti-arthritic effects of crocin on type II collagen-induced arthritis (CIA) in Wistar rats. MATERIALS AND METHODS:The CIA rat model was established and randomly divided into five groups with or without crocin treatment (10, 20 or 40?mg/kg), which was started on day 21 after arthritis induction and persisted for 36 days. The symptoms and molecular mechanisms of CIA and crocin-treated CIA rats were compared and investigated. RESULTS:CIA rats presented severe RA symptoms, including high arthritis score, paw swelling, joint inflammation, bone erosion, chondrocyte death, cartilage destruction, enhanced expressions of matrix metalloproteinase (MMP) and pro-inflammatory cytokines. However, crocin could mitigate these symptoms. Crocin (40?mg/kg) exhibited the most efficient therapeutic function on CIA rats: the histological scores of joint inflammation, bone erosion, chondrocyte death, cartilage surface erosion, and bone erosion of CIA rats receiving 40?mg/kg crocin treatment were comparable to the normal rats. MMP-1, -3 and -13 protein expression levels of CIA rats with 40?mg/kg crocin treatment were decreased to levels similar to normal rats. Moreover, crocin could also inhibit the expression of TNF-?, IL-17, IL-6 and CXCL8 in serum and ankle tissues of CIA rats. CONCLUSIONS:In summary, crocin exhibits therapeutic potential for RA, by mitigating the symptoms and inhibiting the pro-inflammatory factor expression.
Project description:The objective of the study was to elucidate the microRNA (miRNA) profile of an enriched human corneal epithelial stem cell (CESC) population in comparison to differentiated central corneal epithelial cells (CCECs) by small RNA sequencing. The CESCs were enriched by differential enzymatic treatment to isolate the basal limbal epithelial cells followed by laser capture microdissection of cells with nucleus to cytoplasm ratio ?0.7, from donor tissues. Small RNA sequencing was carried out using Illumina NextSeq. 500 platform and the validation of differentially expressed miRNAs by quantitative real-time PCR (qPCR) and locked nucleic acid miRNA in-situ hybridization (LNA-ISH). The sequencing identified 62 miRNAs in CESCs and 611 in CCECs. Six miRNAs: hsa-miR-21-5p, 3168, 143-3p, 10a-5p, 150-5p and 1910-5p were found to be significantly upregulated in enriched CESCs, which was further confirmed by qPCR and LNA-ISH. The expression of hsa-miR-143-3p was exclusive to clusters of limbal basal epithelial cells. The targets of the upregulated miRNAs were predicted to be associated with signaling pathways -Wnt, PI3K-AKT, MAPK and pathways that regulate pluripotency of stem cells, cell migration, growth and proliferation. Further studies are essential to elucidate their functional role in maintenance of stemness. The findings of the study also hypothesize the inherent potential of hsa-miR-143-3p to serve as a biomarker for identifying CESCs.
Project description:Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized with heterotopic ossification of the axis joints ligaments, resulting in joint disability. MicroRNAs (miRNAs) are regulators of mRNAs that play a crucial role in the AS pathological process. Here, we showed that the level of miR-17-5p was significantly higher in fibroblasts and ligament tissues from AS patients as compared to the non-AS individuals. Knockdown of the miR-17-5p from the fibroblasts derived from AS patients exhibited decreased osteogenic differentiation and ossification. On the other hand, AS patient-derived fibroblasts overexpressing miR-17-5p displayed the increased osteogenesis. Furthermore, inhibition of miR-17-5p ameliorated osteophyte formation, and the sacroiliitis phenotype in AS rats received emulsified collagen. Mechanistically, miR-17-5p regulated osteogenic differentiation by targeting the 3' UTR of ankylosis protein homolog (ANKH). Also, downregulation of miR-17-5p slowed AS progression through regulation of cytokines, such as dickkopf-1 (DKK1) and vascular endothelial growth factor (VEGF). In conclusion, our findings reveal a role of the miR-17-5p-ANKH axis in the regulation of heterotopic ossification, which is essential for therapeutic intervention in heterotopic ossification in AS.
Project description:Epidemiological studies show an association between rheumatoid arthritis (RA) and periodontal disease. Porphyromonas gingivalis (P.gingivalis) is a well-known pathogen in periodontitis. This study investigated the pathogenic effects of P.gingivalis on autoimmune arthritis in vivo. Collagen-induced arthritis (CIA) mice were intraperitoneally injected with W83 and 2561 strains of P.gingivalis. Infection with P.gingivalis exacerbated arthritis score in CIA mice. Synovial inflammation and bone destruction in CIA mice infected with P.gingivalis were more severe than in uninfected CIA mice. Both W83 and 2561 strains were more pro-arthritic after arthritis symptom was fully activated. Interestingly, only W83 strain was arthritogenic before autoimmune reaction initiated. Citrullination was detected in synovial tissue of CIA mice and CIA mice inoculated with P.gingivalis, but not in normal control mice. The citrullinated area was greater in P.gingivalis-infected CIA mice than in non-infected CIA mice. This study showed that P.gingivalis exacerbated disease in a mouse model of autoimmune arthritis and increased the expression of citrullinated antigens in the synovium. The arthritogenic effects of P.gingivalis were at least in part, dependent upon the bacterial strain with or without fimbriae expression, route and time of infection. P.gingivalis-mediated citrullination may explain the possible link between periodontal disease and RA.
Project description:Cysteine-rich 61 (Cyr61 or CCN1), a secreted protein from the CCN family, is an important proinflammatory cytokine. Migration and infiltration of mononuclear cells to inflammatory sites play a critical role in the pathogenesis of rheumatoid arthritis (RA). Monocyte chemoattractant protein-1 (MCP-1/CCL2) is the key chemokine that regulates migration and infiltration of monocytes. Here, we examined the role of CCN1 in monocyte migration, and CCL2 expression in osteoblasts. We found higher levels of CCN1 and CCL2 in synovial fluid from RA patients compared with levels from non-RA controls. We also found that the CCN1-induced increase in CCL2 expression is mediated by the MAPK signaling pathway and that miR-518a-5p expression was negatively regulated by CCN1 via the MAPK cascade. In contrast, inhibition of CCN1 expression with lentiviral vectors expressing short hairpin RNA ameliorated articular swelling, cartilage erosion, and infiltration of monocytes in the ankle joints of mice with collagen-induced arthritis. Our study describes how CCN1 promotes monocyte migration by upregulating CCL2 expression in osteoblasts in RA disease. CCN1 could serve as a potential target for RA treatment.
Project description:Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial tissue inflammation and joint destruction associated with the activation of angiogenesis. Exosomes, which play a role in cell-to-cell communication as carriers of genetic information, transfer microRNAs (miRNAs or miRs) between cells and have been studied as delivery vehicles for therapeutic molecules. The aim of the current study was to investigate the therapeutic effect of mesenchymal stem cell (MSC)-derived miR-150-5p exosomes on joint destruction in RA. The expression and secretion of miR-150-5p, matrix metalloproteinase (MMP) 14, and vascular endothelial growth factor (VEGF) in RA patients and fibroblast-like synoviocytes (FLS) were examined by quantitative RT-PCR, ELISA, and Western blotting. Immunohistochemistry was used to assess angiogenesis. MSCs were transfected with an miR-150-5p expression plasmid, and MSC-derived exosomes were harvested. The effect of MSC-derived miR-150-5p exosomes (Exo-150) on MMP14 and VEGF expression was examined. The effects of Exo-150 on cell migration and invasion in cytokine-stimulated FLS from RA patients were examined by HUVEC tube formation and transwell assays. The effect of Exo-150 in vivo was examined in a collagen-induced arthritis mouse model. Exo-150 decreased migration and invasion in RA FLS and downregulated tube formation in HUVECs by targeting MMP14 and VEGF. Injection of Exo-150 reduced hind paw thickness and the clinical arthritic scores in collagen-induced arthritis mice. Exo-150 reduced joint destruction by inhibiting synoviocyte hyperplasia and angiogenesis. Exosomes facilitate the direct intracellular transfer of miRNAs between cells and represent a potential therapeutic strategy for RA.
Project description:SCOPE:We investigate the postprandial modulation of cardiovascular-related microRNAs elicited by extra virgin olive oil (EVOOs) containing different levels of their own polyphenols. METHODS AND RESULTS:It is randomized, postprandial, parallel, double-blind study. Twelve healthy participants consumed 30 mL of EVOO containing low (L-EVOO; 250 mg total phenols kg-1 of oil), medium (M-EVOO; 500 mg total phenols kg-1 of oil), and high (H-EVOO; 750 mg total phenols kg-1 of oil) enriched EVOOs. Postprandial plasma microRNAs levels are analyzed by real-time quantitative PCR. The results show that L-EVOO intake is associated with decreased let-7e-5p and miR-328a-3p levels and increased miR-17-5p and miR-20a-5p, concentrations. M-EVOO decreases plasma let-7e-5p and increases miR-17-5p, miR-20a-5p, and miR-192-5p levels. Finally, H-EVOO decreases let-7e-5p, miR-10a-5p, miR-21-5p, and miR-26b-5p levels. CONCLUSION:During the postprandial state, the levels of let-7e-5p decrease with EVOO regardless of polyphenol content suggesting a general response to the fatty acid composition of EVOO or/and the presence of at least 250 mg polyphenol kg-1 olive oil. Moreover, the miR-17-92 cluster increases by low and medium polyphenol content suggesting a role in fatty acid metabolism and nutrient sensing. Thus, postprandial modulation of circulating microRNAs levels could be a potential mechanism for the cardiovascular benefits associated with EVOO intake.
Project description:Background:Cinnamomum cassia iswidely used as a traditional medicinal plant for the treatment of rheumatoid arthritis. Objective:The present study aimed to assess the anti-arthritic activity of C. cassia bark hydroalcoholic extract (CCHE) in different arthritic animal models. Methods:In formaldehyde model, sub-plantar administration of 0.1?ml of formaldehyde (2% v/v) into the right hind paws of Wistar albino rats on days 0 and 3. The rats were divided into six groups as follows: normal control, disease control, indomethacin group (3?mg/kg, p.o.) and three groups, treated with 50, 100 and 200?mg/kg CCHE (p.o.). Joint diameter was measured, and ankle joints were collected for MDA and GSH measurements. In complete Freund's adjuvant (CFA)-induced arthritis model, CFA was injected into the sub-plantar surface of the right hind paw in rats. Joint diameter was measured, and serum TNF-? and IL-1? were measured. Histopathological and immunohistochemical analyses were also performed. Results:CCHE treatment significantly (p?<?0.01) reduced MDA levels and joint swelling in a concentration-dependent manner in rats with formaldehyde-induced arthritis, in which GSH levels were elevated (p?<?0.01). In rats with CFA-induced arthritis, CCHE treatment significantly reduced joint swelling as well as IL-1? and TNF-? levels (p?<?0.01). TNF-? receptor expression was decreased in rats treated with indomethacin or CCHE. Conclusion:Based on these findings, it can be concluded that C. cassia possesses anti-arthritic properties.