Brucine alleviates fibroblast-like synoviocytes dysfunction and inflammation by regulating YY1 during rheumatoid arthritis.

Brucine is a weak alkaline indole alkaloid with wide pharmacological activities and has been identified to protect against rheumatoid arthritis (RA) process. Circular RNAs (circRNAs) are also reported to be involved in the pathogenesis of RA. Here, we aimed to probe the role and mechanism of Brucine and circ_0139658 in RA progression. The fibroblast-like synoviocytes of RA (RA-FLSs) were isolated for functional analysis. Cell proliferation, apoptosis, invasion, migration, as well as inflammatory response were evaluated by CCK-8 assay, EdU assay, flow cytometry, transwell assay, and ELISA analysis, respectively. qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. The binding between miR-653-5p and circ_0139658 or Yin Yang 1 (YY1), was verified using dual-luciferase reporter and RNA pull-down assays. Brucine suppressed the proliferation, migration, and invasion of RA-FLSs, and alleviated inflammation by reducing the release of pro-inflammatory factors and macrophage M1 polarization. RA-FLSs showed increased circ_0139658 and YY1 levels and decreased miR-653-5p levels. Circ_0139658 is directly bound to miR-653-5p to regulate YY1 expression. Brucine treatment suppressed circ_0139658 and YY1 expression but increased YY1 expression in RA-FLSs. Functionally, circ_0139658 overexpression reversed the suppressing effects of Brucine on RA-FLS dysfunction and inflammation. Moreover, circ_0139658 silencing alleviated the dysfunction and inflammation in RA-FLSs, which were reverted by YY1 overexpression. Brucine suppressed the proliferation, migration, invasion, and inflammation in RA-FLSs by decreasing YY1 via circ_0139658/miR-653-5p axis.

Zhang Q ，Wang G ，Xu B 《-》

m(6)A-mediated lncRNA MAPKAPK5-AS1 induces apoptosis and suppresses inflammation via regulating miR-146a-3p/SIRT1/NF-κB axis in rheumatoid arthritis.

Wen J ，Liu J ，Wan L ，Jiang H ，Xin L ，Sun Y ，Fang Y ，Wang X ，Wang J ... -  《-》

Circ_0083964 knockdown impedes rheumatoid arthritis progression via the miR-204-5p-dependent regulation of YY1.

Rheumatoid arthritis (RA) is a chronic inflammatory disease. Abnormal proliferation and inflammation of fibroblast-like synoviocytes (FLSs) are the main pathological features of the disease. Accumulating studies have identified that circular RNAs (circRNAs) were involved in the progression of RA. Our study was to assess the function and mechanism of circ_0083964 in RA. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were utilized to test the level of circ_0083964, miR-204-5p and YY1. Counting Kit-8 (CCK-8) assay, EdU assay, flow cytometry, transwell assay and wound-healing assay were utilized to test cell viability, proliferation, apoptosis, invasion and migration. Cell inflammation was estimated with enzyme-linked immunosorbent assay (ELISA) kits. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to identify the target relationship between miR-204-5p and circ_0083964 or YY1. Circ_0083964 was highly expressed in RA synovial tissues and RA-FLSs. Circ_0083964 downregulation constrained proliferation, metastasis and inflammation and facilitated apoptosis in RA-FLSs. Furthermore, circ_0083964 served as a sponge of miR-204-5p, and rescue experiments proved that miR-204-5p deficiency overturned the suppressive impacts of circ_0083964 silencing on RA-FLSs progression. Additionally, we also verified that YY1 could be targeted by miR-204-5p, and its overexpression rescued the repressive impact of miR-204-5p introduction on RA-FLSs development. Besides that, we revealed that circ_0083964 mediated YY1 expression by regulating miR-204-5p. Circ_0083964 inhibition confined RA development by sponging miR-204-5p to hamper the YY1 level, which will provide a theoretical basis for the treatment of RA.

Xiang L ，Yang W ，Wang F ，Liu G ... -  《Journal of Orthopaedic Surgery and Research》

Circular RNA circ_0008360 Inhibits the Proliferation, Migration, and Inflammation and Promotes Apoptosis of Fibroblast-Like Synoviocytes by Regulating miR-135b-5p/HDAC4 Axis in Rheumatoid Arthritis.

Circular RNAs (circRNAs) have been demonstrated to play crucial roles in the development and progression of many diseases, including rheumatoid arthritis (RA). However, the functions and molecular mechanism of circ_0008360 in RA remain unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of circ_0008360, microRNA-135b-5p (miR-135b-5p), and histone deacetylase 4 (HDAC4). Cell Counting Kit-8 (CCK-8) assay, wound healing assay, and flow cytometry analysis were performed to assess cell proliferation, migration, and apoptosis, respectively. Inflammatory response was evaluated by enzyme-linked immunosorbent assay (ELISA). The interaction between miR-135b-5p and circ_0008360 or HDAC4 was predicted by bioinformatics analysis and verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) and RNA pull-down assays. Western blot assay was used to detect the protein expression of HDAC4 and proliferating cell nuclear antigen (PCNA). The expression of circ_0008360 was downregulated in RA synovial tissues and RA fibroblast-like synoviocytes (RA-FLSs). Circ_0008360 suppressed the proliferation, migration, and inflammation and promoted apoptosis of RA-FLSs, and circ_0008360 knockdown showed opposite effects. Moreover, miR-135b-5p was a direct target of circ_0008360, and miR-135b-5p could reverse the effects of circ_0008360 on proliferation, migration, inflammation, and apoptosis in RA-FLSs. Furthermore, HDAC4 was a downstream target of miR-135b-5p, and miR-135b-5p accelerated the proliferation, migration, and inflammation and suppressed apoptosis of RA-FLSs by targeting HDAC4. In addition, circ_0008360 positively regulated HDAC4 expression by sponging miR-135b-5p. Circ_0008360 inhibited the proliferation, migration, and inflammation and facilitated apoptosis of RA-FLSs by sponging miR-135b-5p and upregulating HDAC4, providing a potential target for prevention and treatment of RA.

Hao J ，Chen Y ，Yu Y 《-》

ALKBH5-Mediated RNA m(6) A Methylation Regulates the Migration, Invasion, and Proliferation of Rheumatoid Fibroblast-Like Synoviocytes.

Fibroblast-like synoviocytes (FLSs) are critical for promoting joint damage in rheumatoid arthritis (RA). N6 -methyladenosine (m6 A) modification plays key roles in various diseases, but its role in the pathogenesis of RA is largely unknown. Here, we investigate increased demethylase ALKBH5 promotion of proliferation, migration, and invasion of RA FLSs via regulating JARID2 expression. ALKBH5 expression in FLSs was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. 5-ethynyl-2'-deoxyuridine, scratch wound healing, and transwell assays were implemented to determine the role of ALKBH5 on RA FLS proliferation, mobility, and migration. Then, m6 A sequencing combined with RNA sequencing was performed to identify the potential targets of ALKBH5. RNA immunoprecipitation and RNA pulldown were then used to validate the interaction between the protein and messenger RNA (mRNA). Collagen-induced arthritis (CIA) and delayed-type hypersensitivity arthritis (DTHA) models were further established to assess the therapeutic potency of ALKBH5 in vivo. We demonstrated that ALKBH5 expression was increased in FLSs and synovium from RA. Functionally, ALKBH5 knockdown inhibited the proliferation, migration, and invasion of RA FLSs, whereas overexpression of ALKBH5 displayed the opposite effect. Mechanistically, ALKBH5 mediated m6 A modification in the JARID2 mRNA and enhanced its mRNA stability in cooperation with IGF2BP3. Intriguingly, the severity of arthritis was attenuated in mice with DTHA and ALKBH5 knockout or rats with CIA and intra-articular injection of ALKBH5 short hairpin RNA. Our findings suggest that ALKBH5-mediated m6 A modification is crucial for synovial hyperplasia and invasion in RA. ALKBH5 might be a potential therapeutic target for RA and even for dysregulated fibroblasts in a wide range of diseases.

Kuang Y ，Li R ，Wang J ，Xu S ，Qiu Q ，Lin S ，Liu D ，Shen C ，Liu Y ，Xu M ，Lin W ，Zhang S ，Liang L ，Xu H ，Xiao Y ... -  《-》