IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

SMSCs-derived sEV overexpressing miR-433-3p inhibits angiogenesis induced by sEV released from synoviocytes under triggering of ferroptosis.

BACKGROUND: Ferroptosis is characterized by accumulation of lipid peroxides that leads to oxidative stress. In progressive rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) suffered from oxidative stress induced by generation of excess reactive oxygen species (ROS) and survived from elevated lipid oxidation. However the phenomenon of abnormal synovial fibroblasts proliferation under ferroptotic stress remain to be explained and the effects of this event on disease progression of RA need to be investigated. METHODS: FLS from RA patients (RA-FLS) were stimulated with LPS as an inflammatory model in vitro, and simultaneously treated with ferroptosis inducer Erastin/RSL3 or inhibitor ferrostatin-1. Besides, small extracellular vesicles (sEV) from the supernatant of RA-FLS culture under Erastin/RSL3 management were isolated. The degree of ferroptosis in cells were evaluated by Lipid-ROS detection via flowcytometry and ferroptosis marker protein expression determined by western bloting. The expression of core component of ESCRT-III CHMP4A and CHMP5 was determined by western bloting, and knockdown of CHMP4A was further performed to detect the influence of ESCRT-III complex on ferroptosis as well as LPS/Erastin induced sEV (LPS/Erastin-sEV) releasing. Moreover, miR-433-3p level in the isolated sEV was evaluated by RT-qPCR and interaction of miR-433-3p with FOXO1/VEGF axis were evaluated. MiR-433-3p was overexpressed in synovial mesenchymal stem cells (SMSCs) via miR-433-3p mimics transfection. RA-FLS was co-cultured with human dermal microvascular endothelial cells (HDMECs). LPS/Erastin-sEV or sEV derived from miR-433-3p-overexpressing SMSCs (miR-433-3p-SMSCs-sEV) were added to the co-culture system, and supernatants from co-culture without sEV were given to HDMECs. Angiogenic activity of HDMECs were identified by transwell test and endothelial tube formation analysis. Erastin-sEV and miR-433-3p-SMSCs-sEV were also administrated in collagen-induced arthritis (CIA) mouse model respectively, and progression of arthritis were evaluated. RESULTS: Ferroptosis of RA-FLS was triggered by LPS/Erastin and accompanied with increased expression of ESCRT-III core components as well as elevated release of sEV from RA-FLS. HDMECs' migration and tube formation in vitro was significantly induced/suppressed by supernatants from co-culture under management of Erastin-sEV/miR-433-3p-SMSCs-sEV due to varied VEGF expression regulated by miR-433-3p targeting FOXO1. MiR-433-3p-SMSCs-sEV could inhibit the Erastin-sEV promoted VEGF expression and mitigated arthritis severity. CONCLUSION: Erastin-sEV could aggravate synovial angiogenesis and promote arthritis progression. Administration of miR-433-3p-SMSCs-sEV may be a potential novel therapeutic method as significant antagonism to Erastin-sEV for RA treatment.

Therapeutic Potential of Exosomal circRNA Derived from Synovial Mesenchymal Cells via Targeting circEDIL3/miR-485-3p/PIAS3/STAT3/VEGF Functional Module in Rheumatoid Arthritis.

BACKGROUND: Synovial inflammation and its associated activation of angiogenesis play critical roles in rheumatoid arthritis (RA). Exosomes, as carriers of genetic information including circular RNAs (circRNAs), have been explored as delivery vehicles for therapeutic molecules. However, the effects of synovial mesenchymal stem cells (SMSCs)-derived exosomal circRNAs and their mechanisms of action in RA progression remain unclear. METHODS: SMSCs-derived exosomes (SMSCs-Exos) were administered to a co-culture of RA fibroblast-like synoviocytes (RA-FLS) and human dermal microvascular endothelial cells (HDMECs) in vitro as well as to a collagen-induced arthritis (CIA) mouse model in vivo. Their effects on VEGF expression and angiogenic activity in vitro and the therapeutic efficacy in vivo were evaluated. Exosomes from circEDIL3-overexpressing SMSCs (Ad-circEDIL3-SMSCs-Exos) were used to further determine the role of circEDIL3 in SMSCs-Exo-based therapy. RESULTS: Both SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos significantly downregulated the expression of VEGF induced by the IL-6/sIL-6R complex in the supernatants of RA-FLS and HDMECs co-culture as well as in the cell lysate of co-cultured RA-FLS, and the extent of reduction was more pronounced in the latter. Subsequent experiments showed that angiogenic activity was significantly downregulated by SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos due to reduced VEGF expression. CircEDIL3 functioned as a sponge for miR-485-3p, which targeted PIAS3. PIAS3 is known to suppress STAT3 activity and reduce downstream VEGF. Injection of SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos reduced synovial VEGF and consequently ameliorated arthritis severity in the CIA mouse model. CONCLUSION: The intracellular transfer of circEDIL3 by SMSCs-Exos may be a potential novel therapeutic strategy for RA.

Angiogenesis is Inhibited by Arsenic Trioxide Through Downregulation of the CircHIPK3/miR-149-5p/FOXO1/VEGF Functional Module in Rheumatoid Arthritis.

Angiogenesis is a crucial event in the pathogenesis of rheumatoid arthritis (RA). Arsenic trioxide (ATO, As2O3) has been reported to inhibit synovial angiogenesis via the vascular endothelial growth factor (VEGF)-centered functional module. However, the exact mechanisms of ATO on VEGF modulation remain unclear. Circular RNAs (circRNAs) are emerging as important regulators in RA, and the detailed mechanisms remain largely unknown. Here, we reported a circRNA (circHIPK3), the expression of which was significantly increased in RA fibroblast-like synoviocytes (RA-FLS) after TNF-α induction. Moreover, VEGF content in the supernatants of a RA-FLS and human dermal microvascular endothelial cell (HDMEC) co-culture as well as in RA-FLS co-cultured was significantly elevated in accordance with circHIPK3 levels. This increased VEGF expression may significantly upregulate endothelial tube formation and transwell migration, as well as microvessel sprouting in the ex vivo aortic ring assay. CircHIPK3 was further illustrated to be a sponge for the forkhead box transcription factor O1 (FOXO1)-targeting miR-149-5p, leading to the changing expression of the downstream VEGF. These networked factors mainly form a functional module regulating angiogenesis in RA-FLS, and the expression of this functional module could be significantly downregulated by ATO with a consistently reduced vascularity in vitro. In the collagen-induced arthritis (CIA) mice model, an intra-articular injection of the adeno-associated virus-si-circHIPK3 or ATO was demonstrated to alleviate the synovial VEGF expression and arthritis severity respectively. Thus, we elucidate a previously unknown mechanism between circRNAs and RA, and ATO has a significant protective effect on RA-FLS and CIA synovium via its inhibition of the angiogenic functional module of circHIPK3/miR-149-5p/FOXO1/VEGF, suggesting great potential for the combination therapy of ATO with circHIPK3 silencing.

Tobacco NtabSPL6-2 can enhance local and systemic resistances of Arabidopsis thaliana to bacterial and fungal pathogens.

NtabSPL6-2 of Nicotiana tabacum was introduced into Arabidopsis by Agrobacterium-mediated floral-dip method. Compared to wild-type Col-0 plants, the arrangement of cauline leaves in NtabSPL6-2 transgenic plants was converted into opposite from simple and alternate, and the margin of rosette leaves was serrated. NtabSPL6-2 transgenic plants possessed a significantly greater fresh weight. Subcellular localization by fusion with GFP confirmed that the encoded product of NtabSPL6-2 existed in the nucleus. The leaves of NtabSPL6-2 transgenic plants exhibited an enhanced capacity to restrain the bacterial reproduction after infection by Pseudomonas syringae, accompanied by higher expression of the pathogenesis-related gene PR1 in the infiltrated leaves, indicating NtabSPL6-2 could improve the defense response of Arabidopsis to P. syringae at the local sites. Similarly, it was confirmed that NtabSPL6-2 could enhance the systemic acquired resistance of Arabidopsis in response to P. syringae. In addition, the area of necrotic plaque appearing on the transgenic leaves inoculated with Botrytis cinerea was smaller and accompanied by an upregulation of PR1 and PR5, indicating NtabSPL6-2 transgenic leaves were less susceptible to the fungal pathogen. Moreover, there was less accumulation of reactive oxygen species (H2O2 and O2-) and malondialdehyde in the local infected sites of transgenic plants, whereas the wild-type Col-0 plants were more oxidatively injured after infestation by B. cinerea.