Mir - 873 Control Mouse Astrocytes Induced By Il - 17 Produce Inflammation And Study The Mechanism Of Chemokines

Objective: To investigate the expression of several inflammatory cytokines andchemokines including regulatory proteins, and analysis of microRNA profiles inastrocytes stimulated with IL-17and in the brain tissue of mice with EAE. Methods:The production of interleukin-6(IL-6), tumor necrosis factor-α (TNF-α), macrophageinflammatory protein-2(MIP-2), monocyte chemotactic protein-1(MCP-1) andMCP-5in the cultured astrocytes of mice stimulated with IL-17(50ng/ml) at differenttimes (3h,6h,12h,24h,48h) was detected by real-time PCR and ELISA. Meanwhile,the expression of A20(also known as tumor necrosis factor α inducible protein3,TNFAIP3) and p-NF-B/p65in astrocytes upon IL-17stimulation was also examinedusing Western blot. Furthermore, miRNA profiles both in the cultured astrocytesstimulated with IL-17(3h,6h) and in the brain tissues of mice with experimentalautoimmune encephalomyelitis (EAE)(14d,20d) were determined by miRNA chips.And the regulatory miRNAs of A20were further identified using Western blot andluciferase assay, respectively. Results:(1) Compared with control group, mRNA andprotein levels of IL-6, TNF-α, MIP-2and MCP-1/5in astrocytes at6h after IL-17stimulation increased significantly, and then reached peak at12h;(2) At the sametime, the expression of A20protein in atrocytes with IL-17stimulation showed aremarkable decrease, whereas the phosphorylation level of NF-B (p-NF-B/p65) displayed significant increase;(3) The miRNA chips revealed that the expression of98miRNAs were up-regulated (at least higher1.5-fold than control) and62miRNAs were down-regulated (at least lower0.5-fold than control) in astrocytestreated with IL-17stimulation for3h and6h. Moreover,90miRNAs wereup-regulated (at least higher1.5-fold than control) and42miRNAs weredown-regulated (at least lower0.5-fold than control) in the brain tissues of EAEmice (at14d and20d). Meantime,36miRNAs were co-upregulated both inastrocytes and in the brain tissue of EAE mice.(4) Analysis of theseup-regulated miRNAs found that the miRNAs namely miR-323-5p, miR-763,miR-141, miR-139-5p and miR-873could bind to wild type (WT) A203’-UTR. Andastrocytes transfected with these miRNAs (miR-323-5p, miR-763, miR-873) showedobvious reduction of A20protein including luciferase reporter activity ofpGL3-Promoter/WT A203’-UTR, especially miR-873. Conclusion: Astrocytesstimulated with IL-17can result in the production and secretion of IL-6, TNF-α,MIP-2and MCP-1/5, including the decrease of A20and the increase of p-NF-B/p65expression. Additionally, miR-323-5p, miR-763and miR-873can decrease theexpression of A20protein and the luciferase activity of pGL3-Promoter/WT A203’-UTR, in particularly miR-873. Objective: To elucidate the regulatory mechanism of miR-873in the production ofseveral inflammatory cytokines and chemokines in astrocytes stimulated with IL-17in vitro. Methods: Astrocytes of C57BL/6mice were cultured and transfected withmiR-873and LNA-anti-miR-873(miR-873inhibitor) for48h, then these cells werestimulated with IL-17(50ng/ml) for6h, and mRNA and protein levels of IL-6, TNF-α,MIP-2and MCP-1/5were determined by real-time PCR and ELISA. At the sametime, the levels of A20and p-NF-B/p65proteins in astrocytes transfected withmiR-873and LNA-anti-miR-873were also measured using Western blot.Additionally, to identify whether A20is a target of miR-873, mutations withinseed sequence’s of miR-873(Mut-miR-873) and the mutant A203’-UTRcomplementary to the seed region of miR-873of luciferase reporter vector(pGL3-Promoter/Mut A20) were constructed and transfected into the culturedastrocytes, then the luciferase activity of A203’-UTR was detected. Moreover,to silence A20gene, small hairpin RNAs of A20(A20shRNA) were synthesisedand transfected into astrocytes. Thereafter, the production of IL-6, TNF-α, MIP-2and MCP-1/5and the expression of p-NF-B/p65were determined. Furthermore,astrocytes were also pretreated with PDTC (an inhibitor to NF-B) for2hfollowed by IL-17stimulation for6h, and then the content of inflammatory cytokinesand chemokines mentioned-above were measured. Results:(1) Astrocytes transfectedwith miR-873could markedly increase the mRNA and protein levels of IL-6, TNF-α,MIP-2and MCP-1/5, but astrocytes with LNA-anti-miR-873transfection could decrease these cytokines production. Meanwhile, astrocytes transfected withmiR-873could remarkbly reduce the expression of A20and enhance theexpression of p-NF-B/p65.(2) When WT-miR-873and pGL3-Promoter/WT A20were co-transfected into mice astrocytes, the luciferase activity ofpGL3-Promoter/WT A203’-UTR was prominently attenuated, but astrocytes withMut-miR-873transfection had no this effect on luciferase activity ofpGL3-Promoter/WT A20. Meantime, the activity of pGL3-Promoter/Mut A203’-UTR in astrocytes co-transfected with WT-miR-873or Mut-miR-873andpGL3-Promoter/Mut A20was not diminished.(3) Astrocytes with A20shRNAtreatments could markedly increase the secretion of inflammatory cytokines andchemokines mentioned-above including p-NF-B/p65expression. At the same time,the levels of these factors were also down-regulated in astrocytes treated withPDTC. Conclusion: miR-873can regulate the production of IL-6, TNF-α, MIP-2and MCP-1/5in astrocytes induced by IL-17stimulation via regulating A20andp-NF-B/p65expression. Objective: To explore the effects of miR-873on regulating the inflammatory changesof the mice with EAE in vivo. Methods: The model of EAE in C57BL/6mice wasfirst established by injection of MOG35-55antigen, and then the levels of mRNA andprotein of miR-873, IL-17, IL-6, TNF-α, MIP-2and MCP-1/5in the brain tissues andin the sera of EAE mice were measured using real-time PCR and ELISA. At the sametime, the levels of A20and pNF-kB/p65protein in the brain tissue of the mice withEAE were detected using Western blot. In addition, the expression vectors ofLV-miR-873and LV-sponge were constructed and injected into the mice through tailvein. Thereafter, the production of inflammatory cytokines and chemokinesmentioned-above from the EAE mice treated with LV-miR-873and LV-sponge wasalso determined by real-time PCR and ELISA. And the levels of A20andp-NF-kB/p65in EAE mice with LV-miR-873or LV-sponge pre-treatment wereexamined using Western blot and immunohistochemistry. Furthermore, the infiltrationof inflammatory cells in the spinal cord including myelin sheath injury of the EAEmice given with the different vectors in advance was observed with H&E or luxol fastblue (LFB) staining under light microscope (LM). Meanwhile, the changes ofmedullary sheath ultrastructure in the mice treated with LV-miR-873or LV-spongewere observed by electron microscope (EM). Results:(1) The levels of miR-873,IL-17, IL-6, TNF-α, MIP-2and MCP-1/5from the mice with EAE were increasedobviously, even on day20(20d) after the EAE model reproduction. And theexpression of A20and p-NF-kB/p65protein in the brain of mice with EAE decreasedor increased significantly, respectively.(2) The content of IL-6, TNF-α, MIP-2and MIP-1/5in the mice treated with LV-miR-873elevated remarkably, and theexpression of A20or p-NF-kB/p65protein down-regulated or up-regulated both inthe brain and spinal cord of EAE mice treated with LV-miR-873, separately.(3) Thenumbers of inflammatory cells in the spinal cord of EAE mice given withLV-miR-873were more than those of other mice, but not in the mice given withLV-sponge under LM. Besides, severe damages in the medullary sheath of the spinalcord in EAE mice pre-treated with LV-miR-873was shown. And the medullarysheath of EAE mice with LV-miR-873infection was found to be ruptured anddisintegrated while only mild loose of the medullary sheath was seen in EAE micetreated with LV-sponge. Conclusion: miR-873, IL-17, IL-6, TNF-α, MIP-2andMIP-1/5can be up-regulated in EAE mice, but the A20expression in mice isdown-regulated. Overexpression of miR-873can not only raise the production ofinflammatory cytokines, chemokines described previously and p-NF-kB/p65, but alsoincrease the infiltration of inflammatory cells and the injury of medullary sheath inmice with EAE. Moreover, knockdown of miR-873in EAE mice treated withLV-sponge in advance can obviously inhibit the infiltration of inflammatory cellsand demyelination of the spinal cord, indicating that miR-873is a key factor tocontrol the inflammatory changes of mice with EAE.