Role Of MiR-125b-5p In UVB-induced Autophagy In SLE

Objective:To detect the effect of UVB on PBMC autophagy and miRNA expression in Jurkat cells and SLE patients.To screen and identify target gene of miR-125b-5p.To investigate the effects of UVB irradiation on autophagy after regulating the expression of miR-125b-5p and its target gene.To explore the role of UVB in pathogenesis of SLE through autophagy and the role of miRNA in this process and its regulatory mechanism.Methods:1.Jurkat cells were exposed to 0-200 mj/cm2 UVB.The expressions of LC3,Beclin-1 and miR-125b-5p were detected by Western-blotting,qPCR and laser scanning confocal fluorescence microscope at 12h,24h and 48h after exposed to UVB.2.PBMCs of SLE patients and normal individuals were irradiated with 50 mj/cm2 UVB.The expressions of autophagy pathway genes(LC3,Beclin-1)were detected by Western-blotting and qPCR at 24h after irradiation.The specifically expressed mRNAs and miRNAs were screened by RNA-seq and miRNA microarray then identified by Western-blotting and qPCR.3,Using bioinformatics teconologies to find autophagy related target gene of miR-125b-5p,and detect its expression in PBMCs of SLE patients by Western-blotting and qPCR.After transfection with miR-125b-5p agomir/antagomir,the effect of upregulation/downregulation miR-125b-5p on target gene was detected by Western-blotting and qPCR.After co-transfecting miR-125b-5p mimic and luciferase miRNA expression reporter vector,exam luciferase ratio to confirm the target site on 3'UTR of the target gene.4.Jurkat cells were transfected with miR-125b-5p agomir/antagomir,UVRAG overexpression vector/siRNA and irradiated with UVB.The expressions of autophagy pathway genes(LC3,Beclin-1,ATG 7 and UVRAG)were detected by Western-blotting,qPCR and laser scanning confocal fluorescence microscope.Results:1.When the dose of UVB was less than 50mj/cm2,the autophagy activation increased with the increase of dose.When the dose of UVB was more than 50mj/cm2,the autophagy activation decreased gradually.The highest autophagy activation was observed in 50mj/cm2 dose group.2.The expression of miR.125b-5p was negatively correlated with the irradiation dose when the irradiation dose was less than 50mj/cm2,while the level of miR-125b-5p was positively correlated with the irradiation dose when the irradiation dose was greater than 50mj/cm2.The expression of miR-125b-5p was the lowest when the irradiation dose was 50mj/cm2.3.There was no significant difference in the expression of autophagy-related genes(Beclin-1,LC3?,ATG 7,UVRAG)in PBMCs from SLE patients and normal controls when UVB was not irradiated.The expression of autophagy-related genes(Beclin-1 LC3?,ATG 7 and UVRAG)in PBMCs of SLE patients was significantly increased after UVB exposure,while normal controls only showed mild changes.4.The expression of miR-125b-5p in PBMCs without UVB in SLE patients was lower than that in normal controls.The expression level of miR-125b-5p in PBMCs of SLE patients and normal controls after UVB irradiation was lower than that before irradiation,and the expression of miR-125b-5p was the lowest in SLE-UVB group compared with other groups.5.The overexpression of miR-125b-5p can reduce the expression of UVRAG,and the inhibition of miR-125b-5p can increase the expression of UVRAG miR-125b-5p down-regulates UVRAG mRNA and protein expression by binding to the target site in 3'UTR of UVRAG 6.Downregulation of miR-125b-5p or upregulation of UVRAG can increase autophagy activation in Jurkat cells;upregulation of miR-125b-5p or downregulation of UVRAG can reduce autophagy activation in Jurkat cells.Conclusion:UVB can induce autophagy in Jurkat cells and regulate the expression of miR-125b-5p,and its effect is related to radiation dose.The UVB downregulates the expression of miR-125b-5p in PBMCs in patients with systemic lupus erythematosus,and up-regulates the expression of UVRAG,thereby promoting autophagy.