Screening Of Mirnas Expression In Ageing Skin Of Mice And Study On Cytobiology Of MiR-302b-3p

Objectives:Model of D-galactose-induced senescence was performed on female Balb/c mice to unveil the differentially expressed miRNAs between ageing group and control group.then the target genes and cell signaling pathways of miRNAs were predicted by searching mi RNA-related databases and DIANA-mirPath database.The target genes and differentially expressed miRNAs were furtherly verified on the primary skin fibroblasts to provide drug targets that may delay skin ageing.Methods:1.D-galactose was subcutaneously injected on female mice for 3 months by 100mg/kg,and control group were injected with equal saline;Skin was isolated from the bottom right of mice,then soaked into the liquid nitrogen for a few minutes immediately.Part of skin were preserved at-80~0C,the other parts were used to perform micro RNA array.2.The target genes and pathways were predicted and utilized by targetscan,miRwalk2.0,DIANA-Tarbase and DIANA-mirPath databases;then miRNA-gene-network was visualized by cytoscape.3.Primary skin fibroblasts were isolated from mice by type I collagenase digestion.The senescent primary cells were induced by 20g/L d-galactose;theprimaryskinfibroblastswereverifiedbycellular immunohistochemistry;Senescence?-Galactosidase Staining Kit was used to detect cell senescence;RT-qPCR was used to verify and measure the expression of mi RNAs and mRNA expression of target genes.4.MiRNA-302b-3p mimics and inhibitor were transfected into primary fibroblasts by liposome;Western blot was used to qualify the expression of JNK2,p-c-jun,stat3,Akt,Bim,Bcl-2 and Gsk-3?after transfection of miRNA-302b-3p mimics and inhibitors;Western blot was used to quatify the expression of JNK2,p-c-jun,stat3,Akt,Bim,Bcl-2 and Gsk-3?after interference of JNK2 by JNK2 siRNA5.Dual-luciferase reporter assay was performed to verify the relationship between mi RNA-302b-3p and JNK2.Results:The results of mi RNA array showed there were 29 differentially expressed miRNAs,including 22 up-regulated miRNAs and 7 down-regulated mi RNAs.miRNA-gene network indicated that miR-139-3p,184-3p,186-3p,208a-5p,291a-5p,291b-5p,302b-3p,326-3p,467c-3p,487b-3p,742-5p,92b-3p closely related with JNK2,Akt1/2/3,Pak7,Trps1,Bcl2l11,Ikzf2.DIANA-mirPath showed the top 10 important signaling pathways were Focal adhesion,Insulin,Regulation of actin cytoskeleton,PI3K-Akt,MAPK,pathway in cancer,Hepatitis B,Axon guidance,Calcium and Wnt signaling pathway.Different expression of 12 miRNAs between control and d-galactose-induced fibroblasts was consistent with the result of miRNA array,and the up-regulated expression of miR-302b-3p was most obvious.The changes of JNK2 and Akt were consistent with prediction when overexpression or knockdown of miR-302b-3p in fibroblasts.The changes of Bcl-2,Bim,Gsk-3?corresponded with the Akt expression.the changes of p-c-jun,Stat3 corresponded with the JNK2expression.Replicative-induced ageing model showed that the changes of JNK2,Gsk-3?,Bcl-2,Bim,p-c-jun and Stat3 were totally consistent with d-galactose-induced ageing model.Conclusion:1.Expression profile of miRNAs showed that there were 29 differentially expressed miRNAs,22 miRNAs were up-regulated and 7 mi RNAs were down-regulatd.And the different expression of miR-302b-3p was most obvious.2.the mechanism of miRNA-302b-3p influencing ageing skin maybe was:1.inhibiting JNK2 and JNK2 signaling pathway;2.Inhibiting AKT and AKT signaling pathway.