Résumé
Objective:To explore the mechanism underlying microRNA (miR) -125a-mediated inhibition of proliferation of keratinocytes.Methods:After 24-hour pretreatment with interleukin (IL) -23, human HaCaT keratinocytes were divided into miR-125a group and miR-NC group transfected with a miR-125a overexpression plasmid and a control plasmid, respectively. Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative ability of HaCaT cells in the two groups at 0, 24, 48 and 72 hours after transfection, real-time fluorescence-based quantitative PCR to determine the mRNA expression of miR-125a and IL-23 receptors (IL-23R) in the two groups 24 hours after transfection, and Western blot analysis to determine the protein expression of IL-23R, Janus kinase 2 (JAK2) , protein kinase B (AKT) and phosphorylated AKT (p-AKT) in the two groups 48 hours after transfection. Dual-luciferase reporter assay was performed to verify the targeting relationship between miR-125a and IL-23R. Comparison of means between two groups was carried out by using t test, and changes in the proliferative ability of HaCaT cells over time were evaluated by using repeated measures analysis of variance. Results:After plasmid transfection, the relative expression of miR-125a was significantly higher in the miR-125a group (6.377 ± 0.745) than in the miR-NC group (0.700 ± 0.222; t=7.305, P=0.002) . At 0, 24 and 48 hours after transfection, there was no significant difference in cellular proliferative ability between the miR-125a group and the miR-NC group ( t=0.663, 0.623 and 1.930, respectively, all P > 0.05) ; at 72 hours after transfection, the cellular proliferative ability was significantly lower in the miR-125a group than in the miR-NC group ( t=4.407, P < 0.05) . The IL-23R mRNA expression was significantly lower in the miR-125a group than in the miR-NC group ( t=3.082, P < 0.05) . Compared with the miR-NC group, the miR-125a group showed significantly decreased protein expression of IL-23R, JAK2 and p-AKT ( t=11.715, 6.996, 12.424, P < 0.001,=0.002, < 0.001, respectively) . Dual-luciferase reporter assay showed targeted binding of miR-125a to IL-23R. Conclusion:MiR-125a may inhibit the proliferation of keratinocytes by negatively regulating the IL-23R/JAK2/AKT signaling pathway.
Résumé
Objective To evaluate the correlation of long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) with disease risk and severity of bronchial asthma as well as plasma microRNA (miRNA)-125a expression in child patients. Methods Seventy children with asthma exacerbation, 70 children with asthma remission and 70 matched healthy controls were consecutively enrolled in this study. Laboratory parameters and lung function indexes of the participants were recorded. LncRNA ANRIL and miRNA-125a expression levels in plasma were determined using qRT-PCR. Tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and IL-17 levels in plasma were determined using enzyme-linked immunosorbent assay (ELISA). Results LncRNA ANRIL expression was the highest in the asthma exacerbation children, followed by asthma remission children and healthy controls. There were significant differences in the expression of lncRNA ANRIL among the three groups (all P<0.01). Receiver operating characteristic curves revealed that lncRNA ANRIL could well differentiate the participants in the three groups. In the children with asthma exacerbation, lncRNA ANRIL expression was associated with disease severity (P=0.001), positively associated with the levels of TNF-α, IL-1β, IL-6 and IL-17 (all P<0.05), while negatively associated with forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) and FEV1 as percentage of predicted (FEV1%Pred) value (both P<0.05). LncRNA ANRIL expression was also positively associated with the levels of TNF-α, IL-1β and IL-17 in the asthma remission children and IL-6 level in healthy controls (all P<0.05). LncRNA ANRIL expression was negatively associated with miRNA-125a expression in all the participants (all P<0.05). Besides, miRNA-125a expression was positively correlated with FEV1/FVC and FEV1%Pred value in the children with asthma exacerbetion (both P<0.001). Conclusion LncRNA ANRIL may serve as a novel biomarker for predicting asthma, asthma acute exacerbation and severity, and inflammation level. It may participate in the development and progression of asthma in children via targeting miRNA-125a..
Résumé
AIM:To investigate the function of microRNA-125a-5p (miR-125a-5p) on epithelial-mesenchy-mal transition ( EMT) of breast cancer cells via GSK-3β/Snail signaling pathway. METHODS:The expression of miR-125a-5p in normal breast epithelial cells and breast cancer cells, as well as the transfection efficiency of miR-125a-5p plas-mid in MDA-MB-231 cells was detected by RT-qPCR. The chemotaxis ability and invasion ability were detected by chemo-taxis assay and Transwell invasion assay. The changes of EMT-related markers, the protein level of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) and the nuclear translocation of Snail were determined by Western blot. RESULTS:The expression of miR-125a-5p in the breast cancer cells was significantly lower than that in the normal breast epithelial cells. The expression of miR-125a-5p was significantly higher in MDA-MB-231/miR-125a-5p cells than that in MDA-MB-231/NC cells. The ability of epithelial growth factor (EGF) at 10 μg/L to induce chemotaxis of MDA-MB-231 cells was the stron-gest. Compared with MDA-MB-231/NC group, stimulation of EGF decreased the invasion ability of MDA-MB-231/miR-125a-5p cells, and resulted in the increase in E-cadherin expression, while significantly decreased the protein levels of vi-mentin and p-GSK-3β. Meanwhile, the nuclear localization of Snail was significantly inhibited. The invasion capacity of MDA-MB-231/miR-125a-5p+GAB2 cells was significantly enhanced compared with MDA-MB-231/miR-125a-5p +Con cells, the expression of E-cadherin was decreased, and the protein levels of vimentin and p-GSK-3β were significantly in-creased, while the nuclear localization of Snail was promoted. CONCLUSION:miR-125a-5p suppresses EMT via GSK-3β/Snail signaling pathway, thus inhibiting the invasion ability of breast cancer cells.