Mechanism of miRNA-1246 targeting MAPK14 in ultraviolet A-induced photoaging of human fibroblasts / 中华皮肤科杂志

ABSTRACT

Objective:To investigate the miRNA-1246 expression in photoaged human fibroblasts (HSFs) induced by ultraviolet A (UVA) , and to evaluate the effect of upregulating miRNA-1246 expression on its target gene MAPK14 and cell aging.Methods:HSFs were isolated from foreskins of healthy children after circumcision in Children′s Hospital of Soochow University, and irradiated with 10 J/cm 2 UVA once a day for 14 consecutive days. Real-time quantitative PCR was performed to determine the expression of miR-1246 immediately after the first irradiation and on days 3, 7 and 14 after the start of irradiation. Some HSFs were divided into 4 groups blank control group receiving no treatment, UVA group irradiated with 10 J/cm 2 UVA for 14 days, miR-1246 group transfected with a lentiviral vector carrying miR-1246, and UVA + miR-1246 group transfected with a lentiviral vector carrying miR-1246 followed by irradiation with UVA. After treatment, the HSFs were collected, methyl thiazolyl tetrazolium (MTT) assay was performed to assess cellular proliferativy activity, β-galactosidase staining to detect senescent cells, RT-PCR and Western blot analysis were conducted to measure the mRNA and protein expression of MAPK14 and matrix metalloproteinase 1 (MMP-1) . One-way analysis of variance was used for comparison of means among multiple groups, and least significant difference (LSD) - t test was used for multiple comparisons. Results:On days 7 and 14, the relative expression of miR-1246 in HSFs was significantly lower in the UVA group (4.69 ± 0.85, 3.59 ± 0.45, respectively) than in the blank control group (8.42 ± 0.75, 7.61 ± 0.49, t = 29.84, 31.93, respectively, both P < 0.01) . After upregulation of miR-1246 and irradiation with UVA, MTT assay showed that the cellular proliferative activity significantly differed among the blank control group, UVA group, miR-1246 group, UVA + miR-1246 group (0.82 ± 0.03, 0.23 ± 0.02, 0.81 ± 0.02, 0.61 ± 0.02, respectively; F = 34.90, P < 0.05) , significantly lower in the UVA group than in the blank control group ( t = 28.14, P < 0.01) , lower in the UVA + miR-1246 group than in the miR-1246 group ( t = 10.61, P < 0.01) , but significantly higher in the UVA + miR-1246 group than in the UVA group ( t = 20.30, P < 0.01) . β-Galactosidase staining showed that the proportion of senescent cells significantly differed among the above 4 groups (3.93% ± 1.11%, 81.29% ± 2.53%, 5.50% ± 1.15%, 54.13% ± 2.09%, respectively; F = 16.14, P < 0.05) , significantly higher in the UVA group than in the blank control group ( t = 48.46, P < 0.01) , higher in the UVA + miR-1246 group than in the miR-1246 group ( t = 35.31, P < 0.01) , but significantly lower in the UVA + miR-1246 group than in the UVA group ( t = 14.32, P < 0.01) . Both RT-PCR and Western blot analysis showed that the mRNA and protein expression of MAPK14 and MMP-1 significantly differed among the above 4 groups (both P < 0.05) , significantly higher in the UVA group than in the blank control group ( P < 0.05) , higher in the UVA + miR-1246 group than in the miR-1246 group ( P < 0.05) , but significantly lower in the UVA + miR-1246 group than in the UVA group ( P < 0.05) . Conclusions:In the senescent HSFs induced by UVA, the expression of miR-1246 is suppressed. Upregulating the expression of miR-1246 can exert anti-photoaging effect by inhibiting the expression of its target gene MAPK14 and aging-related protein MMP-1.