Abstract: 【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/cm2 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/cm2 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.

Key words: Fibroblasts, Ultraviolet rays, MicroRNAs, Mitogen-activated protein kinase 14, Matrix metalloproteinase 1, miR-1246