Role of miR-146a in ultraviolet A-induced photoaging of human skin fibroblasts and its mechanism
10.3760/cma.j.issn.0412-4030.2016.03.011
- VernacularTitle:MiR-146a 在长波紫外线诱导人皮肤成纤维细胞光老化中作用机制的研究
- Author:
Xiao LI
;
Wei LI
;
Qinghua YANG
;
Zhiheng LI
;
Guixiong GU
- Publication Type:Journal Article
- Keywords:
Fibroblasts;
Ultraviolet rays;
Skin aging;
MicroRNAs;
Smad4 protein;
miR-146a
- From:Chinese Journal of Dermatology
2016;(3):197-202
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate miR-146a-Smad4 expression during ultraviolet A(UVA)-induced photoaging of human skin fibroblasts (HSFs), and to evaluate effects of up-regulation of miR-146a expression on its target gene Smad4 and cell photoaging. Methods HSFs were isolated from the prepuce, and subjected to primary culture and maintained up to 10th passage. Then, the HSFs were classified into 4 groups: blank control group receiving no treatment, UVA group irradiated with 10 J/cm2 UVA, miR-146a group transfected with a lentiviral vector expressing miR-146a, UVA+ miR-146a group transfected with the lentiviral vector expressing miR-146a followed by UVA radiation. Real time PCR was performed to measure miR-146a expression in HSFs in the UVA group on day 0, 3, 7 and 14 after UVA radiation.Fluorescence microscopy was carried out to estimate transfection efficiency on day 7 and 14 in the miR-146a group after transfection, and real time PCR was performed to quantify miR-146a expression in these cells. Methyl thiazolyl tetrazolium (MTT)assay was conducted to evaluate proliferative activity of HSFs, real time PCR to quantify mRNA expressions of photoaging-related genes p53, p21 and p16, and Western blot analysis to measure Smad4 protein expression in these cells. Statistical analysis was carried out by using repeated-measures analysis of variance and factorial design analysis of variance. Results Repeated-measures analysis of variance showed that the expression of miR-146a decreased over time in both the UVA group and blank control group(F = 213.840, P < 0.01), and significantly lower in the UVA group than in the blank control group (F = 52.55, P < 0.01), with the difference between the two groups increasing over time. After transfection with the lentiviral vector expressing miR-146a-Smad4, HSFs showed a strong fluorescence intensity of miR-146a. The expression level of miR-146a was significantly higher in the miR-146a group than in the blank control group on day 7 and 14 after transfection(10.31 ± 0.17 vs. 8.33 ± 0.13 on day 7, 9.65 ± 0.19 vs. 7.86 ± 0.11 on day 14, F =42.49, P < 0.01), but insignificantly different between day 7 and 14 in the miR-146a group (P > 0.05). Factorial design analysis of variance showed that UVA radiation had an inhibitory effect on the proliferative activity of HSFs (P < 0.01), which was significantly lower in the UVA group than in the blank control group(P < 0.01), and lower in the UVA + miR-146a group than in the miR-146a group (P < 0.01). The lentivirus-mediated up-regulation of miR-146a expression also affected cellular proliferative activity (P < 0.01), which was significantly higher in the UVA + miR-146a group than in the UVA group(P < 0.01), but insignificantly different between the miR-146a group and blank control group(P > 0.05). Real time PCR and Western blot analysis both revealed that UVA radiation could increase the expressions of p53, p21 and p16 mRNAs as well as Smad4 protein(all P < 0.01). Concretely speaking, the expressions of p53, p21, p16 mRNAs and Smad4 protein were all significantly higher in the UVA group than in the blank control group (all P < 0.01), and higher in the UVA +miR-146a group than in the miR-146a group (all P < 0.01), but significantly lower in the UVA + miR-146a group than in the UVA group (all P < 0.01), and insignificantly different between the blank control group and miR-146a group (all P >0.05). Conclusion The expression of miR-146a is inhibited in UVA-induced photoaged HSFs, and its up-regulation may counteract cell photoaging by suppressing Smad4 expression in, and promoting proliferation of, photoaged HSFs.
