The Mechanism Of MiR-146a-Smad4 In Photoaged Primary Human Fibroblasts Irradiated With Ultraviolet A

Objective:To investigate the effect of ultraviolet A(UVA)radiation on the expression of mi R-146 a by human dermal fibroblasts,and to explore the effect of up-regulation of mi R-146 a expression on its target gene Smad4 and photoaged cells proliferation.Methods:Dermal fibroblasts were isolated from the foreskins of boys,and subjected to primary culture and subculture.After 10 or less passages,the fibroblasts were collected and divided into several groups to be irradiated with 10J/cm2 UVA followed by 0,3,7,14 days of additional culture,or be irradiated with 10J/cm2 UVA followed by 14 days,which could mimic UVA irradiation-induced photoaging of skin,meanwhile,lentivirus-mediated mi R-146 a overexpression was performed,with those receiving no treatment serving as the control group. Subsequently,cells were collected,real time PCR was performed to detect the expressions of mi R-146 a m RNA and p53,p21 and p16 m RNA,which are photoaging related gene. MTT were performed and OD570 values were determined to evaluate the proliferation ability of UVA-irradiated control group or mi R-146 a overexpressing fibroblasts. Wsetern blot were performed to detect the expression of Smad4 protein.Results:Repeated-measures analysis of variance showed that the expression of mi R-146 a 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 mi R-146a-Smad4,HSFs showed a strong fluorescence intensity of mi R-146 a. The expression level of mi R-146 a was significantly higher in the mi R-146 a 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 mi R-146 a 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 + mi R-146 a group than in the mi R-146 a group(P < 0.01). The lentivirus-mediated up-regulation of mi R-146 a expression also affected cellular proliferative activity(P < 0.01),which was significantly higher in the UVA + mi R-146 a group than in the UVA group(P < 0.01),but insignificantly different between the mi R-146 a 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 m RNAs as well as Smad4 protein(all P < 0.01). Concretely speaking,the expressions of p53,p21,p16 m RNAs 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 + mi R-146 a group than in the mi R-146 a group(all P < 0.01),but significantly lower in the UVA + mi R-146 a group than in the UVA group(all P < 0.01),and insignificantly different between the blank control group and mi R-146 a group(all P > 0.05).Conclusion:The expression of mi R-146 a was inhibited in UVA irradiation-induced photoaging cells,up-regulating mi R-146 a could suppress the expression of Smad4,promoting fibroblasts cells proliferation,which may antagonize UVA irradiation-induced photoaging.