Abstract: Song Xiaojing, Peng Yating, Chen Haiyan, Zheng Yue, Xu Qingfang, Gong Zijian, Lu Chun, Lai Wei Department of Dermatology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China Corresponding author: Lai Wei, Email: drlaiwei@163.com 【Abstract】 Objective To evaluate the effect of miRNA-29 （miR-29） family on the synthesis of collagen Ⅰand Ⅲ in chronically photodamaged （photoaged） skin. Methods Some cultured human dermal fibroblasts （HDFs） were divided into 2 groups: non-irradiated group receiving no treatment, and chronic photodamage group treated with repetitive ultraviolet A （UVA） radiation, which served as a chronically photodamaged cell model and was verified by flow cytometry and β-galactosidase staining. Western blot analysis was performed to determine the protein of collagenⅠand Ⅲ, and real-time fluorescence-based quantitative PCR （qRT-PCR） to measure of 3 members of the miR-29 family （miR-29a-3p, miR-29b-3p and miR-29c-3p） in the above 2 groups. The differentially expressed miR-29c-3p between the above 2 groups was chosen for further functional tests. Some HDFs were divided into 4 groups to be transfected with fluorescein-labelled miR-29c-3p mimics （over group）, inhibitors （inhibition group）, and their control RNA oligonucleotides （negative control group and inhibitor control group） respectively. The transfection efficiency was evaluated by the proportion of fluorescent cells, and the relative of miR-29c-3p in the above 4 groups was measured by qRT-PCR for evaluating the RNA interference efficiency. qRT-PCR was conducted to determine the mRNA of collagen typeⅠα1 （COL1A1） and collagen type Ⅲ α1 （COL3A1） genes, and Western blot analysis to measure the protein of collagenⅠand Ⅲ. Results Compared with the non-irradiated group, the chronic photodamage group showed significantly increased proportion of senescent cells （36.47% ± 3.20% vs. 12.56% ± 1.46%, P < 0.01） and G1-phase cells （71.70% ± 2.43% vs. 41.89% ± 1.86%, P < 0.01）, but significantly decreased proportion of S-phase cells （10.63% ± 0.36% vs. 36.48% ± 1.31%, P < 0.01）, which indicated that the chronically photodamaged cell model was established successfully. The protein of collagenⅠ and Ⅲ was significantly lower in the chronic photodamage group （0.40 ± 0.19 and 0.52 ± 0.10） than in the non-irradiated group （1.00 ± 0.12 and 1.00 ± 0.10, respectively, both P < 0.01）. The of miR-29c-3p was significantly higher in the chronic photodamage group than in the non-irradiated group （4.42 ± 2.05 vs. 0.89 ± 0.10, P < 0.05）, while there were no significant differences in the of miR-29a-3p or miR-29b-3p between the 2 groups （both P > 0.05）. Twenty-four hours after transfection, the over group and inhibition group both showed more than 90% transfection efficiency which met the interference requirements. The of miR-29c-3p was significantly higher in the over group than in the negative control group （224.17 ± 2.00 vs. 2.45 ± 0.34, P < 0.01）, but significantly lower in the inhibition group than in the inhibitor control group （0.20 ± 0.08 vs. 2.24 ± 0.14, P < 0.01）, suggesting that a RNA interference model was successfully established. The mRNA of COL1A1 and COL3A1 and the protein of collagenⅠand Ⅲ were significantly lower in the over group than in the negative control group and inhibition group （all P < 0.05）, and significantly higher in the inhibition group than in the inhibitor control group （all P < 0.01）. Conclusion The of miR-29c-3p is up-regulated in chronically photodamaged HDFs, likely by regulating the mRNA of COL1A1 and COL3A1 and the protein of collagenⅠand Ⅲ.

Key words: Ultraviolet rays, Fibroblasts, MicroRNAs, Collagen type I, Collagen type III, Photoaging, miR-29