miRNA-29c-3p对体外慢性光损伤皮肤成纤维细胞COL1A1和COL3A1基因表达及I、III型胶原蛋白合成的影响

Objective To evaluate the effect of miRNA-29 (miR-29) family on the synthesis of collagen Ⅰ and 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 expression of collagenⅠand Ⅲ, and real-time fluorescence-based quantitative PCR (qRT-PCR) to measure expression 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 (overexpression 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 expression 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 expression of collagen type Ⅰα1 (COL1A1) and collagen type Ⅲ α1 (COL3A1) genes, and Western blot analysis to measure the protein expression of collagen Ⅰ and 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 0.05) . Twenty-four hours after transfection, the overexpression group and inhibition group both showed more than 90% transfection efficiency which met the interference requirements. The expression of miR-29c-3p was significantly higher in the overexpression 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 expression of COL1A1 and COL3A1 and the protein expression of collagen Ⅰ and Ⅲ were significantly lower in the overexpression 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 expression of miR-29c-3p is up-regulated in chronically photodamaged HDFs, likely by regulating the mRNA expression of COL1A1 and COL3A1 and the protein expression of collagenⅠand Ⅲ. Key words: Ultraviolet rays; Fibroblasts; MicroRNAs; Collagen type I; Collagen type III; Photoaging; miR-29