Effects of miRNA-29c-3p on the expression of collagen type Ⅰ α1 and collagen type Ⅲ α1 genes and the synthesis of collagen Ⅰ and Ⅲ in chronically photodamaged human dermal fibroblasts in vitro
10.3760/cma.j.issn.0412-4030.2017.12.003
- VernacularTitle: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
- Author:
Xiaojing SONG
1
;
Yating PENG
;
Haiyan CHEN
;
Yue ZHENG
;
Qingfang XU
;
Zijian GONG
;
Chun LU
;
Wei LAI
Author Information
1. 中山大学附属第三医院皮肤科
- Keywords:
Ultraviolet rays;
Fibroblasts;
MicroRNAs;
Collagen type Ⅰ;
Collagen type Ⅲ;
Photoaging;
miR-29
- From:
Chinese Journal of Dermatology
2017;50(12):869-874
- CountryChina
- Language:Chinese
-
Abstract:
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 collagen Ⅰ and 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 Ⅰ α1 (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 ＜ 0.01) 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 expression of collagen Ⅰ 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 expression 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 expression of miR-29a-3p or miR-29b-3p between the 2 groups (both 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 collagen Ⅰ and Ⅲ.
