RESUMO
Objective: To investigate the expression and function of collagen type â ©â ¦ α1 (COL17α1) in aging mouse skin and its effect on the stemness and proliferation of human epidermal stem cells (ESCs), and to explore the mechanism of related microRNA (miR) in intervening the expression of COL17α1 of human ESC. Methods: The method of experimental research was used. Twelve 2-month-old (young) and twelve 24-month-old (aged) male C57BL/6J mice were selected, and full-thickness skin samples from their upper back were taken for follow-up detection. After hematoxylin-eosin staining of the full-thickness skin samples of young mice and aged mice, the structure of the epidermis was observed and the thickness of the epidermis was measured; the morphology of epidermal basement membrane and hemidesmosomes were observed by transmission electron microscopy, and the hemidesmosomes were counted; the mRNA and protein expressions of COL17α1 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively, and the protein expression and distribution of COL17α1 was observed and detected by immunofluorescence method. The fresh foreskin tissue discarded after surgery was obtained from 3 healthy men aged 20-30 years who underwent circumcision at the Fourth Medical Center of PLA General Hospital, ESCs were extracted and well-grown cells were wsed for follow-up experiments. According to the random number table (the same grouping method below), ESCs were divided into blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group with corresponding treatment. After 48 hours of culture, the mRNA expression of COL17α1 was detected by real-time fluorescent quantitative RT-PCR, the protein expressions of COL17α1 and cytokeratin 14 (CK14) were detected by Western blotting, and the cell proliferation level was detected by cell counting kit 8. miRs that might act on the 3' non-coding region of COL17α1 mRNA were screened through DIANA, miRTarBase, miRNAMap, TargetScan, and microRNA databases. The ESCs were divided into negative control group transfected with miR mimic negative control and each miR mimic group transfected with each of the previously screened miR mimics. Forty-eight hours after transfection, the protein expression of COL17α1 was detected by Western blotting. Based on the sequencing data set GSE114006 in Gene Expression Omnibus (GEO), the GEO2R tool was used to statistically analyze the expression of the previously screened miRs that could cause the reduction of COL17α1 protein expression in the skin of 30 young (18-25 years old) and 30 elderly (>70 years old) human skins. The full-thickness skin samples of young mice and aged mice were taken, and the expressions of increased miRs in the aforementioned aged human skin were detected by real-time fluorescent quantitative RT-PCR. Two batches of human ESCs were taken, the first batch was divided into COL17α1 wild type+miR-203b-3p negative control group and COL17α1 wild type+miR-203b-3p mimic group, and the second batch was divided into COL17α1 mutant+miR-203b-3p negative control group and COL17α1 mutant+miR-203b-3p mimic group. Each group of ESC was transfected with corresponding sequences respectively. Forty-eight hours later, the luciferase reporter gene detection kit was used to detect the gene expression level of COL17α1. The number of samples in the tissue experiment was 6, and the number of samples in the cell experiment was 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, least significant difference test or Dunnett's test, Mann-Whitney U test or Kruskal-Wallis H test. Results: Compared with those of young mice, the boundary between the epidermis and the dermis of the aged mice skin was blurred and the cell layers were less, and the thickness of epidermis was significantly thinner (Z=-2.88, P<0.01); the morphology of basement membrane was discontinuous, with less unevenly distributed hemidesmosomes at the epidermis-dermis junction, and the number of hemidesmosomes was significantly reduced (Z=-2.91, P<0.01); the mRNA and protein expression levels of COL17α1 in the skin of aged mice were significantly decreased (with t values of 10.61 and 6.85, respectively, P<0.01). Compared with those of young mice, the protein expression of COL17α1 in the basal layer of epidermis and the bulb of hair follicle in the skin of aged mice was significantly decreased (Z=-2.24, P<0.05). After 48 hours of culture, the protein expression levels of COL17α1 in ESCs of blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group were 1.00±0.27, 1.12±0.21, 1.13±0.23, and 0.42±0.18, respectively. Compared with those of blank control group, the mRNA and protein expression levels of COL17α1, the protein expression level of CK14, and the proliferation level of ESCs in transfection reagent control group and empty vector plasmid group did not change significantly (P>0.05), while these indexes in COL17α1 knockdown plasmid group were significantly decreased (P<0.05 or P<0.01). miR-203a-3p, miR-203b-3p, miR-512-5p, miR-124-3p, miR-28-5p, miR-590-3p, and miR-329-5p might bind to the 3' non-coding region of COL17α1 mRNA. Forty-eight hours after transfection, compared with 1.000±0.224 in negative control group, the protein expression level of COL17α1 in ESCs of miR-329-5p mimic group, miR-203b-3p mimic group, and miR-203a-3p mimic group decreased significantly (0.516±0.188, 0.170±0.025, and 0.235±0.025, with t values of 3.17, 5.43, and 5.07, respectively, P<0.05 or P<0.01). Only the expression level of miR-203b-3p in the skin of the elderly was significantly higher than that of the young (t=3.27, P<0.01). The expression level of miR-203b-3p in the skin of aged mice was significantly higher than that of young mice (Z=-2.88, P<0.01). Forty-eight hours after transfection, the gene expression level of COL17α1 in ESCs of COL17α1 wild type+miR-203b-3p mimic group was significantly lower than that of COL17α1 wild type+miR-203b-3p negative control group (t=7.66, P<0.01). The gene expression level of COL17α1 in ESCs of COL17α1 mutant+miR-203b-3p mimic group was similar to that of COL17α1 mutant+miR-203b-3p negative control group (P>0.05). Conclusions: The mRNA and protein expression levels of COL17α1 decrease with age increasing in mice, which may lead to the detachment of mouse ESC from the epidermal basement membrane. Decreased expression of COL17α1 can inhibit the expression of CK14 and ESC proliferation, which may be responsible for the thinning of the epidermis and slower wound healing in aged human skin. The increased expression of miR-203b-3p in aged mouse skin can target and bind to the 3' non-coding region of COL17α1 mRNA, hindering the post-transcriptional translation process, thus resulting in decreased COL17α1 protein expression.