Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism
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摘要:
目的 探讨ⅩⅦ型胶原蛋白α1（COL17α1）在小鼠衰老皮肤中的表达与作用及其对人表皮干细胞（ESC）干性和增殖能力的影响，并且探讨相关微小RNA（miR）干预人ESC中COL17α1表达的机制。 方法 采用实验研究方法。取2个月龄（青年）和24个月龄（老年）雄性C57BL/6J小鼠各12只，取其胸背部全层皮肤标本进行后续检测。对青年小鼠和老年小鼠全层皮肤标本，行苏木精-伊红染色后观察表皮层结构并测量表皮厚度，用透射电子显微镜观察表皮基底膜形态和半桥粒并行半桥粒计数，分别采用实时荧光定量反转录PCR（RT-PCR）法与蛋白质印迹法检测COL17α1的mRNA与蛋白表达，采用免疫荧光法观测COL17α1的蛋白表达与分布。取于解放军总医院第四医学中心行包皮切除手术的3名20~30岁健康男性术后弃用的新鲜包皮组织，提取ESC，取生长良好的细胞进行后续实验。按随机数字表法（分组方法下同）将ESC分为进行相应处理的空白对照组、转染试剂对照组、空载体质粒组、敲低COL17α1质粒组，培养48 h后，采用实时荧光定量RT-PCR法检测COL17α1的mRNA表达，采用蛋白质印迹法检测COL17α1和细胞角蛋白14（CK14）的蛋白表达，采用细胞计数试剂盒8法检测细胞增殖水平。通过DIANA、miRTarBase、miRNAMap、TargetScan、microRNA数据库筛选可能作用于COL17α1 mRNA 3'非编码区的miR。将ESC分为转染miR模拟物阴性对照物的阴性对照组和转染前述筛选出的各miR的模拟物的各miR模拟物组，转染后48 h，通过蛋白质印迹法检测COL17α1的蛋白表达。基于基因表达综合数据库（GEO）的miR测序数据集GSE114006，使用GEO2R工具统计分析前述筛选出的可造成COL17α1蛋白表达下降的miR在30名青年（18~25岁）人和30名老年（>70岁）人皮肤中的表达。取青年小鼠和老年小鼠全层皮肤标本，采用实时荧光定量RT-PCR法检测前述老年人皮肤中表达增多的miR的表达。取2批ESC，第1批分为COL17α1野生型+miR-203b-3p阴性对照组与COL17α1野生型+miR-203b-3p模拟物组，第2批分为COL17α1突变型+miR-203b-3p阴性对照组与COL17α1突变型+miR-203b-3p模拟物组，分别转染对应序列，48 h后，采用荧光素酶报告基因检测试剂盒检测COL17α1的基因表达水平。组织实验中样本数均为6，细胞实验中样本数均为3。对数据行独立样本t检验、单因素方差分析、LSD检验或Dunnett检验、Mann-Whitney U检验或Kruskal-Wallis H检验。 结果 与青年小鼠比较，老年小鼠皮肤表皮层与真皮层分界模糊且细胞层次较少，表皮层厚度明显变薄（Z=-2.88，P<0.01），基底膜形态不连续，表皮-真皮连接处半桥粒较少且分布不均，半桥粒数量明显减少（Z=-2.91，P<0.01），COL17α1的mRNA和蛋白表达水平均明显降低（t值分别为10.61、6.85，P<0.01）。与青年小鼠比较，老年小鼠皮肤表皮基底层和毛囊球部的COL17α1蛋白表达均明显减少（Z=-2.24，P<0.05）。培养48 h后，空白对照组、转染试剂对照组、空载体质粒组、敲低COL17α1质粒组ESC中COL17α1的蛋白表达水平分别为1.00±0.27、1.12±0.21、1.13±0.23、0.42±0.18。相较于空白对照组，转染试剂对照组和空载体质粒组ESC中COL17α1的mRNA和蛋白表达水平、CK14的蛋白表达水平及ESC增殖水平均未发生明显变化（P>0.05），敲低COL17α1质粒组ESC中这些指标均明显降低（P<0.05或P<0.01）。miR-203a-3p、miR-203b-3p、miR-512-5p、miR-124-3p、miR-28-5p、miR-590-3p、miR-329-5p可能结合COL17α1 mRNA的3'非编码区。转染48 h后，与阴性对照组的1.000±0.224比较，miR-329-5p模拟物组、miR-203b-3p模拟物组和miR-203a-3p模拟物组ESC中COL17α1的蛋白表达水平明显降低（0.516±0.188、0.170±0.025、0.235±0.025，t值分别为3.17、5.43、5.07，P<0.05或P<0.01）。老年人皮肤中仅miR-203b-3p表达水平明显高于青年人（t=3.27，P<0.01）。老年小鼠皮肤中miR-203b-3p表达水平明显高于青年小鼠（Z=-2.88，P<0.01）。转染后48 h，COL17α1野生型+miR-203b-3p模拟物组ESC中COL17α1的基因表达水平明显低于COL17α1野生型+miR-203b-3p阴性对照组（t=7.66，P<0.01），COL17α1突变型+miR-203b-3p模拟物组ESC中COL17α1的基因表达水平与COL17α1突变型+miR-203b-3p阴性对照组相近（P>0.05）。 结论 COL17α1的mRNA和蛋白表达水平随小鼠年龄增长而减少，可能导致小鼠ESC脱离表皮基底膜。COL17α1的表达减少可抑制CK14的表达与ESC增殖，这可能是老年人皮肤中表皮层变薄和创面愈合减慢的原因。小鼠衰老皮肤中表达增多的miR-203b-3p可以靶向结合COL17α1 mRNA的3'非编码区，阻碍转录后翻译过程，造成COL17α1蛋白表达减少。 Abstract:Objective To investigate the expression and function of collagen type ⅩⅦ 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. -
Key words:
- Aging /
- Skin /
- Hemidesmosomes /
- Collagen type XVII α1 /
- Epidermal stem cells /
- microRNA
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参考文献
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1 青年小鼠与老年小鼠全层皮肤中表皮层结构和厚度观察 苏木精-伊红×200。1A.青年小鼠表皮层厚度较厚，表皮层与真皮层分界明显并且细胞层次较多；1B.老年小鼠表皮层较图1A薄，表皮层与真皮层间分界模糊且细胞层次较图1A少
2 青年小鼠与老年小鼠全层皮肤中基底膜和半桥粒情况。2A、2B.分别为青年小鼠、老年小鼠，图2A中皮肤基底膜形态完整（黑框处），图2B中皮肤基底膜形态断续（黑框处） 透射电子显微镜×1 500；2C、2D.分别为图2A、2B方框处放大图，图2C中含大量半桥粒结构且均匀分布在表皮-真皮连接处，图2D中半桥粒较少且分布不均 透射电子显微镜×3 750
注：箭头指示半桥粒
4 青年小鼠与老年小鼠全层皮肤中COL17α1蛋白的表达与分布 Alexa Fluor 488-4',6-二脒基-2-苯基吲哚×200。4A、4B、4C.分别为青年小鼠细胞核染色、COL17α1染色、细胞核与COL17α1染色重叠图片，细胞核完整，COL17α1蛋白表达较多，主要分布在表皮基底层和毛囊球部；4D、4E、4F.分别为老年小鼠细胞核染色、COL17α1染色、细胞核与COL17α1染色重叠图片，细胞核完整，COL17α1蛋白表达较图4B少
注：细胞核阳性染色为蓝色，ⅩⅦ型胶原蛋白α1（COL17α1）阳性染色为绿色
5 采用蛋白质印迹法检测的4组人表皮干细胞培养48 h后CK14和COL17α1的蛋白表达
注：CK14为细胞角蛋白14，COL17α1为ⅩⅦ型胶原蛋白α1，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4分别指示空白对照组、转染试剂对照组、空载体质粒组、敲低COL17α1质粒组
6 采用蛋白质印迹法检测的8组人表皮干细胞中COL17α1的蛋白表达
注：COL17α1为ⅩⅦ型胶原蛋白α1，GAPDH为3-磷酸甘油醛脱氢酶，miR为微小RNA；条带上方1、2、3、4、5、6、7、8分别指示阴性对照组、miR-512-5p模拟物组、miR-124-3p模拟物组、miR-28-5p模拟物组、miR-203a-3p模拟物组、miR-590-3p模拟物组、miR-329-5p模拟物组、miR-203b-3p模拟物组
表1 4组人表皮干细胞培养48 h后COL17α1和CK14的相对表达量比较（
组别 样本数 COL17α1 mRNA COL17α1蛋白 CK14蛋白 空白对照组 3 1.00±0.11 1.00±0.27 1.00±0.19 转染试剂对照组 3 1.09±0.12 1.12±0.21 1.20±0.27 空载体质粒组 3 0.97±0.09 1.13±0.23 1.17±0.21 敲低COL17α1质粒组 3 0.55±0.08a 0.42±0.18b 0.39±0.12a 注：COL17α1为ⅩⅦ型胶原蛋白α1，CK14为细胞角蛋白14；与空白对照组比较，aP<0.01,bP<0.05 
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