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浙江大学学报（医学版）
浙江大学学报（医学版）  2017, Vol. 46 Issue (6): 609-617    DOI: 10.3785/j.issn.1008-9292.2017.12.06
原著     
微小RNA222靶向调控基质金属蛋白酶1促进增生性瘢痕组织成纤维细胞生长
张谊1(),张璃1,张启瑜2,洪炜龙3,林孝华1,*()
1. 温州医科大学第一附属医院皮肤科, 浙江 温州 325000
2. 温州医科大学第一附属医院肝胆外科, 浙江 温州 325000
3. 温州医科大学第一附属医院外科实验室, 浙江 温州 325000
microRNA-222 regulates proliferation and apoptosis of fibroblasts in hypertrophic scar via matrix metalloproteinase 1
ZHANG Yi1(),ZHANG Li1,ZHANG Qiyu2,HONG Weilong3,LIN Xiaohua1,*()
1. Department of Dermatology, the First Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China
2. Department of Hepatobiliary Surgery, the First Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China
3. Department of Surgical Laboratory, the First Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China
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摘要：

目的: 观察微小RNA（miR-）222在增生性瘢痕（HS）组织中的表达，并研究其调控成纤维细胞生长的机制。方法: 采用实时定量RT-PCR检测36例患者HS和正常组织中miR-222的表达；MTT法、流式细胞术和蛋白质印迹法分别检测成纤维细胞的增殖能力、生长周期、凋亡和相关蛋白表达。双荧光素酶报告分析确定miR-222的靶基因。结果: 与正常皮肤组织相比，HS组织中miR-222表达显著上调（P < 0.05）。上调miR-222的表达可显著提高成纤维细胞的增殖能力，增加增殖细胞核抗原（PCNA）、Ⅰ型和Ⅲ型胶原α1的mRNA和蛋白表达，上调S期细胞比例和细胞周期相关蛋白的表达，下调成纤维细胞的凋亡率和凋亡相关蛋白的表达。MMP1是miR-222的靶基因，miR-222通过绑定MMP1-3'UTR区负向调控MMP1在成纤维细胞的表达。MMP1可逆转miR-222的部分促纤维化作用。结论: miR-222通过负调控其靶基因MMP1的表达来实现其调控成纤维细胞的生长和凋亡。miR-222/MMP1信号通路可能成为HS诊断和治疗的生物学标志物和靶点。
关键词： 微RNAs/药理学;  基质金属蛋白酶1;  成纤维细胞;  瘢痕, 肥大性/病因学;  瘢痕, 肥大性/治疗;  细胞增殖    
Abstract:

Objective: To explore the effect of microRNA(miR)-222 on cell proliferation and apoptosis of fibroblasts in hypertrophic scar (HS) and the underlying mechanisms. Methods: The expression of miR-222 in the HS and the normal skin tissues was detected by real-time RT-PCR. The HS fibroblasts were transfected with miR-222 mimic and miR-222 inhibitor respectively. The cell viability was tested with MTT assay, cell cycle distribution and apoptosis were detected with flow cytometry and the expression levels of proliferation, apoptosis and cell cycle related proteins were determined with Western blot. Direct target of miR-222 was evaluated by dual-luciferase reporter assay. Results: miR-222 was significantly up-regulated in HS tissues compared with normal skin tissues(P < 0.05). Overexpression of miR-222 enhanced the cell viability of HS fibroblasts; increased mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), collagen alpha-1 (Ⅰ) chain (Col1A1) and collagen alpha-1 (Ⅲ) chain (Col3A1); increased cell population in S phase and protein expressions of cyclin D1, cyclin E1 and cyclin-dependent kinases 1 (CDK1); inhibited cell apoptosis and reduced protein expressions of caspase-3/9. Overexpression of MMP1 attenuated the effects of miR-222 on the cell viability and apoptosis in fibroblasts, reduced expression levels of PCNA, cyclin D1 and the expression of caspase-3 was increased. Conclusion: miR-222 enhances cell proliferation and inhibits cell apoptosis of HS fibroblasts through negative regulation of MMP1, which suggests that miR-222 and MMP1 might be used as novel biomarkers and targets in diagnostic and therapeutic approaches for HS.
Key words: MicroRNAs/pharmacology    Matrix metalloproteinase 1    Fibroblasts    Cicatrix, hypertrophic/etiology    Cicatrix, hypertrophic/therapy    Cell proliferation
收稿日期: 2017-06-28 出版日期: 2017-12-25
CLC:  R62  
基金资助: 浙江省医药卫生科技计划(2015KYB244);浙江省教育厅高教重中之重学科项目(2008-255)
通讯作者: 林孝华     E-mail: 409705470@qq.com;wzlinxiaohua@163.com
作者简介: 张谊(1981-), 男, 硕士, 主治医师, 主要从事皮肤外科学研究; E-mail:409705470@qq.com; https://orcid.org/0000-0001-9548-1916
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引用本文:

张谊,张璃,张启瑜,洪炜龙,林孝华. 微小RNA222靶向调控基质金属蛋白酶1促进增生性瘢痕组织成纤维细胞生长[J]. 浙江大学学报（医学版）, 2017, 46(6): 609-617.

ZHANG Yi,ZHANG Li,ZHANG Qiyu,HONG Weilong,LIN Xiaohua. microRNA-222 regulates proliferation and apoptosis of fibroblasts in hypertrophic scar via matrix metalloproteinase 1. J Zhejiang Univ (Med Sci), 2017, 46(6): 609-617.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2017.12.06    或    http://www.zjujournals.com/med/CN/Y2017/V46/I6/609

基因名称 引物序列(5′ \begin{document}$\to $\end{document} 3′)
miR-222 上游：CGCAGCTACATCTGGCTACTG
下游：GTGCAGGGTCCGAGGT
COL1A1 上游：CGAAGACATCCCACCAATCAC
下游：GATCGCACAACACCTTGCC
COL3A1 上游：CCTGGTCCTTGCTGTGGTGGTGT
下游：GCAGTTTCTAGCGGGGTTTTTACG
MMP1 上游：GGACACCAACTATTGCTTCAG
下游：ATGTCCTTGGGGTATCCGTGTAG
U6 上游：CGCTTCGGCAGCACATATAC
下游：CAGGGGCCATGCTAATCTT
表 1  定时定量RT-PCR引物序列
图 1  转染miR-222模拟物和miR-222抑制物后成纤维细胞中miR-222表达量比较
图 2  增生性瘢痕组织成纤维细胞中Col1A1和Col3A1蛋白表达电泳图
图 3  转染miR-222模拟物和miR-222抑制物后增生性瘢痕组织成纤维细胞中Col1A1、Col3A1蛋白和mRNA表达水平的变化
图 4  转染miR-222模拟物和miR-222抑制物后增生性瘢痕组织成纤维细胞增殖数变化
图 5  增生性瘢痕组织成纤维细胞中增殖细胞核抗原(PCNA)蛋白表达电泳图
图 6  转染miR-222模拟物和miR-222抑制物后增生性瘢痕组织成纤维细胞生长周期
图 7  增生性瘢痕组织成纤维细胞中cyclin D1、cyclin E1和CDK1蛋白表达电泳图
图 8  转染miR-222模拟物和miR-222抑制物后增生性瘢痕组织成纤维细胞中cyclin D1、cyclin E1和CDK1蛋白表达量变化
图 9  增生性瘢痕组织成纤维细胞中caspase-3和caspase-9蛋白表达电泳图
图 10  转染miR-222模拟物和miR-222抑制物后增生性瘢痕组织成纤维细胞中caspase-3和caspase-9蛋白表达量变化
图 11  增生性瘢痕组织成纤维细胞基质金属蛋白酶1(MMP1)表达电泳图
图 12  转染miR-222模拟物和miR-222抑制物后增生性瘢痕组织成纤维细胞中基质金属蛋白酶1(MMP1)mRNA和蛋白表达的变化
图 13  转染MMP1后增生性瘢痕组织成纤维细胞中PCNA、cyclin D1和caspase-3蛋白表达电泳图
图 14  转染MMP1后增生性瘢痕组织成纤维细胞中PCNA、cyclin D1和caspase-3蛋白水平变化
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