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J Zhejiang Univ (Med Sci)  2017, Vol. 46 Issue (6): 609-617    DOI: 10.3785/j.issn.1008-9292.2017.12.06
    
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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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 wordsMicroRNAs/pharmacology      Matrix metalloproteinase 1      Fibroblasts      Cicatrix, hypertrophic/etiology      Cicatrix, hypertrophic/therapy      Cell proliferation     
Received: 28 June 2017      Published: 25 December 2017
CLC:  R62  
Corresponding Authors: LIN Xiaohua     E-mail: 409705470@qq.com;wzlinxiaohua@163.com
Cite this article:

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.

URL:

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


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

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 
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
Tab 1 Primer sequences for real-time RT-PCR
Fig 1 Expression of miR-222 in hypertrophic scar fibroblasts transfected with miR-222 mimic or miR-222 inhibitor
Fig 2 Electropherogram of Col1A1, Col3A1 protein expression in hypertrophic scar fibroblasts
Fig 3 Protein and mRNA expressions of Col1A1 and Col3A1 in hypertrophic scar fibroblasts transfected with miR-222 mimic or miR-222 inhibitor
Fig 4 Cell viability of hypertrophic scar fibroblasts transfected with miR-222 mimic or miR-222 inhibitor
Fig 5 Electropherogram of proliferating cell nuclear antigen (PCNA) expression in hypertrophic scar fibroblasts
Fig 6 Cell cycle of hypertrophic scar fibroblasts transfected with miR-222 mimic or miR-222 inhibitor
Fig 7 Electropherogram of cyclin D1, cyclin E1 and CDK1 expression in hypertrophic scar fibroblasts
Fig 8 Protein expressions of cyclin D1, cyclin E1 and CDK1 in hypertrophic scar fibroblasts transfected with miR-222 mimic or miR-222 inhibitor
Fig 9 Electropherogram of caspase-3/9 in hypertrophic scar fibroblasts
Fig 10 Protein expressions of caspase-3/9 in hypertrophic scar fibroblasts transfected with miR-222 mimic or miR-222 inhibitor
Fig 11 Electropherogram of matrix metalloproteinase 1 protein expression in hypertrophic scar fibroblasts
Fig 12 Protein and mRNA expression of matrix metalloproteinase 1 in hypertrophic scar fibroblasts transfected with miR-222 mimic or miR-222 inhibitor
Fig 13 Electropherogram of PCNA, cyclin D1 and caspase-3 protein expression in hypertrophic scar fibroblasts transfected with miR-222 mimic and pcDNA-MMP1
Fig 14 Protein expressions of PCNA, cyclin D1 and caspase-3 in hypertrophic scar fibroblasts transfected miR-222 mimic and miR-222 mimic+MMP1
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