miR-203过表达对人瘢痕成纤维细胞增殖的影响-现代医学

miR-203过表达对人瘢痕成纤维细胞增殖的影响

单位：1. 陕西省人民医院烧伤整形医学美容外科, 陕西西安 710068;
2. 陕西省人民医院神经内二科, 陕西西安 710068

分类号：R619.6

出版年·卷·期（页码）：2017·36·第五期（655-660）

摘要：

目的：探讨miR-203过表达对人瘢痕成纤维细胞增殖的影响及其机制。方法：组织块法培养原代人瘢痕成纤维细胞6、12、24和48 h；miR-203模拟物（miR-203 mimics）转染人瘢痕成纤维细胞并培养24 h；采用CCK-8检测细胞的存活率；流式细胞术Annexin V/PI双染法检测细胞凋亡；双荧光素酶报告基因检测miR-203与p53基因家族成员截短型p63（ΔNp63）的靶标关系；qRT-PCR检测miR-203和ΔNp63 mRNA的表达水平；Western blotting检测ΔNp63和P53的蛋白表达水平。结果：miR-203在瘢痕成纤维细胞中表达明显降低（P < 0.01），且呈时间依赖性；转染miR-203 mimics后作用24 h，miR-203表达水平显著增加（P < 0.01）；miR-203过表达使细胞存活率降低至42%（P < 0.01），细胞凋亡率增高至45%（P < 0.01）；miR-203与ΔNp63存在直接靶标关系；miR-203过表达可显著抑制ΔNp63的mRNA和蛋白表达水平，同时显著上调P53的蛋白表达水平（P < 0.01）。结论：miR-203过表达可抑制人瘢痕成纤维细胞增殖，其机制可能是通过靶向抑制ΔNp63的表达进而上调P53的蛋白表达。

Objective: To investigate the effect of miR-203 overexpression on the cell proliferation of human keloid fibroblasts and further elucidate its molecular mechanism. Methods: Human keloid fibroblasts were cultured by tissue cultivation in vitro for 6,12,24 and 48 h. Human keloid fibroblasts were transfected with miR-203 mimics and incubated for 24 h. Cell survival was detected by CCK-8 assay. Cell apoptosis was tested with Annexin V/propidium iodide (PI) double staining method. The target relationship between miR-203 and delta p63 (ΔNp63), a novel member of p53 gene family, was analyzed using dual-luciferase reporter assay.The miR-203 expression level was examined by qRT-PCR.The protein expression levels of ΔNp63 and P53 were detected by Western blotting.Results: The expression of miR-203 was significantly decreased in human keloid fibroblasts in a time-dependent manner (P<0.01).Transfection with miR-203 mimics for 24 h significantly increased miR-203 expression level (P<0.05) and CCK-8 results showed that miR-203 overexpression markedly decreased cell survival rate of human keloid fibroblasts to 42% (P<0.05).Annexin V/PI double staining assay showed that miR-203 overexpression significantly increased cell apoptosis to 45%(P<0.05).Dual-luciferase reporter assay showed that the miR-203 and ΔNp63 had a directly targeting relationship.Overexpression of miR-203 significantly inhibited the protein expression of ΔNp63 as well as increased the protein expression of P53. Conclusion: Overexpression of miR-203 can inhibit the cell proliferation of human keloid fibroblasts and the potential underlying mechanism may be associated with targeting and suppressing ΔNp63 expression leading to promoting P53 protein expression that may inhibit the formation of keloid.

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