miR-222通过MMP1对增生性瘢痕成纤维细胞的调控机制研究

Micro RNA-222 regulates the proliferation of fibroblasts in hypertrophic scar via matrix metalloproteinase 1

目的观察成纤维胶原酶1(MMP1)和miR-222在增生性瘢痕(HS)成纤维细胞中的表达水平,探讨miR-222对HS发生、发展的调控机制。方法选取36例HS患者的HS组织,并各留取HS旁正常组织作为对照。分离培养出HS组织与正常组织的成纤维细胞;采用RT-PCR法检测成纤维细胞MMP1 m RNA和miR-222表达水平;采用Western blot法检测成纤维细胞MMP1蛋白表达水平;采用MTT法检测成纤维细胞增殖情况;采用双荧光素酶报告实验检测miR-222是否可调控MMP1基因。结果与正常组织比较,HS组织成纤维细胞MMP1 m RNA、蛋白表达水平均下调(均P<0.05),miR-222表达水平上调(P<0.05)。与空白对照成纤维细胞比较,HS组织转染MMP1过表达质粒后的成纤维细胞MMP1 m RNA、蛋白表达水平均上调(均P<0.05),增殖速度明显减慢(P<0.05);转染antagomiR-222质粒后的成纤维细胞miR-222表达水平下调(P<0.05),MMP1 m RNA表达水平上调(P<0.05),增殖速度明显减慢(P<0.05)。双荧光素酶报告实验结果表明miR-222能与MMP1基因的3′-UTR区相结合,从而调控其表达。结论 miR-222在HS组织中存在过表达,miR-222或通过负调控其靶基因MMP1的表达而发挥其调控成纤维细胞增殖和凋亡的作用。

Objective To determine the expressions of matrix metalloproteinase 1 (MMP1) and miR - 222 in fibroblasts ofhypertrophic scar (HS), and investigate the regulatory mechanism of miR- 222 on the occurrence and development of HS.Methods HS tissues and HS- adjacent normal tissues were both collected from 36 HS patients. Primary fibroblasts were obtained.qRT- PCR was used to measure the level of mRNA of MMP1 and miR- 222. Western blotting was carried out to determine MMP1protein expression. MTT assay was employed to detect the proliferation of fibroblasts. Dual luciferase reporter assay wasperformed to identify the binding of miR- 222 with MMP1 mRNA. Results MMP1 mRNA/protien were significantly downregulatedand miR- 222 was significantly upregulated in HS fibroblasts, compared with control fibroblasts (P <0.05). The expression ofMMP1mRNA/protein were significantly upregulated (P<0.05),and the cell proliferation abilities was significantly inhibited in HSfibroblasts after transfected with pcDNAMMP1(P<0.05). miR- 222 was downregulated (P<0.05), MMP1 mRNA was upregulatedand the cell proliferation abilities was inhibited (P<0.05)in HS fibroblasts after transfected with antagomiR- 222,Dual- LuciferaseReporter assay showed miR- 222 regulated the expression of MMP1 by binding with its 3′- UTR. Conclusion miR- 222overexpressed in HS fibroblasts, miR- 222 may regulate the proliferation and apoptosis of fibroblasts in HS via negatively regulating the expression of target gene MMP1.