miR-148b-3p调控瘢痕疙瘩来源的成纤维细胞增殖的机制研究

Mechanism of miR-148b-3p regulating proliferation of keloid derived fibroblasts

目的分析miR-148b-3p对瘢痕疙瘩来源的成纤维细胞增殖的影响。方法通过Real-time PCR分析人瘢痕疙瘩来源成纤维细胞(HKF)和正常人成纤维细胞(NFs)中miR-148b-3p和SPARC的表达水平。通过Western blot检测细胞中富含半胱氨酸的酸性分泌蛋白(SPARC)蛋白表达水平。利用细胞计数试剂盒8(CCK-8)法和平板克隆形成分析miR-148b-3p和SPARC对HKF增殖的影响。使用在线分析软件预测miR-148b-3p的靶基因,随后构建荧光素酶报告基因质粒,通过荧光素酶报告基因法分析miR-148b-3p与靶基因的靶向结合位点。结果miR-148b-3p在HKF中低表达,SPARC在HKF中高表达。在HKF细胞中转染miR-148b-3p能够下调SPARC的表达,并抑制细胞增殖。在线分析软件预测miR-148b-3p能够靶向结合SPARC的3′-UTR;双荧光素酶报告基因的结果进一步证实miR-148b-3p能够靶向作用于SPARC的3′-UTR。在转染miR-148b-3p的HKF中再转染SPARC真核表达质粒,能够抵消miR-148b-3p的作用,恢复细胞增殖能力。结论miR-148b-3p通过靶向作用于SPARC的3′-UTR,抑制其表达,抑制HKF增殖。

Objective To analyze the effect of miR-148b-3p on the proliferation of keloid derived fibroblasts.Methods The expression levels of miR-148b-3p and SPARC in human keloid derived fibroblasts(HKF)and normal human fibroblasts(NFS)were analyzed by real time PCR.The expression level of SPARC protein was detected by Western blot.The effects of miR-148b-3p and SPARC on HKF proliferation were analyzed by CCK-8 method and plate clone formation.The target gene of miR-148b-3p was predicted using online analysis software,and then the luciferase reporter plasmid was constructed.The targeted binding site of miR-148b-3p and the target gene was analyzed by luciferase reporter gene method.Results miR-148b-3p was low expressed in HKF and SPARC was high expressed in HKF.Transfection of miR-148b-3p in HKF cells could down regulate the expression of SPARC and inhibit cell proliferation.Online analysis software predicted that miR-148b-3p could target the 3′-UTR binding SPARC;The results of dual luciferase reporter gene further confirmed that miR-148b-3p could target the 3′-UTR of SPARC.Transfection of SPARC eukaryotic expression plasmid into HKF transfected with miR-148b-3p could counteract the effect of miR-148b-3p and restore cell proliferation.Conclusion miR-148b-3p can inhibit the proliferation of HKF by targeting the 3′-UTR of SPARC and inhibiting its expression.