Wnt5a对肝星状细胞增殖、迁移和衰老的调控作用

The regulation of Wnt5a on proliferation,migration and senescence of hepatic stellate cells

目的寻找抗纤维化的治疗方案和治疗靶点具有重要意义。文章旨在探讨Wnt蛋白家族成员5a(Wnt5a)对肝星状细胞(HSCs)增殖、克隆形成、迁移能力以及衰老的影响。方法构建胆汁淤积性肝纤维化大鼠模型,利用芯片分析差异表达的基因。用不同浓度的TGF-β1刺激肝星状细胞,将实验分为3组,分别为只加完全培养基的对照组以及用5μg/L和10μg/L TGF-β1刺激的处理组,通过qRT-PCR、Western blot和免疫荧光检测平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原α1(COL1A1)和Wnt5a的表达。Wnt5a的siRNA(si-Wnt5a)转染LX-2细胞后,通过qRT-PCR和Western Blot检测LX-2细胞中α-SMA、COL1A1、Wnt5a的mRNA和蛋白表达;通过CCK-8、集落形成、Transwell、划痕、β-半乳糖苷酶染色分别检测LX-2细胞的增殖、集落形成、迁移能力以及细胞的衰老情况。利用生物信息学网站预测与Wnt5a有靶向关系的miRNAs,STRING数据库分析与Wnt5a有相互作用的蛋白,并用Cytoscape绘制蛋白-蛋白互作图。结果BDL大鼠芯片结果显示Wnt5a是纤维化肝组织中显著上调的基因之一。10μg/L TGF-β1处理细胞后,LX-2细胞中α-SMA[(10.81±1.82)、(1.57±0.12)]、COL1A1[(9.83±3.27)、(1.41±0.11)]、Wnt5a[(8.17±0.90)、(1.29±0.03)]mRNA和蛋白表达升高(均P<0.05)。Wnt5a的敲降抑制了LX-2细胞中α-SMA、COL1A1和Wnt5a mRNA和蛋白的表达,抑制了LX-2细胞的增殖能力、细胞集落形成能力和迁移能力(均P<0.05),而促进了细胞的衰老(P<0.05)。生物信息学网站预测结果显示Wnt5a与34个miRNAs有靶向关系,STRING数据库分析表明Wnt5a与10个蛋白相互作用密切。结论Wnt5a通过改变细胞的增殖、迁移、衰老等生物学功能,进而实现促进肝纤维化的作用。

Objective Liver fibrosis is a reversible pathological reparative response generated by liver injury,therefore it is important to find therapeutic options and targets against fibrosis.This study aims to investigate the effects of Wnt protein family member 5a(Wnt5a)on the proliferation,clonogenesis,migration and senescence of hepatic stellate cells(HSCs).Methods A rat model of cholestatic liver fibrosis was established,and the differentially expressed genes were analyzed by microarray.Hepatic stellate cells were stimulated with different concentrations of TGF-β1.The experiment was divided into three groups:control group with complete culture medium and treatment group with 5μg/L and 10μg/L TGF-β1 stimulation,and then the expressions ofα-smooth muscle actin(α-SMA),collagen type Iα1(COL1A1)and Wnt5a were detected by qRT-PCR,Western Blot and Immunofluorescence.After Wnt5a siRNA(si-Wnt5a)was transfected into LX-2 cells,the mRNA and protein expressions ofα-SMA,COL1A1 and Wnt5a in LX-2 cells were detected by qRT-PCR and Western Blot.The proliferation,colony formation,migration capacity,and cellular senescence of LX-2 cells were examined by CCK-8,colony formation,Transwell,wounding healing andβ-galactosidase staining,respectively.Bioinformatics websites were used to predict miRNAs which have a target relationship with Wnt5a;STRING database and Cytoscape were used to draw the protein-protein interaction network of Wnt5a.Results Microarray results in BDL rats revealed that Wnt5a was one of the significantly upregulated genes in fibrotic liver tissue.The mRNA and protein expressions ofα-SMA[(10.81±1.82),(1.57±0.12)],COL1A1[(9.83±3.27),(1.41±0.11)]and Wnt5a[(8.17±0.90),(1.29±0.03)]were increased in LX-2 cells treated with 10μg/L TGF-β1(all P<0.05).Knockdown of Wnt5a suppressed the mRNA and protein expression ofα-SMA,COL1A1 and Wnt5a in LX-2 cells,and inhibited the proliferation,colony formation and migration of LX-2 cells(all P<0.05)and promoted cell senescence(P<0.05).Bioinformatics websites prediction showed that Wnt5a has a target relationship with 34 miRNAs,and STRING database analysis showed that Wnt5a interacts closely with 10 proteins.Conclusion Wnt5a promotes liver fibrosis by altering the biological functions of cells such as proliferation,migration,and senescence.