miR-223-3p调控人肝星状细胞增殖及迁移的作用及分子机制

Role and molecular mechanism of miR-223-3p in regulating the proliferation and migration of human hepatic stellate cells

目的探讨miR-223-3p抑制人肝星状细胞LX-2增殖、迁移的作用及分子机制。方法以TGF-β1诱导活化的LX-2细胞为研究对象,分为空白对照组、miR-223-3p mimics组、NC mimics组,分别采用空白溶剂、miR-223-3p mimics、模拟物对照(NC mimics)转染干预24,48 h。采用CCK-8法检测miR-223-3p mimics对LX-2细胞增殖活性的影响,划痕实验检测miR-223-3p mimics对LX-2细胞迁移的抑制作用。采用miRNA靶基因预测数据库Targetscan、miRanda预测miR-223-3p的靶基因,并利用双荧光素酶报告基因实验验证靶基因,RT-qPCR和Western blot法分别检测miR-223-3p mimics对NLRP3、Caspase-1、IL-1βmRNA及蛋白表达的影响。结果与空白组及NC mimics相比,miR-223-3p mimics组LX-2细胞增殖和迁移能力显著降低(P<0.05)。靶基因预测结果显示NLRP3与miR-223-3p碱基序列存在8个连续的碱基互补配对,表明NLRP3可能为miR-223-3p的下游靶基因。双荧光素酶报告基因实验显示,NLRP3为miR-223-3p的靶基因。实时荧光定量PCR及Western blot检测结果显示,与空白组及阴性对照NC mimics组相比,miR-223-3p mimics组LX-2细胞中NLRP3、Caspase-1、IL-1βmRNA及蛋白的表达显著降低(P<0.05)。结论miR-223-3p通过靶向作用于NLRP3/Caspase-1/IL-1β信号通路进而抑制人肝星状细胞株LX-2的增殖及迁移。

Objective To investigate the role and mechanism of miR-223-3p in inhibiting the proliferation and migration of human hepatic stellate cells LX-2.Methods TGF-β1-induced activated LX-2 cells were divided into blank control group,miR-223-3p mimics group and NC mimics group,and transfected with blank solvent,miR-223-3p mimics,and mimics control(NC mimics)for 24,48 h,respectively.CCK-8 method was used to detect the effect of miR-223-3p mimics on the proliferative activity of LX-2 cells,and the scratch assay was used to detect the inhibitory effect of miR-223-3p mimics on the migration of LX-2 cells.The miRNA target gene prediction database Targetscan and miRanda were used to predict the target gene of miR-223-3p,and the target gene was verified using dual luciferase reporter gene assay.RT-qPCR and Western blot assay were performed to detect the effect of miR-223-3p mimics on NLRP3,Caspase-1,IL-1βmRNA and protein expression.Results Compared with blank group and NC mimics group,the proliferation and migration abilities of LX-2 cells were significantly reduced in miR-223-3p mimics group(P<0.05).The target gene prediction results showed there were eight consecutive base complementary pairings between NLRP3 and miR-223-3p base sequences,indicating that NLRP3 may be the downstream target gene of miR-223-3p.Dual luciferase reporter gene assay showed that NLRP3 was the target gene of miR-223-3p.The results of RT-qPCR and Western blot assay showed that the expression of NLRP3,Caspase-1,IL-1βmRNA and protein in LX-2 cells in miR-223-3p mimics group was significantly lower than that in blank group and NC mimics group(P<0.05).Conclusion miR-223-3p targets the NLRP3/Caspase-1/IL-1βsignaling pathway to inhibit the proliferation and migration of human hepatic stellate cell line LX-2.