microRNA-223-3p靶向微管相关蛋白1B抑制肝星状细胞活化的机制研究
DOI: 10.3969/j.issn.1001-5256.2023.12.015
利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:谢文涛负责实验操作,撰写论文;鱼康康负责课题设计,指导并修改论文;程琦负责指导实验,修改论文;李宁负责课题设计,指导撰写论文并定稿。
Mechanism of microRNA-223-3p inhibiting hepatic stellate cell activation by targeting microtubule-associated protein 1B
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摘要:
目的 探讨microRNA-223-3p (miR-223-3p)对肝星状细胞活化的影响及机制。 方法 选取人肝星状细胞系LX2细胞为研究对象,采用TGF-β刺激LX2细胞的方式构建肝星状细胞活化模型,通过实时荧光定量PCR检测miR-223-3p在肝星状细胞活化过程中表达水平变化;转染miR-223-3p mimic至LX2细胞,通过实时荧光定量PCR、蛋白免疫印迹法(Western Blot)及细胞免疫荧光明确miR-223-3p对肝星状细胞活化的调控作用;通过双荧光素酶报告基因实验验证miR-223-3p与靶基因MAP1B的关系;转染MAP1B的siRNA至LX2细胞,通过Western Blot明确抑制MAP1B表达对肝星状细胞活化的影响;转染miR-223-3p至LX2细胞,通过实时荧光定量PCR和Western Blot验证miR-223-3p对MAP1B的调控作用。计量资料两组间比较采用成组t检验。 结果 与静息状态相比,肝星状细胞处于活化状态时,miR-223-3p表达水平下降(t=9.12,P<0.001)。过表达miR-223-3p能抑制肝星状细胞活化标志物平滑肌肌动蛋白及Ⅰ型胶原的mRNA(t值分别为8.35、12.23,P值均<0.01)和蛋白(t值分别为16.24、20.90,P值均<0.001)表达水平。双荧光素酶报告基因实验证实MAP1B是miR-223-3p的潜在靶基因。与对照组相比,过表达miR-223-3p的LX2上MAP1B的mRNA表达(t=5.95,P<0.01)及蛋白表达(t=11.12,P<0.001)水平均显著降低。 结论 miR-223-3p可通过靶向MAP1B抑制肝星状细胞活化。 Abstract:Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on hepatic stellate cell (HSC) activation and its mechanism. Methods Human HSC LX2 cells were selected for the study, and LX2 cells were stimulated by TGF-β to establish a model of HSC activation; quantitative real-time PCR was used to measure the change in the expression level of miR-223-3p during HSC activation. After LX2 cells were transfected with miR-223-3p mimic, quantitative real-time PCR, Western blot, and immunofluorescence assay were used to clarify the regulatory effect of miR-223-3p on HSC activation, and dual-luciferase reporter assay was used to verify the association between miR-223-3p and the target gene MAP1B. After LX2 cells were transfected with MAP1B siRNA, Western blot was used to clarify the influence of inhibiting MAP1B expression on HSC activation; after LX2 cells were transfected with miR-223-3p, quantitative real-time PCR and Western blot were used to verify the regulatory effect of miR-223-3p on MAP1B. The independent-samples t test was used for comparison of continuous data between two groups. Results HSC in the activated state had a significant reduction in the expression level of miR-223-3p compared with those in the resting state (t=9.12, P<0.001). Overexpression of miR-223-3p inhibited the mRNA and protein expression levels of the markers for HSC activation alpha-smooth muscle actin and collagen type Ⅰ (mRNA expression: t=8.35 and 12.23, both P<0.01; protein expression: t=16.24 and 20.90, both P<0.001). The dual-luciferase reporter assay confirmed that MAP1B was a potential target gene of miR-223-3p. Compared with the control group, LX2 cells with miR-223-3p overexpression had significant reductions in the mRNA and protein expression levels of MAP1B (mRNA expression: t=5.95, P<0.01; protein expression: t=11.12, P<0.001). Conclusion This study shows that miR-223-3p can inhibit HSC activation by targeting MAP1B. -
Key words:
- Hepatic Fibrosis /
- Microtubule-Associated Proteins /
- Hepatic Stellate Cells /
- MicroRNAs
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