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

谢文涛; 鱼康康; 程琦; 李宁

doi:10.3969/j.issn.1001-5256.2023.12.015

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

复旦大学附属华山医院感染科，上海市传染病与生物安全应急响应重点实验室，国家传染病医学中心，上海 200040

基金项目:

国家自然科学基金 (8200033704);

国家自然科学基金 (81770565)

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。

详细信息

通信作者:
李宁， lining_hs@fudan.edu.cn （ORCID： 0000-0002-4375-2857）

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出版历程
- 收稿日期: 2022-12-09
- 录用日期: 2023-05-19
- 出版日期: 2023-12-12

Mechanism of microRNA-223-3p inhibiting hepatic stellate cell activation by targeting microtubule-associated protein 1B

Department of Infectious Diseases，Huashan Hospital，Fudan University；Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response；National Medical Center for Infectious Diseases，Shanghai 200040，China

Research funding:

National Natural Science Fundation of China (8200033704);

National Natural Science Fundation of China (81770565)

More Information

Corresponding author: LI Ning， lining_hs@fudan.edu.cn （ORCID： 0000-0002-4375-2857）

摘要

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。
- 肝纤维化 /
- 微管相关蛋白质类 /
- 肝星状细胞 /
- 微小RNAs
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.
- Hepatic Fibrosis /
- Microtubule-Associated Proteins /
- Hepatic Stellate Cells /
- MicroRNAs

HTML全文

图 1 miR-223-3p在HSC活化后表达下降

注： a，生物信息学比较静息态HSC与活化态HSC中miR-223-3p表达水平；b，Western Blot验证TGF-β激活HSC；c，实时荧光定量PCR验证miR-223-3p在HSC活化前后表达水平变化。

Figure 1. miR-223-3p expression decreased during HSC activation

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图 2 miR-223-3p对 HSC活化的影响

注： a、b，实时荧光定量PCR验证过表达miR-223-3p对HSC活化标志物mRNA水平的影响；c，Western Blot验证过表达miR-223-3p对HSC活化标志物的影响。

Figure 2. Effect of miR-223-3p on the activation of HSC

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图 3 细胞免疫荧光验证过表达miR-223-3p对HSC活化标志物的影响

注：比例尺为200 μm。

Figure 3. Effect of overexpression of miR-223-3p on hepatic stellate activation markers verified by cellular immunofluorescence

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图 4 MAP1B是miR-223-3p的靶基因

注： a，miR-223-3p与MAP1B 3′UTR存在互补结合位点；b，双荧光素酶报告基因验证MAP1B为miR-223-3p的靶基因。

Figure 4. MAP1B is the target gene for miR-223-3p

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图 5 抑制MAP1B对HSC活化的影响

注： a，Western Blot验证抑制MAP1B的表达对HSC活化标志物的影响；b、c，过表达miR-223-3p抑制MAP1B mRNA和蛋白水平的表达。

Figure 5. Effect of MAP1B inhibition on HSC activation

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