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miR-29c-3p/IGF1分子轴对肝星状细胞活化,增殖和凋亡的作用机制

张梁 王保全 雷喜锋 王旭 柯阳 张玮

张梁, 王保全, 雷喜锋, 王旭, 柯阳, 张玮. miR-29c-3p/IGF1分子轴对肝星状细胞活化,增殖和凋亡的作用机制[J]. 昆明医科大学学报, 2023, 44(9): 7-14. doi: 10.12259/j.issn.2095-610X.S20230926
引用本文: 张梁, 王保全, 雷喜锋, 王旭, 柯阳, 张玮. miR-29c-3p/IGF1分子轴对肝星状细胞活化,增殖和凋亡的作用机制[J]. 昆明医科大学学报, 2023, 44(9): 7-14. doi: 10.12259/j.issn.2095-610X.S20230926
Liang ZHANG, Baoquan WANG, Xifeng LEI, Xu WANG, Yang KE, Wei ZHANG. Effect of miR-29c-3p/IGF1 Molecular Axis on Activation,Proliferation and Apoptosis of Hepatic Stellate Cells[J]. Journal of Kunming Medical University, 2023, 44(9): 7-14. doi: 10.12259/j.issn.2095-610X.S20230926
Citation: Liang ZHANG, Baoquan WANG, Xifeng LEI, Xu WANG, Yang KE, Wei ZHANG. Effect of miR-29c-3p/IGF1 Molecular Axis on Activation,Proliferation and Apoptosis of Hepatic Stellate Cells[J]. Journal of Kunming Medical University, 2023, 44(9): 7-14. doi: 10.12259/j.issn.2095-610X.S20230926

miR-29c-3p/IGF1分子轴对肝星状细胞活化,增殖和凋亡的作用机制

doi: 10.12259/j.issn.2095-610X.S20230926
基金项目: 云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目(202001AY070001-147)
详细信息
    作者简介:

    张梁(1985~),男,陕西渭南人,医学学士,主治医师,主要从事胃肠肿瘤及各类肝炎的临床治疗与基础研究工作

    通讯作者:

    柯阳,E-mail:keyang1218@126.com

    张玮,E-mail:Zwww2168864@163.com

  • 中图分类号: R735.2

Effect of miR-29c-3p/IGF1 Molecular Axis on Activation,Proliferation and Apoptosis of Hepatic Stellate Cells

  • 摘要:   目的  探讨miR-29c-3p通过IGF-1对肝星状细胞(hepatic stellate cells,HSCs)活化,增殖和凋亡的影响。  方法  原代培养小鼠HSCs,并通过免疫荧光检测HSCs标志物ɑ-SMA表达。双荧光素酶报告基因实验验证miR-29c-3p和IGF-1的靶向关系。TGF-β激活HSCs,并且外源性调控miR-29c-3p和IGF-1的表达水平后,分别采用Westernbolt,CCK-8,克隆形成实验和流式细胞术检测活化HSCs中活化相关蛋白(ɑ-SMA,DDR2,FN1,ITGB1和GFAP)的表达,增殖,克隆形成数和凋亡。  结果  ɑ-SMA阳性表达表明成功分离小鼠HSCs。miR-29c-3p mimic可降低野生型IGF-1的荧光素酶活性,但是对突变型IGF-1没有影响。过表达miR-29c-3p和低表达IGF-1能减少ɑ-SMA,DDR2,FN1和ITGB1表达,增加GFAP的表达,并且降低HSCs的增殖活力和克隆形成数,上调其凋亡比例。  结论  miR-29c-3p通过靶向抑制IGF-1表达,进而抑制HSCs活化和增殖,并促进其凋亡。
  • 图  1  成功分离HSCs,并且miR-29c-3p在不同状态的HSCs中差异表达

    A:采用IF检测HSCs标志物ɑ-SMA是否表达;B:TGF-β处理HSCs前后,WB检测活化相关蛋白(ɑ-SMA,DDR2,FN1,ITGB1和GFAP)的表达;C:在静息及激活状态HSCs中,miR-29c-3p的表达差异。**P < 0.01,***P < 0.001。

    Figure  1.  Successful isolation of HSCs and differential expression of miR-29c-3p in HSCs of different status.

    图  2  miR-29c-3p抑制HSCs的活化和增殖,并促进其凋亡

    A:采用RT-qPCR检测miR-29c-3p mimic的转染效率;B:通过WB检测miR-29c-3p对活化相关蛋白(ɑ-SMA,DDR2,FN1,ITGB1和GFAP)表达的影响;C:CCK-8试剂盒检测不同组别中TGF-β激活的HSCs增殖活力;D:克隆形成实验检测miR-29c-3p对活化HSCs的克隆形成数的影响;E:流式细胞术检测活化HSCs的凋亡比例。**P < 0.01,***P < 0.001。

    Figure  2.  miR-29c-3p inhibits the activation and proliferation of HSCs and promotes their apoptosis.

    图  3  IGF-1是miR-29c-3p下游靶标mRNA

    A:Starbase数据库预测得到的miR-29c-3p与IGF-1的潜在3’ UTR结合序列(上),并且突变IGF-1的3’ UTR结合序列后,双荧光素酶报告基因实验验证miR-29c-3p与IGF-1的靶向关系(下);B:活化的HSCs中分别转染miR-29c-3p inhibitor和miR-29c-3p mimic,采用WB检测IGF-1的表达变化。**P < 0.01。

    Figure  3.  IGF-1 is a downstream target mRNA of miR-29c-3p.

    图  4  miR-29c-3p通过IGF-1抑制HSCs的活化和增殖,并促进其凋亡

    A: miR-29c-3p inhibior的转染效率;B:通过WB检测得到的sh-IGF-1的转染效率;C:不同组别HSCs中活化相关蛋白(ɑ-SMA,DDR2,FN1,ITGB1和GFAP)表达的表达变化;D:CCK-8实验检测活化的HSCs增殖活力;E:克隆形成实验得到的不同组别中活化HSCs的克隆形成数;F:流式细胞术检测活化HSCs的凋亡比例。与sh-NC组比较,aP < 0.05,aaP < 0.01,aaaP < 0.001;与sh-IGF-1组比较,bP < 0.05,bbP < 0.01,bbbP < 0.001;*P < 0.05,***P < 0.001。

    Figure  4.  miR-29c-3p inhibits the activation and proliferation of HSCs and promotes their apoptosis through IGF-1.

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  • 收稿日期:  2023-04-26
  • 网络出版日期:  2023-09-22
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