郝家祺, 文静, 刘春城, 蔡禄. 微小RNA-18a靶向Notch2抑制NIH-3T3细胞外基质相关基因的表达[J]. 环境与职业医学, 2022, 39(7): 786-791. DOI: 10.11836/JEOM21336
引用本文: 郝家祺, 文静, 刘春城, 蔡禄. 微小RNA-18a靶向Notch2抑制NIH-3T3细胞外基质相关基因的表达[J]. 环境与职业医学, 2022, 39(7): 786-791. DOI: 10.11836/JEOM21336
HAO Jiaqi, WEN Jing, LIU Chuncheng, CAI Lu. Inhibiting effect of miR-18a on expression of extracellular matrix-related genes of NIH-3T3 by targeting Notch2[J]. Journal of Environmental and Occupational Medicine, 2022, 39(7): 786-791. DOI: 10.11836/JEOM21336
Citation: HAO Jiaqi, WEN Jing, LIU Chuncheng, CAI Lu. Inhibiting effect of miR-18a on expression of extracellular matrix-related genes of NIH-3T3 by targeting Notch2[J]. Journal of Environmental and Occupational Medicine, 2022, 39(7): 786-791. DOI: 10.11836/JEOM21336

微小RNA-18a靶向Notch2抑制NIH-3T3细胞外基质相关基因的表达

Inhibiting effect of miR-18a on expression of extracellular matrix-related genes of NIH-3T3 by targeting Notch2

  • 摘要: 背景 矽肺主要病理特征是肺部纤维化。矽肺发生发展过程中多种 miRNAs 具有调控作用。

    目的 利用成纤维细胞系,探究微小RNA-18a(miR-18a)对细胞外基质相关基因表达的影响,并对其作用机制进行验证。

    方法 在成纤维细胞系NIH-3T3细胞中转染miR-18a模拟物以及神经源性基因座Notch同源蛋白2(Notch2)基因的小干扰RNA(siRNA)。通过应用实时定量逆转录聚合酶链反应(qRT-PCR)技术检测Acta2Col1a1Notch2基因mRNA表达变化,通过蛋白质印迹技术检测α-平滑肌肌动蛋白(α-SMA)及Notch2蛋白的表达变化,利用双荧光素酶报告载体在人胚肾细胞HEK293T细胞中验证miR-18a调控Notch2基因的直接作用位点。

    结果 qRT-PCR检测结果表明,在NIH-3T3细胞中,miR-18a模拟物过表达36 h抑制了Col1a1以及Acta2的mRNA表达(P < 0.05),同时利用蛋白质印迹技术检测发现,miR-18a模拟物过表达48 h后α-SMA蛋白表达丰度降低。通过qRT-PCR未检测到过表达miR-18a 36 h对于 Notch2基因表达的影响,通过蛋白质印迹技术检测发现过表达miR-18a模拟物 36 h可以在蛋白水平上抑制Notch2的表达。双荧光素酶报告载体检测结果发现,在HEK293T细胞中,无论是过表达miR-18a模拟物或者抑制物 24 h均证明了Notch2是 miR-18a 的直接靶基因。当Notch2被抑制36 h时,qRT-PCR检测发现Acta2Col1a1基因表达下调(P < 0.05);蛋白质印迹技术检测表明α-SMA在蛋白水平也受到抑制。

    结论 本研究发现miR-18a可以通过直接作用于靶基因Notch2的3’UTR而抑制其表达,从而抑制成纤维细胞系NIH-3T3细胞外基质相关基因的表达。

     

    Abstract: Background The main pathological feature of silicosis is pulmonary fibrosis. Multiple miRNAs regulate the development of silicosis.

    Objective Using a fibroblast cell line, to explore the effect of miR-18a on the expression of extracellular matrix-related genes, and verify the mechanism.

    Methods The fibroblast cell line NIH-3T3 cells were transfected with miR-18a mimics or neurogenic locus notch homolog protein 2 (Notch2) small interfering RNA (siRNA). The mRNA expression changes ofActa2, Col1a1, and Notch2 were detected by real-time quantitative reverse transcription PCR (qRT-PCR), α-smooth muscle actin (α-SMA) and Notch2 were also detected at the protein level by Western blotting. To verify whether miR-18a could directly act on the complementary sequences of the Notch2 gene, human embryonic kidney HEK293T cells and the psiCHECKTM-2 vector were used.

    Results The results of qRT-PCR showed that in NIH-3T3 cells, the over-expression of miR-18a mimics for 36 h inhibited the mRNA expression of Col1a1 and Acta2 (P<0.05). The results of Western blotting showed that the protein expression abundance of α-SMA was decreased at 48 h of miR-18a mimics over-expression. The qRT-PCR results showed that the over-expression of miR-18a for 36 h had no significant effect onNotch2 gene expression, but the Western blotting results showed that the over-expression of miR-18a mimics inhibited the expression of Notch2 at the protein level. The results of the dual luciferase reporter vector assay showed that in HEK293T cells, both over-expressed miR-18a mimics and inhibitors for 24 h demonstrated that Notch2 is a direct target gene of miR-18a. When Notch2 was inhibited for 36 h, the qRT-PCR results showed that Acta2 and Col1a1 were down-regulated (P < 0.05), and the results of Western blotting showed that α-SMA protein was also inhibited.

    Conclusion The findings indicate that miR-18a could inhibit the expression of extracellular matrix-related genes of NIH-3T3 cells by directly acting on the 3’UTR of target gene Notch2.

     

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