Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran.
Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Biol Res. 2020 Jun 5;53(1):25. doi: 10.1186/s40659-020-00293-4.
Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition.
We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT.
Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and β-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl. The gene expression of VEGF, vimentin, and β-catenin and protein level of β-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of β-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant.
bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.
缺氧诱导因子-1（HIF-1）被认为是对低氧水平或缺氧反应最活跃的转录因子。HIF-1 结合不同基因启动子中的低氧反应元件（HRE）序列，主要通过 bHLH 结构域激活基因转录，特别是那些参与血管生成和 EMT 的基因。鉴于 bHLH 在将 HIF-1 结合到 HRE 序列中的关键作用，我们假设 bHLH 可能是缺氧条件下有前途的靶向候选物。
我们在 pIRES2-EGFP 载体中插入了一个抑制性 bHLH（ibHLH）结构域，并将对照载体或设计的构建体转染到 HEK293T 细胞中。ibHLH 结构域由 HIF-1a 和 Arnt 的 bHLH 结构域组成，能够与 HIF-1 竞争结合 HRE 序列。然后用 200µM 氯化钴（CoCl）处理转染细胞 48 小时以诱导缺氧。进行实时 PCR 和 Western blot 以评估 ibHLH 对涉及血管生成和 EMT 的基因和蛋白的影响。
成功诱导了 HEK293T 细胞系中的缺氧，因为在用 200µM CoCl 处理未转染的 HEK293T 细胞后，VEGF、波形蛋白和β-连环蛋白的基因表达显著增加。在转染对照或 ibHLH 载体的细胞中，VEGF、波形蛋白和β-连环蛋白的基因表达以及β-连环蛋白的蛋白水平在缺氧时显著降低。然而，与对照载体相比，ibHLH 对这些基因和β-连环蛋白的蛋白水平均无效。我们还观察到，与对照载体相比，ibHLH 过表达对 N-钙粘蛋白的基因和蛋白表达具有更强的抑制作用。然而，这没有统计学意义。
bHLH 已被报道为参与 HIF 与 DNA 结合活性的重要结构域。然而，我们发现靶向该结构域不足以抑制内源性 HIF-1 转录活性。进一步研究 HIF-1 的关键结构域的功能对于开发特异性 HIF-1 抑制剂是必要的。
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