DNMT3A 受 Stat5a 下调调控，并通过抑制 Jak2 细胞中 miR-17-5p/Cdkn1a 轴来介导 G0/G1 期阻滞。
Dnmt3a is downregulated by Stat5a and mediates G0/G1 arrest by suppressing the miR-17-5p/Cdkn1a axis in Jak2 cells.
机构信息
Tongji University School of Medicine, Shanghai, 200092, China.
Department of Gastroenterology, Tongji Hospital of Tongji University, Shanghai, 200065, China.
出版信息
BMC Cancer. 2021 Nov 13;21(1):1213. doi: 10.1186/s12885-021-08915-0.
BACKGROUND
Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2 mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2 mutation and its effects on downstream signaling pathways in cMPNs.
METHODS
Specimens of Jak2 positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes.
RESULTS
Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2 positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2 positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest.
CONCLUSIONS
Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2 cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.
背景
尽管在经典骨髓增殖性肿瘤（cMPN）患者中经常报道 Dnmt3a 异常，但很少有研究探讨 Jak2 突变如何调节 Dnmt3a。在这项研究中，我们研究了 Jak2 突变如何调节 Dnmt3a 及其对 cMPN 下游信号通路的影响。
方法
收集 Jak2 阳性 cMPN 患者和正常对照的标本。使用小鼠 BaF3 细胞系构建细胞模型。通过双荧光素酶报告基因检测和染色质免疫沉淀（ChIP）-qPCR 检测 Stat5a 对 Dnmt3a 转录活性的影响。软琼脂集落形成实验和细胞计数实验检测细胞增殖。BrdU 染色和流式细胞术检测细胞周期分布。Western blot 和定量逆转录 PCR（qPCR）检测基因表达水平。
结果
首先，Western blot 和 qPCR 的结果表明，与对照样本相比，Jak2 阳性样本中 Dnmt3a 下调。然后我们探讨了其背后的机制，发现 Dnmt3a 是 Stat5a 的下游靶标，在 Jak2 阳性样本中，异常激活的 Stat5a 与 Dnmt3a 启动子结合，抑制 Dnmt3a 的转录和翻译。我们进一步揭示了 Dnmt3a 启动子第一个外显子上游约 800bp 的区域，该区域包含 Stat5a 的γ激活序列（GAS）基序，是 Stat5a 结合的特定位点。软琼脂集落形成实验、细胞计数实验、BrdU 染色和流式细胞术发现，Jak2-BaF3 细胞中的 Dnmt3a 通过抑制 miR-17-5p/Cdkn1a 轴介导的 Cdkn1a，显著影响细胞增殖能力和细胞周期分布，导致 G0/G1 期阻滞。
结论
Jak2 细胞中 Stat5a 与 Dnmt3a 启动子结合，下调 Dnmt3a 的转录和翻译。Dnmt3a 启动子上的 GAS 基序是 Stat5a 结合的精确位点。Dnmt3a 通过调节 miR-17-5p/Cdkn1a 轴导致 G0/G1 期阻滞。Stat5a/Dnmt3a/miR-17-5p/Cdkn1a 轴可能为 cMPN 提供治疗靶点。
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