Division of Hematology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam Road, Pokfulam, Hong Kong.
Cell Commun Signal. 2021 May 27;19(1):62. doi: 10.1186/s12964-021-00707-0.
miR-1250 is localised to the second intron of AATK at chromosome 17q25. As a CpG island is present at the putative promoter region of its host gene, AATK, we postulated that the intronic miR-1250-5p is a tumor suppressor miRNA co-regulated with its host gene, AATK, by promoter DNA methylation in non-Hodgkin's lymphoma (NHL).
AATK/miR-1250 methylation was studied in healthy controls, including ten normal peripheral blood buffy coats and eleven normal tonsils, ten lymphoma cell lines, and 120 primary lymphoma samples by methylation-specific PCR (MSP). The expression of miR-1250-5p and AATK was investigated by quantitative real-time PCR. Tumor suppressor properties of miR-1250-5p were demonstrated by over-expression of precursor miR-1250-5p in lymphoma cells. The target of miR-1250-5p was verified by luciferase reporter assay.
AATK/miR-1250 methylation was absent in healthy peripheral blood and tonsils, but detected in five (50%) NHL cell lines. AATK/miR-1250 methylation correlated with repression of miR-1250-5p and AATK in NHL cell lines. In completely methylated SU-DHL-6 and SUP-T1 cells, treatment with 5-AzadC led to promoter demethylation and re-expression of both miR-1250-5p and AATK. In primary lymphoma samples, AATK/miR-1250 was frequently methylated in B-cell lymphoma (n = 41, 44.09%) and T-cell lymphoma (n = 9, 33.33%) with a comparable frequency (P = 0.318). In SU-DHL-6 and SU-DHL-1 cells, restoration of miR-1250-5p resulted in decreased cellular proliferation by MTS assay, increased cell death by trypan blue staining and enhanced apoptosis by annexin V-PI assay. Moreover, MAPK1 and WDR1 were verified as direct targets of miR-1250-5p by luciferase assay. In 39 primary NHLs, miR-1250-5p expression was shown to be inversely correlated with each of MAPK1 (P = 0.05) and WDR1 (P = 0.031) by qRT-PCR. Finally, in SU-DHL-1 cells, overexpression of miR-1250-5p led to repression of MAPK1 and WDR1 at both transcript and protein levels, with downregulation of phospho-ERK2 by Western-blotting and inhibition of SDF-1-dependent cell migration by transwell assay.
miR-1250-5p is a novel tumor suppressive intronic miRNA co-regulated and silenced by promoter DNA methylation of its host gene AATK in NHL. MAPK1 and WDR1 are novel miR-1250-5p direct targets rendering inhibition of MAPK/ERK signaling and SDF-1-dependent cell migration, hence implicated in survival and dissemination of lymphoma. Video Abstract.
miR-1250 定位于 17q25 染色体上的 AATK 第二内含子。由于其宿主基因 AATK 的启动子区域存在 CpG 岛，我们推测内含子 miR-1250-5p 是一种肿瘤抑制 miRNA，其表达受到非霍奇金淋巴瘤（NHL）中启动子 DNA 甲基化的调控。
采用甲基化特异性 PCR（MSP）法检测健康对照组（包括 10 例正常外周血血涂片和 11 例正常扁桃体）、10 种淋巴瘤细胞系和 120 例原发性淋巴瘤样本中的 AATK/miR-1250 甲基化情况。通过实时定量 PCR 检测 miR-1250-5p 和 AATK 的表达。通过在淋巴瘤细胞中过表达前体 miR-1250-5p 来证明 miR-1250-5p 的肿瘤抑制特性。通过荧光素酶报告基因检测验证 miR-1250-5p 的靶标。
健康外周血和扁桃体中未检测到 AATK/miR-1250 甲基化，但在 5 种（50%）NHL 细胞系中检测到。AATK/miR-1250 甲基化与 NHL 细胞系中 miR-1250-5p 和 AATK 的抑制相关。在完全甲基化的 SU-DHL-6 和 SUP-T1 细胞中，用 5-AzadC 处理可导致启动子去甲基化和 miR-1250-5p 和 AATK 的重新表达。在原发性淋巴瘤样本中，B 细胞淋巴瘤（n=41，44.09%）和 T 细胞淋巴瘤（n=9，33.33%）中 AATK/miR-1250 频繁甲基化，频率相当（P=0.318）。在 SU-DHL-6 和 SU-DHL-1 细胞中，miR-1250-5p 的恢复导致 MTS 测定的细胞增殖减少、台盼蓝染色的细胞死亡增加和 annexin V-PI 测定的细胞凋亡增强。此外，通过荧光素酶测定验证 MAPK1 和 WDR1 是 miR-1250-5p 的直接靶标。在 39 例原发性 NHL 中，通过 qRT-PCR 显示 miR-1250-5p 的表达与 MAPK1（P=0.05）和 WDR1（P=0.031）呈负相关。最后，在 SU-DHL-1 细胞中，miR-1250-5p 的过表达导致 MAPK1 和 WDR1 的转录和蛋白水平下调，Western blot 检测到磷酸化 ERK2 下调，transwell 测定抑制 SDF-1 依赖性细胞迁移。
miR-1250-5p 是一种新型肿瘤抑制性内含子 miRNA，其宿主基因 AATK 的启动子 DNA 甲基化可调控其表达并使其沉默。MAPK1 和 WDR1 是 miR-1250-5p 的新型直接靶标，可抑制 MAPK/ERK 信号通路和 SDF-1 依赖性细胞迁移，从而影响淋巴瘤的存活和扩散。视频摘要。
