Tumor necrosis factor-related apoptosis-inducing ligand is a novel transcriptional target of runt-related transcription factor 1
Tumor necrosis factor-related apoptosis-inducing ligand is a novel transcriptional target of runt-related transcription factor 1
复制标题
DOI：
10.3892/ijo.2021.5296
复制
发表时间：
2021-12
影响因子：
5.2
通讯作者：
Tatsushi Yoshida;K. Yamasaki;K. Tadagaki;Y. Kuwahara;Akifumi Matsumoto;Adèm Ejub Sofovic;Noriko Kondo;T. Sakai;Tsukasa Okuda
中科院分区：
文献类型：
--
作者：
Tatsushi Yoshida;K. Yamasaki;K. Tadagaki;Y. Kuwahara;Akifumi Matsumoto;Adèm Ejub Sofovic;Noriko Kondo;T. Sakai;Tsukasa Okuda
Runt-related transcription factor 1（RUNX 1），又称急性髓细胞白血病1（AML 1），是人类白血病中常见的基因组异常。RUNX 1编码一种转录因子，可以调节造血基因的表达。此外，肿瘤坏死因子相关凋亡诱导配体（TRAIL）在恶性肿瘤的免疫监视中发挥重要作用。然而，TRAIL表达的调控机制仍有待充分阐明。在本研究中，十四酰佛波醇13-乙酸酯处理的巨核细胞分化的K562细胞被用来检查RUNX 1对TRAIL表达的影响。对RUNX 1和核心结合因子β（CBFβ）表达载体共转染的细胞进行TRAIL启动子的荧光素酶测定系列，以评估TRAIL转录调控的性质。还对TRAIL启动子的RUNX 1共有序列与重组RUNX 1和CBFβ蛋白进行了电泳迁移率变动分析。对急性髓性白血病患者的TRAIL表达进行BloodSpot数据库分析。通过逆转录-定量PCR（RT-qPCR）评估用RUNX 1表达载体和RUNX 1 siRNA转染的K562细胞中TRAIL、其受体死亡受体4和5以及RUNX 1的表达。还通过RT-qPCR在用RUNX 1-ETO siRNA转染的Kasumi-1细胞和用RUNX 1-ETO表达质粒转染的KG-1细胞中测量了TRAIL和RUNX 1-ETO表达。用TRAIL和HDAC抑制剂丁酸钠或丙戊酸处理Kasumi-1、KG-1、SKNO-1和K562细胞，进行细胞计数、乳酸脱氢酶测定和流式细胞术分析细胞周期。本研究表明RUNX 1是TRAIL的转录调节因子。最初发现，人白血病细胞巨核细胞分化后TRAIL表达的诱导是RUNX 1依赖性的。随后，发现RUNX 1的过表达通过激活其启动子活性来增加TRAIL mRNA的表达。进一步的分析表明，RUNX 1以间接的方式调节TRAIL的表达，因为RUNX 1保留了在所有可能的RUNX 1共有位点突变后激活该启动子的能力。此外，在携带t（8;21）易位的白血病细胞中，TRAIL表达减少，其中RUNX 1-ETO嵌合蛋白干扰正常的RUNX 1功能。发现重组TRAIL蛋白的外源性处理诱导白血病细胞死亡。总之，本研究提供了一种新的机制，即TRAIL是RUNX 1的靶基因，RUNX 1-ETO抑制了TRAIL的表达。这些结果表明，TRAIL是一种有希望的药物，用于临床治疗t（8;21）AML。
Runt-related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13-acetate-treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co-transfected with RUNX1 and core-binding factor β (CBFβ) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFβ proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription-quantitative PCR (RT-qPCR). TRAIL and RUNX1-ETO expression was also measured in Kasumi-1 cells transfected with RUNX1-ETO siRNA and in KG-1 cells transfected with RUNX1-ETO expression plasmid, both by RT-qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi-1, KG-1, SKNO-1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1-dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1-ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1-ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.