Pan Di, Yang Wanwan, Zeng Yao, Li Wenjun, Wang Kaizhen, Zhao Li, Li Jia, Ye Yuting, Guo Qinglong
Department of Physiology, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing 211198, Jiangsu, China; State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, China.
Department of Physiology, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing 211198, Jiangsu, China.
Cell Signal. 2021 Aug;84:110038. doi: 10.1016/j.cellsig.2021.110038. Epub 2021 May 11.
Drug resistance is an important cause of death for most patients with chronic myeloid leukemia (CML). The bone marrow microenvironment is believed to be mainly responsible for resistance to BCR-ABL tyrosine kinase inhibitors. The mechanism involved, however, is still unclear.
Bioinformatic analysis from GEO database of AKR1C3 was utilized to identify the AKR1C3 expression in CML cells under bone marrow microenvironment. Western blot and qPCR were performed to detect the AKR1C3 expression in two CML cell lines K562 and KU812 cultured +/- bone microenvironment derived stromal cells. CCK-8, soft agar colony assay, and Annexin V/PI assay were performed to detect the sensitivity of CML cells (K562 and KU812) to Imatinib under a gain of or loss of function of AKR1C3 treatment. The CML murine model intravenous inoculated with K562-OE-vector and K562-OE-AKR1C3 cells were established to estimate the effect of AKR1C3 inhibitor Indomethacin on Imatinib resistance. The bioinformatic analysis of miRNA databases was used to predict the potential miRNAs targeting AKR1C3. And the luciferase assay was utilized to validate the target relationship between miR-379-5p and AKR1C3. And, the soft agar colony assay and Annexin V/PI were used to validate the effect of miR-379-5p in AKR1C3 induced Imatinib resistance.
In present study, we investigated AKR1C3 was highly expressed in CML under bone marrow microenvironment. AKR1C3 decreased Imatinib activity in K562 and KU812 cells, while inhibition of AKR1C3 could enhance Imatinib sensitivity in vitro study. Furthermore, murine model results showed combination use of AKR1C3 inhibitor Indomethacin effectively prolong mice survival, indicating that AKR1C3 is a promising target to enhance Imatinib treatment. Mechanically, AKR1C3 was found to be suppressed by miR-379-5p, which was down-expression in bone marrow microenvironment. Besides, we found miR-379-5p could bind AKR1C3 3'UTR but not degrade its mRNA level. Further, gain of miR-379-5p rescued the imatinib resistance induced by AKR1C3 overexpression in CML cells.
Altogether, our study identifies a novel signaling regulation of miR-379-5p/AKR1C3/EKR axis in regulating IM resistance in CML cell, and provides a scientific base for exploring AKR1C3 as a biomarker in impeding IM resistance in CML.
耐药是大多数慢性髓性白血病（CML）患者死亡的重要原因。骨髓微环境被认为是导致对BCR-ABL酪氨酸激酶抑制剂耐药的主要原因。然而，其中涉及的机制仍不清楚。
利用来自GEO数据库的关于AKR1C3的生物信息学分析，来鉴定骨髓微环境下CML细胞中AKR1C3的表达。进行蛋白质免疫印迹法（Western blot）和定量聚合酶链反应（qPCR），以检测在有/无骨髓微环境来源的基质细胞培养条件下，两种CML细胞系K562和KU812中AKR1C3的表达。进行细胞计数试剂盒-8（CCK-8）、软琼脂集落试验和膜联蛋白V/碘化丙啶（Annexin V/PI）试验，以检测在AKR1C3功能获得或缺失处理下，CML细胞（K562和KU812）对伊马替尼的敏感性。建立静脉接种K562-OE-载体和K562-OE-AKR1C3细胞的CML小鼠模型，以评估AKR1C3抑制剂吲哚美辛对伊马替尼耐药性的影响。利用miRNA数据库的生物信息学分析来预测靶向AKR1C3的潜在miRNA。并利用荧光素酶试验来验证miR-379-5p与AKR1C3之间的靶向关系。此外，使用软琼脂集落试验和Annexin V/PI试验来验证miR-379-5p在AKR1C3诱导的伊马替尼耐药中的作用。
在本研究中，我们调查发现AKR1C3在骨髓微环境下的CML中高表达。AKR1C3降低了K562和KU812细胞中伊马替尼的活性，而在体外研究中抑制AKR1C3可增强伊马替尼的敏感性。此外，小鼠模型结果显示，联合使用AKR1C3抑制剂吲哚美辛可有效延长小鼠生存期，表明AKR1C3是增强伊马替尼治疗效果的一个有前景的靶点。从机制上来说，发现AKR1C3受到miR-379-5p的抑制，而miR-379-5p在骨髓微环境中表达下调。此外，我们发现miR-379-5p可以结合AKR1C3的3'非翻译区（3'UTR），但不会降低其mRNA水平。进一步研究发现，miR-379-5p的增加挽救了CML细胞中由AKR1C3过表达诱导的伊马替尼耐药性。
总之，我们的研究确定了miR-379-5p/AKR1C3/EKR轴在调节CML细胞对伊马替尼耐药中的一种新的信号调节机制，并为探索将AKR1C3作为阻碍CML中伊马替尼耐药的生物标志物提供了科学依据。
