长链非编码RNA-PAX8-AS1沉默通过miR-378g/ERBB2轴降低髓系白血病细胞活力、增强细胞凋亡并抑制阿霉素耐药性。
LncRNA-PAX8-AS1 Silencing Decreases Cell Viability, Enhances Apoptosis, and Suppresses Doxorubicin Resistance in Myeloid Leukemia via the miR-378g/ERBB2 Axis.
作者信息
Song Xiaolu, Chen Yirui, Peng Ye, Wang Xiaogang, Zheng Sujie, Shi Fangfang, Lan Jianping
机构信息
Cancer Center, Department of Hematology, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, Zhejiang 310014, China.
Laboratory Medicine Center, Department of Clinical Laboratory, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, Zhejiang 310014, China.
出版信息
Evid Based Complement Alternat Med. 2022 Oct 6;2022:2295044. doi: 10.1155/2022/2295044. eCollection 2022.
OBJECTIVE
Considering the role of lncRNAs reported as regulators in acute myeloid leukemia (AML) progression, the current research aims to investigate the role of PAX8-AS1 in chemo-resistant AML.
METHODS
Human AML cells HL60 and human doxorubicin (ADM)-resistant AML cells (HL60/ADM cells) were used to establish models of chemo-sensitive AML and refractory/recurrent AML, respectively. CCK-8 assay and flow cytometry were used to determine cell resistance to ADM, viability, and apoptosis. PAX8-AS1, miR-378g, and ERBB2 expressions in the models and/or AML patients were quantified via qRT-PCR or Western blot. The miRNA/mRNA axis targeted by PAX8-AS1 was analyzed using Starbase, TargetScan, or GEO and validated through a dual-luciferase reporter assay. The expressions of Bcl-2, Bax, and C Caspase-3 in cells were quantitated by Western blot.
RESULTS
The highly expressed PAX8-AS1 was observed in AML patients and HL60 cells, which was more evident in refractory/recurrent AML patients and HL60/ADM cells. Compared with that in ADM-treated parental HL60 cells, the viability of ADM-treated HL60/ADM cells remained strong. PAX8-AS1 overexpression increased viability and Bcl-2 expression, while diminishing apoptosis, Bax, and C Caspase-3 expressions in HL60 cells. However, the abovementioned aspects were oppositely impacted by PAX8-AS1 silencing in HL60/ADM cells. PAX8-AS1 directly targeted miR-378g, whose expression pattern is opposite to that of PAX8-AS1 in AML. MiR-378g upregulation abrogated the effects of PAX8-AS1 overexpression on HL60 cells. MiR-378g downregulation offset PAX8-AS1 silencing-induced effects on HL60/ADM cells. Moreover, ERBB2 was recognized as the target of miR-378g, with a higher expression in HL60/ADM cells than in HL60 cells.
CONCLUSION
PAX8-AS1 silencing decreases cell viability, enhances apoptosis, and suppresses ADM resistance in AML via regulating the miR-378g/ERBB2 axis.
目的
鉴于长链非编码RNA（lncRNAs）在急性髓系白血病（AML）进展中作为调节因子的作用，本研究旨在探究PAX8-AS1在化疗耐药AML中的作用。
方法
分别用人AML细胞HL60和人阿霉素（ADM）耐药AML细胞（HL60/ADM细胞）建立化疗敏感AML模型和难治/复发AML模型。采用CCK-8法和流式细胞术检测细胞对ADM的耐药性、活力及凋亡情况。通过qRT-PCR或蛋白质印迹法对模型和/或AML患者中PAX8-AS1、miR-378g和ERBB2的表达进行定量分析。利用Starbase、TargetScan或GEO分析PAX8-AS1靶向的miRNA/mRNA轴，并通过双荧光素酶报告基因检测进行验证。通过蛋白质印迹法定量检测细胞中Bcl-2、Bax和Caspase-3的表达。
结果
在AML患者和HL60细胞中观察到PAX8-AS1高表达，在难治/复发AML患者和HL60/ADM细胞中更明显。与ADM处理的亲本HL60细胞相比，ADM处理的HL60/ADM细胞活力仍然很强。PAX8-AS1过表达增加了HL60细胞的活力和Bcl-2表达，同时减少了细胞凋亡、Bax和Caspase-3表达。然而，在HL60/ADM细胞中，PAX8-AS1沉默对上述方面产生了相反的影响。PAX8-AS1直接靶向miR-378g，其在AML中的表达模式与PAX8-AS1相反。miR-378g上调消除了PAX8-AS1过表达对HL60细胞的影响。miR-378g下调抵消了PAX8-AS1沉默对HL60/ADM细胞的影响。此外，ERBB2被确认为miR-378g的靶标，在HL60/ADM细胞中的表达高于HL60细胞。
结论
PAX8-AS1沉默通过调节miR-378g/ERBB2轴降低AML细胞活力，增强细胞凋亡，并抑制AML对ADM的耐药性。
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