Chen Jianying, Li Yuhua, Xie Xiaoling
Department of Rheumatology, Hunan Provincial People's Hospital, Changsha, Hunan 410012, P.R. China.
Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510006, P.R. China.
Exp Ther Med. 2020 Aug;20(2):1169-1175. doi: 10.3892/etm.2020.8771. Epub 2020 May 19.
The present study aimed to investigate the effects of microRNA (miR)-425 on the proliferation of chronic lymphocytic leukaemia (CLL) cells and the possible underlying mechanisms. The expression of miR-425 was determined in the B lymphocytes of CLL patients and in normal B lymphocytes by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, MEC-1 cells transfected with miR-425 negative control (NC) or miR-425 mimic were examined. The cell proliferation of different groups was evaluated using an MTT assay, and cell cycle distribution was evaluated using flow cytometry analysis. A dual-luciferase reporter assay was used to verify whether Bruton's tyrosine kinase (BTK) was a target of miR-425. Furthermore, the expression levels of BTK, phospholipase Cγ2 (PLCγ2), Ki-67 and proliferating cell nuclear antigen (PCNA) were determined by RT-qPCR and western blotting. The results revealed that the expression of miR-425 was significantly downregulated in B lymphocytes obtained from CLL patients as compared with that in normal B lymphocytes. When cells were transfected with miR-425 mimic, the proliferation of MEC-1 cells was significantly inhibited at 24, 48 and 72 h compared with the proliferation of control cells. Additionally, the ratio of G0/G1 cells was significantly increased and the ratio of G2/M cells was significantly decreased in miR-425-overexpressing cells compared with that in control cells. The luciferase reporter assay revealed that miR-425 binds to the 3'-untranslated region of BTK mRNA. Finally, BTK, PLCγ2, Ki-67 and PCNA expression was significantly inhibited at the mRNA and protein level in cells where miR-425 was upregulated. In conclusion, miR-425 inhibits the proliferation of MEC-1 cells, potentially by inhibiting BTK/PLCγ2 signalling, and Ki-67 and PCNA expression levels. These results provide a deeper insight for understanding the development of CLL and suggest a potential novel target for the treatment of CLL patients.
本研究旨在探讨微小RNA（miR）-425对慢性淋巴细胞白血病（CLL）细胞增殖的影响及其潜在机制。通过逆转录-定量聚合酶链反应（RT-qPCR）检测CLL患者B淋巴细胞及正常B淋巴细胞中miR-425的表达。此外，对转染了miR-425阴性对照（NC）或miR-425模拟物的MEC-1细胞进行检测。采用MTT法评估不同组细胞的增殖情况，通过流式细胞术分析评估细胞周期分布。使用双荧光素酶报告基因检测法验证布鲁顿酪氨酸激酶（BTK）是否为miR-425的靶标。此外，通过RT-qPCR和蛋白质印迹法检测BTK、磷脂酶Cγ2（PLCγ2）、Ki-67和增殖细胞核抗原（PCNA）的表达水平。结果显示，与正常B淋巴细胞相比，CLL患者来源的B淋巴细胞中miR-与425的表达显著下调。当细胞转染miR-425模拟物时，与对照细胞相比，MEC-1细胞在24、48和72小时的增殖受到显著抑制。此外，与对照细胞相比，miR-425过表达细胞中G0/G1期细胞比例显著增加，G2/M期细胞比例显著降低。荧光素酶报告基因检测显示，miR-425与BTK mRNA的3'-非翻译区结合。最后，在miR-425上调的细胞中，BTK、PLCγ2、Ki-67和PCNA在mRNA和蛋白质水平的表达均受到显著抑制。总之，miR-425可能通过抑制BTK/PLCγ2信号通路以及Ki-67和PCNA的表达水平来抑制MEC-1细胞的增殖。这些结果为深入了解CLL的发病机制提供了依据，并为CLL患者的治疗提出了潜在的新靶点。
