Department of Hematology, The First Affiliated Hospital of Zhejiang Chinese Medical Universi¬ty, Hangzhou, Zhejiang Province, China.
Department of Orthopedics, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang Province, China.
J Biol Regul Homeost Agents. 2019;33(3):721-729.
This study aimed to investigate the mechanism underlying the inhibitory effect of tumor suppressor gene miR-186 and zinc finger protein 545 (ZNF545) on the proliferation of multiple myeloma (MM) cells. CD138 magnetic beads were used to isolate different types of myeloma cell lines (KM3, U266, RPMI-8226, and H929), which were then infected by lentivirus carrying the miR-186 gene. Using uninfected myeloma cells as the control, MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide] assay was performed to calculate the rate of cell proliferation at different time points. In addition, the correlation between the expression of Jagged 1 and miR-186 was analyzed by real-time Polymerase Chain Reaction (PCR). Furthermore, the effect of 5-Aza-2-deoxycytidine and acetylase inhibitor Trichomycin A (TSA) on the expression of ZNF545 and proliferation/apoptosis of MM cells was investigated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blotting (WB), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] cell proliferation assay, and Annexin V-FITC/PI staining. Compared with the control group, the proliferation of miR-186-overexpressing U266 and RPMI-8226 cells was significantly decreased. In cell cloning experiments, miR-186 decreased the number of U266 and RPMI-8226 clones while reducing the protein expression of Jagged 1. The expression level of ZNF545 in myeloma patients was also reduced to some extent. ZNF545 protein also promoted the apoptosis of myeloma cells. By inhibiting the proliferation of myeloma cells, miR-186 gene and ZNF protein may be used as tumor suppressors in the treatment of myeloma.
本研究旨在探讨肿瘤抑制基因 miR-186 和锌指蛋白 545（ZNF545）对多发性骨髓瘤（MM）细胞增殖抑制作用的机制。使用 CD138 磁珠分离不同类型的骨髓瘤细胞系（KM3、U266、RPMI-8226 和 H929），然后用携带 miR-186 基因的慢病毒感染。以未感染的骨髓瘤细胞作为对照，采用 MTT [3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四唑溴盐，噻唑蓝四唑溴盐]法计算不同时间点的细胞增殖率。此外，通过实时聚合酶链反应（PCR）分析 Jagged 1 和 miR-186 之间的表达相关性。进一步采用逆转录聚合酶链反应（RT-PCR）、Western blot（WB）、MTS [3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑]细胞增殖检测、Annexin V-FITC/PI 染色研究 5-Aza-2-脱氧胞苷和乙酰酶抑制剂 Trichomycin A（TSA）对 ZNF545 的表达和 MM 细胞增殖/凋亡的影响。与对照组相比，miR-186 过表达 U266 和 RPMI-8226 细胞的增殖明显减少。在细胞克隆实验中，miR-186 减少了 U266 和 RPMI-8226 克隆的数量，同时降低了 Jagged 1 的蛋白表达。骨髓瘤患者 ZNF545 的表达水平也有所降低。ZNF545 蛋白也促进了骨髓瘤细胞的凋亡。通过抑制骨髓瘤细胞的增殖，miR-186 基因和 ZNF 蛋白可作为骨髓瘤治疗中的肿瘤抑制因子。
