Zhou Nan, Cao Shu-Xing, Luo Jian-Min, Liu Xiao-Jun, Yang Lin
Department of Hematology,The Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Provine, China.
Department of Orthopedics, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Provine, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct;30(5):1482-1489. doi: 10.19746/j.cnki.issn.1009-2137.2022.05.027.
To study the expression of miR-21 in multiple myeloma (MM) cell lines and plasma cells of patients, and explore the mechanism of miR-21 in MM.
Bone marrow samples from 30 patients with MM and 18 healthy controls were collected. The plasma cells were separated by magnetic beads. MM cell lines (MM1.S cells, RPMI-8226 cells and U266 cells) were cultured. The expression level of miR-21 was detected by real-time fluorescent quantitative PCR (qRT-PCR). After transfection with hsa-miR-21 mimics and hsa-miR-21 inhibitor, the proliferation of MM cells was detected by CCK-8 and cell cloning assay. The target genes regulated by miR-21 were predicted by bioinformatics website. The binding sites of miR-21 and KLF5 were detected by luciferase reporter gene assay. The expression of KLF5 were detected by Western blot and qRT-PCR after hsa-miR-21 mimics and hsa-miR-21 inhibitor were transfected into RPMI-8226 cells. KLF5 plasmid with 3'UTR knockout was synthesized and cotransfected into RPMI-8226 cells with hsa-miR-21 mimics, and the proliferation of MM cells was detected by CCK-8 and cell cloning assay.
Compared with healthy donors, the expression level of miR-21 in plasma cells of patients with MM was significantly increased (P<0.001); the expression of miR-21 in MM cell lines MM1.S, RPMI-8226 and U266 was significantly higher than that in control group (P<0.05). After hsa-miR-21 mimics transfection, the proliferation and the number of colony formation of MM cells was significantly increased, while the proliferation and the number of colony formation of MM cells was decreased after hsa-miR-21 inhibitor transfection (P<0.01). The results of luciferase reporter gene assay showed that miR-21 could bind to 3'UTR of KLF5, and the expression level of KLF5 protein was significantly decreased after hsa-miR-21 mimics transfection. After 3'UTR-knockout KLF5 plasmid and hsa-miR-21 mimics were cotransfected into RPMI-8226 cells, the proliferation of the cells was significantly decreased.
MiR-21 may be involved in regulating the proliferation of MM cells by inhibiting the expression of KLF5.
研究微小RNA-21（miR-21）在多发性骨髓瘤（MM）细胞系及患者浆细胞中的表达情况，探讨miR-21在MM中的作用机制。
收集30例MM患者及18例健康对照者的骨髓样本，采用磁珠法分离浆细胞，培养MM细胞系（MM1.S细胞、RPMI-8226细胞和U266细胞）。采用实时荧光定量聚合酶链反应（qRT-PCR）检测miR-21的表达水平。转染hsa-miR-21模拟物和hsa-miR-21抑制剂后，采用细胞计数试剂盒-8（CCK-8）法和细胞克隆实验检测MM细胞的增殖情况。通过生物信息学网站预测miR-21调控的靶基因，采用荧光素酶报告基因实验检测miR-21与Krüppel样因子5（KLF5）的结合位点。将hsa-miR-21模拟物和hsa-miR-21抑制剂转染至RPMI-8226细胞后，采用蛋白质免疫印迹法和qRT-PCR检测KLF5的表达。构建3'非翻译区（3'UTR）敲除的KLF5质粒，与hsa-miR-21模拟物共转染至RPMI-8226细胞，采用CCK-8法和细胞克隆实验检测MM细胞的增殖情况。
与健康供者相比，MM患者浆细胞中miR-21的表达水平显著升高（P<0.001）；MM细胞系MM1.S、RPMI-8226和U266中miR-21的表达显著高于对照组（P<0.05）。转染hsa-miR-21模拟物后，MM细胞的增殖及克隆形成数量显著增加，转染hsa-miR-21抑制剂后，MM细胞的增殖及克隆形成数量减少（P<0.01）。荧光素酶报告基因实验结果显示，miR-21可与KLF5的3'UTR结合，转染hsa-miR-21模拟物后，KLF5蛋白表达水平显著降低。将3'UTR敲除的KLF5质粒与hsa-miR-21模拟物共转染至RPMI-8226细胞后，细胞增殖显著降低。
miR-21可能通过抑制KLF5的表达参与调控MM细胞的增殖。
