Objective: 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.
Methods: 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.
Results: 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.
Conclusion: MiR-21 may be involved in regulating the proliferation of MM cells by inhibiting the expression of KLF5.
题目: MiR-21靶向调控KLF5促进多发性骨髓瘤细胞增殖的机制研究.
目的: 探讨多发性骨髓瘤（MM）细胞中miR-21的表达及其作用机制。.
方法: 从30例MM患者及20例健康者骨髓标本中分选出浆细胞，检测miR-21的表达水平；选取MM1.S、RPMI-8226和U266细胞，用qRT-PCR方法检测miR-21的水平；转染hsa-miR-21 mimics和hsa-miR-21 inhibitor后通过CCK-8和细胞克隆实验检测MM细胞增殖情况；预测miR-21调控的靶基因，通过荧光酶素报告基因实验检测miR-21与KLF5的结合位点；利用Western blot和qRT-PCR方法检测转染hsa-miR-21 mimics和hsa-miR-21 inhibitor后KLF5蛋白的表达水平；合成缺失3′UTR的KLF5质粒转染进入过表达miR-21的RPMI-8226细胞，利用CCK-8和细胞克隆实验检测MM细胞增殖情况。.
结果: MM患者浆细胞中miR-21表达水平较正常对照组显著升高（P<0.001）；MM1.S、RPMI-8226和U266细胞中miR-21的表达较对照组也显著升高（P<0.05）。转染hsa-miR-21 mimics后细胞增殖和克隆形成数增加，转染hsa-miR-21 inhibitor后细胞增殖能力明显减弱和克隆形成数明显减少（P<0.01）；荧光酶素报告基因实验结果显示miR-21直接结合KLF5的3′UTR，转染hsa-miR-21 mimics后KLF5蛋白的表达水平明显减低，转染hsa-miR-21 inhibitor后明显升高；将缺失3′UTR的KLF5质粒转染进入miR-21过表达的RPMI-8226细胞后，细胞增殖和克隆形成数显著减弱。.
结论: miR-21可能通过抑制KLF5的表达参与调控MM细胞增殖。.
Keywords: KLF5; cell proliferation; miR-21; multiple myeloma.