Objective: To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism.
Methods: The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively.
Results: Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells.
Conclusion: TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.
题目: TRIM31基因沉默对U266细胞增殖及凋亡的影响及其机制.
目的: 探讨三结构域蛋白31（TRIM31）基因沉默对多发性骨髓瘤细胞增殖及凋亡的影响及其可能机制.
方法: 以正常人骨髓浆细胞为对照，用RT-qPCR和Western blot实验检测人多发性骨髓瘤细胞株（U266、RPMI-8226、NCI-H929和KMS-11）中TRIM31 mRNA和蛋白表达水平。用RT-qPCR法检测含有shRNA-TRIM31的重组慢病毒载体及其对照载体感染之U266细胞中TRIM31 mRNA的水平；用CCK-8、软琼脂克隆形成实验和流式细胞术分别检测细胞增殖活性、克隆形成能力和凋亡率；Western blot检测细胞中TRIM31、cleaved-caspase-3、BCL-2、Bax、p-Akt（Ser473）、Akt和PI3K（p110α）等蛋白的表达水平。用PI3K/Akt信号通路特异性抑制剂LY294002和TRIM31-shRNA慢病毒联合干预U266细胞，用CCK-8、流式细胞术检测细胞增殖活性和凋亡率，Western blot检测细胞中p-Akt（Ser473）和Akt蛋白表达水平.
结果: 与正常人骨髓浆细胞比较，U266、RPMI-8226、NCI-H929和KMS-11细胞中TRIM31 mRNA和蛋白表达水平显著升高（P<0.001），其中U266细胞最为显著；慢病毒感染后，U266细胞中TRIM31 mRNA和蛋白水平显著降低（P<0.001）；沉默TRIM31可显著抑制U266细胞增殖（P<0.05），降低细胞克隆形成能力，提高细胞凋亡率，并上调cleaved-caspase-3和Bax蛋白水平，下调BCL-2、p-Akt（Ser473）和PI3K（p110α）蛋白水平，而对Akt蛋白无显著影响；LY294002干预显著增强TRIM31基因沉默对U266细胞增殖的抑制作用以及细胞凋亡的促进作用.
结论: TRIM31基因沉默可抑制U266细胞增殖，并促进其凋亡，其机制可能与PI3K/Akt信号通路抑制有关.