摘要
目的探索c-Myc/miR-27b-3p/Bcl-2轴促进多发性骨髓瘤(multiple myeloma,MM)存活的分子机制。方法以骨髓间充质干细胞(bone marrow stem cells,BMSCs)为对照组,qPCR法测定人MM细胞系RPMI8226和U266细胞中MM相关基因和microRNAs(miRNAs)的相对表达水平。TargetScan和双荧光素酶报告基因方法分别预测和验证miR-27b-3p和Bcl-2的直接序列互作关系。通过分离14名新疆医科大学第五附属医院临床确诊为MM的患者的原代MM细胞,qPCR法测定其中c-Myc、Bcl-2的mRNA及miR-27b-3p的相对表达水平;Pearson相关性分析3者的相关性。构建腺病毒过表达c-Myc(c-Myc OE)或Bcl-2(Bcl-2 OE)载体以及miR-27b-3p抑制物(miR-27b-3p inhibitor)载体并转染BMSCs。蛋白质印迹测定BMSCs中p-STAT3、STAT3、p-JAK2和JAK2的表达水平。结果与BMSCs细胞相比,c-Myc和Bcl-2 mRNA在RPMI8226和U266细胞中均显著上调(P<0.01),miR-27b-3p在RPMI8226和U266细胞中均显著下调(P<0.01)。荧光素酶报告基因结果显示miR-27b-3p直接靶向Bcl-2的3′-UTR区域。qPCR及Pearson相关性分析结果显示,c-Myc mRNA与miR-27b-3p RNA相对表达水平、miR-27b-3p RNA与Bcl-2 mRNA相对表达水平呈线性负相关(P<0.01);而c-Myc mRNA与Bcl-2 mRNA相对表达水平呈线性正相关(P<0.01)。与BMSCs组相比,BMSCs+c-Myc OE组、BMSCs+miR-27b-3p inhibitor组、BMSCs+Bcl-2 OE组细胞中p-STAT3、STAT3、p-JAK2、JAK2的表达水平均升高(P<0.05)。结论MM通过上调c-Myc的表达抑制miR-27b-3p从而激活Bcl-2,促进MM细胞存活。
Objective To explore the molecular mechanisms of c-Myc/miR-27b-3p/Bcl-2 axis on promoting multiple myeloma(MM)cell survival.Methods The relative expression levels of MM-related genes and microRNAs(miRNAs)were measured in human MM cell lines RPMI8226 and U266 cells by using qPCR analysis.TargetScan and dual-luciferase gene reporter assay were used to predict and verify the targeting relationship between miR-27b-3p and Bcl-2,respectively.The primary MM cells were isolated from 14 patients with MM recruited from the Fifth Affiliated Hospital of Xinjiang Medical University.The mRNA expression levels of c-Myc,Bcl-2,and the expression level of miR-27b-3p in primary MM cells were detected by qPCR.Pearson correlation analysis was used to analyze the relationship among their expression levels.Adenovirus vectors with c-Myc or Bcl-2 overexpression(Bcl-2 OE,c-Myc OE),or miR-27b-3p inhibitor(miR-27b-3p inhibitor)were constructed and transfected into the BMSCs.The expression levels of p-STAT3,STAT3,p-JAK2,and JAK2 in BMSCs were detected by Western blotting.Results Compared with those in the BMSCs,the mRNA expression levels of c-Myc and Bcl-2 were upregulated in the RPMI8226 cells and the U266 cells(P<0.01),and miR-27b-3p was downregulated in the RPMI8226 cells and the U266 cells(P<0.01).Dual-luciferase gene reporter assay results showed that miR-27b-3p was directly targeted to the Bcl-23′-UTR.qPCR and Pearson correlation analysis showed that the relative expression levels of c-Myc mRNA and miR-27b-3p,miR-27b-3p and Bcl-2 mRNA were linearly and negatively correlated(P<0.01),while the relative expression levels of c-Myc mRNA and Bcl-2 mRNA were linearly and positively correlated(P<0.01).Compared with those in the BMSCs group,the expression levels of p-STAT3,STAT3,p-JAK2,and JAK2 in the BMSCs+c-Myc OE group,the BMSCs+miR-27b-3p inhibitor group,and the BMSCs+Bcl-2 OE group were all increased(P<0.05).Conclusion c-Myc upregulation and miR-27b-3p inhibition activated the Bcl-2 to promote cell survival in MM.
作者
薛琴
田新玮
王静
郑琦丽
艾比巴·阿不都拉
XUE Qin;TIAN Xinwei;WANG Jing;ZHENG Qili;Aibiba Abudula(Department of Rheumatology,Immunology and Hematology,the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi,830000,China)
出处
《医学分子生物学杂志》
2025年第4期332-338,共7页
Journal of Medical Molecular Biology
基金
新疆维吾尔自治区自然科学基金(No.2020D01C327)。
