微小RNA-335-3p靶向小鼠双微体基因调控骨髓瘤细胞的增殖和凋亡  

MiR-335-3p regulates proliferation and apoptosis of myeloma cells by targeting MDM2 gene

作　　者：于云祥[1] 龚泰芳[1] 刘小涛[1] 柯文[1] 李彬彬[1] YU Yunxiang;GONG Taifang;LIU Xiaotao;KE Wen;LI Binbin(Department of Orthopedics,Taihe Hospital,Shiyan,Shiyan,Hubei 442000,China)

机构地区：[1]十堰市太和医院骨一科,湖北十堰442000

出　　处：《安徽医药》2021年第12期2491-2495,共5页Anhui Medical and Pharmaceutical Journal

摘　　要：目的探讨微小RNA(miR)-335-3p对骨髓瘤细胞增殖和凋亡的影响及作用机制。方法2018年5月至2019年1月,培养正常骨髓细胞MNC和骨髓瘤细胞KM3和U266,实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测细胞中miR-335-3p表达水平。将KM3细胞分为miR-NC组(转染模拟对照序列)、miR-335-3p组[转染miR-335-3p模拟物(miR-335-3p mimics)]、pcDNA3.1+miR-335-3p组(共转染空载体和miR-335-3p mimics)和pcDNA3.1-MDM2+miR-335-3p组[共转染小鼠双微体基因(MDM2)过表达载体和miR-335-3p mimics],MTT法检测细胞增殖,流式细胞仪检测细胞凋亡的影响,蛋白质印迹法(Western blotting)检测细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖性激酶抑制剂1A(P21)、B淋巴细胞瘤-2(Bcl-2)蛋白和Bcl-2相关X(Bax)蛋白表达。双荧光素酶报告基因实验验证KM3细胞中miR-335-3p与MDM2调控关系。结果骨髓瘤细胞KM3和U266中miR-335-3p表达水平分别为0.24±0.01、0.38±0.02,显著低于正常骨髓细胞MNC中的miR-335-3p表达水平0.77±0.03(均P<0.05)。与miR-NC组比较,miR-335-3p组KM3细胞吸光度[48 h:(0.60±0.03)比(0.99±0.06);72 h:(0.82±0.05)比(1.67±0.07)]、细胞中cyclin D1和Bcl-2蛋白表达均降低(均P<0.05),细胞凋亡率[(24.31±0.99)%比(8.13±0.83)%]、细胞中P21和Bax蛋白表达均升高(均P<0.05)。miR-335-3p在KM3细胞中负调控MDM2表达。与pcDNA3.1+miR-335-3p组比较,pcDNA3.1-MDM2+miR-335-3p组KM3细胞吸光度[48 h:(0.80±0.05)比(0.63±0.04);72 h:(1.28±0.06)比(0.88±0.05)]、细胞中cyclin D1和Bcl-2蛋白表达均升高(均P<0.05),凋亡率[(15.34±0.66)%比(23.98±1.41)%]、细胞中P21、Bax蛋白表达均降低(均P<0.05)。结论miR-335-3p可能通过下调MDM2表达抑制骨髓瘤细胞的增殖,并诱导细胞凋亡。Objective To explore the effects of microRNA(miR)-335-3p on proliferation and apoptosis of myeloma cells and its pos-sible mechanism.Methods The study started from May 2018 and ended in January 2019.Normal bone marrow cells MNC and myelo-ma cells KM3 and U266 were cultured,and the levels of miR-335-3p were detected by real-time fluorescent quantitative reverse tran-scription polymerase chain reaction(qRT-PCR).The KM3 cells were assigned into miR-NC group(mimics control),miR-335-3p group(miR-335-3p mimics),pcDNA3.1+miR-335-3p group(miR-335-3p mimics+empty vector)and pcDNA3.1-MDM2+miR-335-3p group[mouse double minute 2(MDM2)overexpression vector+miR-335-3p mimics].And then the cell proliferation was detected by MTT,the apoptosis was detected by flow cytometry.The protein expressions of cyclin D1,P21,Bcl-2 and Bax in cells were detected by West-ern blotting.The dual luciferase reporter gene assay verified the relationship between miR-335-3p and MDM2 in KM3 cells.Results The expression levels of miR-335-3p in myeloma cells KM3 and U266 were 0.24±0.01 and 0.38±0.02,respectively,which were signifi-cantly lower than that in normal bone marrow cells 0.77±0.03(both P<0.05).Compared with the miR-NC group,the absorbance of KM3 cells in the miR-335-3p group[48 h:(0.60±0.03)vs.(0.99±0.06);72 h:(0.82±0.05)vs.(1.67±0.07)]and the protein expressions of cy-clin D1 and Bcl-2 were decreased(all P<0.05),but the apoptosis rates[(24.31±0.99)%vs.(8.13±0.83)%]and the protein expressions of P21 and Bax were increased(all P<0.05).miR-335-3p negatively regulated the expression of MDM2 expression in KM3 cells.Com-pared with the pcDNA3.1+miR-335-3p group,the absorbance of KM3 cells in the pcDNA3.1-MDM2+miR-335-3p group[48 h:(0.80±0.05)vs.(0.63±0.04);72 h:(1.28±0.06)vs.(0.88±0.05)]and the protein expressions of cyclin D1 and Bcl-2 were increased(all P<0.05),but the apoptosis rate[(15.34±0.66)%vs.(23.98±1.41)%]and the protein expressions of P21 and Bax were decreased(all P<0.05).Con⁃clusion miR-335-3p may inhibit the prolifera

关 键 词：多发性骨髓瘤 微小RNA-335-3p 小鼠双微体基因 细胞增殖 细胞凋亡 

分 类 号：R733.3[医药卫生—肿瘤]