shRNA靶向敲低Cx43抑制人多发性骨髓瘤细胞MM.1S  

Inhibiton of targeted knockdown of Cx43 by shRNA on human multiple myeloma cell line MM.1S

作　　者：蒋小端[1] 鱼强[1] JIANG Xiaoduan;YU Qiang(Department of Blood Rheumatology and Immunology,Dongfeng General Hospital of Sinopharm Affiliated to Hubei Medical College,Shiyan 442000,China)

机构地区：[1]湖北医药学院附属国药东风总医院血液风湿免疫科,湖北十堰442000

出　　处：《生物技术》2023年第6期727-731,共5页Biotechnology

摘　　要：[目的]探究shRNA靶向敲低Cx43基因表达对人多发性骨髓瘤细胞株MM.1S生存的影响及其机制。[方法]针对Cx43 mRNA不同位点构建sh/Cx43-1、sh/Cx43-2、sh/Con重组质粒,感染体外培养的MM.1S细胞,RT-PCR或Western Blotting检测Cx43 mRNA或蛋白表达,CCK-8、流式细胞术检测细胞增殖、凋亡情况,Western Blotting检测磷脂酸肌醇-3-激酶(PI3K)、丝苏氨酸蛋白激酶(AKT)通路蛋白表达。[结果]sh/Cx43-1组、sh/Cx43-2组Cx43 mRNA或蛋白均低于sh/Con组,且sh/Cx43-1组低于sh/Cx43-2组(0.18±0.05 vs 0.75±0.07,0.21±0.03 vs 0.81±0.06,P<0.05);培养24h时各组细胞增殖水平无明显差异(P>0.05),培养48 h、72 h时sh/Cx43-1组、sh/Cx43-2组细胞增殖水平均低于sh/Con组(0.42±0.05 vs 0.58±0.09,1.02±0.10 vs 1.08±0.13,P<0.05);sh/Cx43-1组、sh/Cx43-2组细胞总凋亡率高于sh/Con组,PI3K、AKT蛋白表达均低于sh/Con组,且sh/Cx43-1组变化较sh/Cx43-2组更明显(P<0.05)。[结论]通过shRNA靶向敲低Cx43基因表达能显著抑制MM.1S细胞增殖,促进其凋亡,这可能与敲低Cx43后PI3K/AKT通路受到抑制有关。[Objective]To investigate the effect of shRNA-targeted knockdown of Cx43 gene expression in multiple myeloma cell line MM.1S survival and possible mechanisms.[Method]sh/Cx43-1,sh/Cx43-2 and sh/Con recombinant plasmids were constructed according to different sites of Cx43 mRNA and infected with MM.1S cells,Cx43 mRNA or protein expression was detected by RT-PCR or Western Blotting,cell proliferation level was detected by CCK-8,apoptosis level was detected by flow cytometry,and phosphatidylinositide 3-kinases(PI3K)and ser-ine-threonine protein kinase(AKT)pathway protein expression was detected by Western Blotting.[Result]Cx43 mRNA or protein in sh/Cx43-1 group and sh/Cx43-2 group were lower than those in sh/Con group,and sh/Cx43-1 group was lower than that in sh/Cx43-2 group(0.18±0.05 vs 0.75±0.07,0.21±0.03 vs 0.81±0.06,P<0.05).There was no significant difference in cell proliferation level between the groups at 24 hours of culture(P>0.05).Cell proliferation level in sh/Cx43-1 group and sh/Cx43-2 group was lower than that in sh/Con group at 48 and 72 hours of culture,and sh/Cx43-1 group was lower than that in sh/Cx43-2 group(0.42±0.05 vs 0.58±0.09,1.02±0.10 vs 1.08±0.13,P<0.05);And the changes were more significant in the sh/Cx43-1 group than in the sh/Cx43-2 group(P<0.05).[Conclusion]Targeted knockdown of Cx43 gene expression by shRNA can significantly inhibit MM.1S cells proliferate and promote their apoptosis,which may be related to the inhibition of PI3K/AKT pathway after knockdown of Cx43.

关 键 词：CX43 多发性骨髓瘤 增殖 凋亡 磷脂酸肌醇-3-激酶 丝苏氨酸蛋白激酶 

分 类 号：R733.3[医药卫生—肿瘤]