MiRNA-19a靶向SMO基因调控Hedgehog信号通路影响骨髓瘤细胞生物学特性的机制研究  

Mechanism of miRNA-19a targeting the SMO gene regulates the hedgehog signalling pathway and affects the biological characteristics of myeloma cells

作　　者：李光宝 尹建文[1] 张轶群[1] 李时中 吴祖同[1] 贺爱军 LI Guang-bao;YIN Jian-wen;ZHANG Yi-qun;LI Shi-zhong;WU Zu-tong;HE Ai-jun(Department of Orthopaedics,the First Affiliated Hospital of Shaoyang University,Shaoyang,Hunan 422000,China)

机构地区：[1]邵阳学院附属第一医院骨科,湖南邵阳422000

出　　处：《中华全科医学》2022年第5期745-748,760,共5页Chinese Journal of General Practice

基　　金：湖南省教育厅科学研究项目(19C1636)。

摘　　要：目的探索miRNA-19a对多发性骨髓瘤细胞增殖、凋亡和转移的影响,及对SMO蛋白和Hedgehog信号通路的影响。方法人骨髓瘤细胞系U266B1、LP-1、RPMI 8226和NCI-H929为研究模型,首先检测4种细胞中SMO蛋白的背景表达,然后使用U266B1细胞作为模型,设计了miRNA-19a inhibitor,以此建立miRNA-19a敲低组、对照质粒组和空载组。探究miRNA-19a敲减后细胞中SMO蛋白含量以及细胞增殖、凋亡和转移的变化。采用MTT法检测细胞毒性,流式细胞术检测细胞凋亡情况,Transwell试验检测细胞侵袭能力。结果SMO蛋白在U266B1、LP-1、RPMI 8226和NCI-H929细胞系中表达量分别为0.925±0.057、0.889±0.067、0.904±0.075、0.507±0.048,其中以NCI-H929细胞系中表达量较低(P<0.05)。MiRNA-19a敲低组、对照质粒组和空载组miRNA-19a含量分别为0.325±0.058、1.158±0.128、1.172±0.131,miRNA-19a敲低组miRNA-19a含量显著降低(P<0.05)。对照质粒组、miRNA-19a敲低组和空载组SMO蛋白表达量分别为0.787±0.104、0.354±0.068、0.807±0.106,miRNA-19a敲低组SMO蛋白含量明显降低(P<0.05)。MTT实验显示,miRNA-19a敲低组细胞增殖OD值在24~72 h内明显低于空载组和对照质粒组(均P<0.05)。凋亡实验显示,miRNA-19a敲低组细胞凋亡率明显高于空载组和对照质粒组(均P<0.05)。Transwell实验显示,miRNA-19a敲低组侵袭细胞数明显低于其他2组(均P<0.05)。结论miRNA-19a/SMO蛋白可能是骨髓瘤细胞系U266B1中控制细胞恶性增长的关键信号,可以作为多发性骨髓瘤治疗的潜在靶点。Objective To explore the effects of miRNA-19a on the proliferation,apoptosis and metastasis of multiple myeloma cells,as well as on smoothened frizzled class receptor(SMO)protein and the hedgehog signalling pathway.Methods Human myeloma cell lines U266b1,LP-1,RPMI 8226 and NCI-H929 were used as the research models.Firstly,the background expression of SMO protein in four kinds of cells was detected.Using U266b1 cells as the model,the miRNA-19a inhibitor was designed,and the miRNA-19a knockdown group,control plasmid group and empty group were established.Changes in the SMO protein content,cell proliferation,apoptosis and metastasis were explored after miRNA-19a knockdown.Cytotoxicity was detected by MTT assay,apoptosis was detected by flow cytometry and cell invasion was detected by Transwell test.Results The expression levels of SMO protein in the U266b1,LP-1,RPMI 8226 and NCI-H929 cell lines were 0.925±0.057,0.889±0.067,0.904±0.075,and 0.507±0.048,respectively,whereas the expression level of SMO protein in the NCI-H929 cell line was low(P<0.05).The miRNA-19a contents in the miRNA-19a knockdown group,control plasmid group and empty group were 0.325±0.058,1.158±0.128 and 1.172±0.131,respectively.The miRNA-19a contents in the miRNA-19a knockdown group decreased significantly(P<0.05).The expression levels of SMO in the control plasmid group,miRNA-19a knockdown group and empty group were 0.787±0.104,0.354±0.068 and 0.807±0.106,respectively.The content of SMO protein in the miRNA-19a knockdown group decreased significantly(P<0.05).MTT assay showed that the OD value of cell proliferation in the miRNA-19a knockdown group was significantly lower than that in the empty group and control plasmid group within 24-72 h(all P<0.05).Apoptosis experiments showed that the apoptosis rate of the miRNA-19a knockdown group was significantly higher than that of the empty group and control plasmid group(all P<0.05).Transwell experiments showed that the number of invasive cells in the miRNA-19a knockdown group was significantly

关 键 词：多发性骨髓瘤 微RNA-19a HEDGEHOG信号通路 SMO蛋白 

分 类 号：R733.3[医药卫生—肿瘤]