摘要
目的:探讨微RNA(microRNA,miRNA,miR)-377对组蛋白去乙酰化酶1(sirtuin 1,SIRT1)的调控作用,及对索拉菲尼敏感性的影响。方法:采用实时荧光定量PCR法检测miR-377 mRNA在30例肝癌组织以及相应癌旁组织中的表达水平,及其在肝癌Huh7和HepG2细胞和正常肝细胞LO2中的表达水平。采用癌症和肿瘤基因图谱(The Cancer Genome Atlas,TCGA)数据库分析miR-377表达对于肝癌患者生存预后的影响。通过脂质体法将miR-377-模拟物(mimics)转入肝癌Huh7细胞,以转入阴性对照-mimics(negative control-mimics,NC-mimics)作为对照组;MTT法检测miR-377过表达后不同浓度的索拉菲尼对Huh7细胞活性的影响。通过Starbase网站分析miR-377的靶向基因,并采用荧光素酶报告基因系统验证miR-377是否直接靶向结合SIRT1基因。采用实时荧光定量PCR和蛋白质印迹法检测转染miR-377-mimics后对SIRT1 mRNA及蛋白表达水平的影响。采用脂质体法将miR-377-mimics和携带有SIRT1全基因的pcDNA3.1-SIRT1同时转入Huh7细胞,并再行索拉菲尼处理;分别用MTT法和克隆形成实验检测对Huh7细胞增殖能力的影响。结果:miR-377在肝癌组织中的表达水平明显低于对应的癌旁正常肝组织(P<0.05),miR-377在肝癌Huh7和HepG2细胞中的表达水平明显低于正常肝LO2细胞(P<0.05)。miR-377低表达组患者的总生存率高于高表达组(P<0.05)。索拉菲尼对miR-377过表达Huh7细胞活性具有明显的抑制作用(P<0.05)。在线生物信息学分析结果和蛋白荧光素酶报告基因系统验证结果显示,miR-377直接靶向结合SIRT1基因;实时荧光定量PCR和蛋白质印迹法检测结果表明,转染miR-377-mimics后Huh7细胞中SIRT1 mRNA和蛋白的表达水平明显下调(P值均<0.05)。而共转染miR-377-mimics和pcDNA3.1-SIRT1后,SIRT1过表达能够明显减弱索拉菲尼对Huh7细胞增殖的抑制作用(P值均<0.05)。结论:miR-377通过抑制SIRT1表达促进肝癌细胞对索拉菲尼的敏感性,可能为肝癌细胞索拉菲尼耐药的治疗提供一条新的思路。
Objective:To investigate the regulation of microRNA(miR)-377 on sirtuin 1(SIRT1),and to explore the effect on the sensitivity of sorafenib.Methods:The expression level of miR-377 was detected by real-time fluorescent quantitative PCR in 30 cases of hepatocellular carcinoma(HCC)and the corresponding adjacent tissues;The expression level of miR-377 in HCC Huh7 and HepG2 cells and normal human liver cells LO2(as the control)was detected by real-time fluorescent quantitative PCR.The Cancer Genome Atlas(TCGA)database bioinformatics was used to analyze the effect of miR-377 on the survival and prognosis of patients with HCC.Huh7 cells were transfected with miR-377-mimics by liposome,Huh7 cells were transfected with negative control(NC)-mimics as the control.The cell viability of Huh7 cells treated with different concentrations sorafenib was detected by MTT method.Starbase was used to analyze the target gene of miR-377,and luciferase reporter gene assay was used to verify whether miR-377 directly targets SIRT1 gene;The expression level of SIRT1 mRNA and protein in Huh7 cells after transfection with miR-377-mimics.After co-transfection of miR-377-mimics and recombinant plasmid pcDNA3.1-SIRT1,the Huh7 cells were treated with sorafenib,the proliferation was detected by MTT method and clone formation assay,respectively.Results:The expression level of miR-377 in HCC tissues and cells was lower than that in normal liver tissues and LO2 cells,the difference was statistically significant(both P<0.05).The overall survival rate of the miR-377 low expression patient group was higher than that of the high expression group(P<0.05).Sorafenib could inhibite the activity of miR-377 overexpressed Huh7 cells(P<0.05).Bioinformatics analysis and luciferase reporter gene assay results showed that miR-377 could target SIRT1 gene.The real-time fluorescent quantitative PCR and Western blotting results showed that after transfection with miR-377-mimics,the expression level of SIRT1 mRNA and protein were significantly decreased(both P<0.05).After co-transfection of miR-377-mimics and pcDNA3.1-SIRT1(SIRT1 overexpression)could significantly reduce the inhibitory effect of sorafenib on the proliferation of Huh7 cells(both P<0.05).Conclusion:MiR-377 can promote the sensitivity of HCC cells to sorafenib by inhibiting SIRT1 expression,and provide some ideas for the treatment of sorafenib resistance.
作者
陈俊
丛秀峰
张婧超
郑伟
CHEN Jun;CONG Xiufeng;ZHANG Jingchao;ZHENG Wei(Department of First Oncology Ward,Shengjing Hospital,China Medical University,Shenyang 110000,Liaoning Province,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2020年第11期748-757,共10页
Tumor
