摘要
目的探讨miR-483对喉癌细胞增殖、凋亡的影响及机制。方法实验分正常组、实验组和对照组,各组设8个复孔,实验组Hep-2细胞转染miR-483 inhibitor,对照组细胞转染miR-483inhibitor NC,正常组仅进行细胞培养未进行任何处理。以实时荧光定量聚合酶链式反应(Real-time PCR)检测转染细胞中miR-483表达情况;以CCK-8法检测Hep-2细胞存活率;以Annexin V-FITC/PI双染流式细胞术检测细胞凋亡情况;进行Hep-2细胞裸鼠成瘤体内实验观察miR-483对喉癌细胞增殖的影响;以Real-time PCR及蛋白质印迹法检测Hep-2细胞Smad4 mRNA及蛋白表达情况。结果Real-time PCR检测结果可见,正常组、实验组和对照组Hep-2细胞中miR-483的表达量分别为1.00±0.00,0.36±0.05,0.96±0.05,干预72 h后,正常组、实验组和对照组Hep-2细胞活率分别为(99.32±0.21)%,(58.66±7.57)%,(99.67±0.42)%,细胞凋亡率分别为(2.36±0.15)%,(15.36±2.47)%,(2.06±0.12)%,正常组和对照组分别与实验组比较,差异均有统计学意义(均P<0.05)。Hep-2细胞接种裸鼠后,干预后第10天正常组、实验组和对照组移植瘤体积分别为(296.58±12.52),(125.46±23.69),(305.24±15.26)mm^3,干预后第30天移植瘤体积分别为(1025.46±23.25),(563.48±22.56),(996.52±24.51)mm^3,自药物干预后第10天,实验组裸鼠移植瘤体积明显小于正常组和对照组(P<0.05)。正常组、实验组和对照组细胞Smad4 mRNA相对表达量为0.16±0.02,0.69±0.11,0.18±0.03,Smad4蛋白相对表达量为0.56±0.13,1.24±0.32,0.33±0.05,与实验组比较,正常组和对照组Smad4 mRNA和蛋白相对表达量均降低(均P<0.05)。结论miR-483可以体内及体外抑制Hep-2细胞增殖,促进其凋亡,其机制可能与上调Smad4基因转录促进蛋白表达有关。
Objective To investigate the effect and mechanism of miR-483 on the proliferation and apoptosis of laryngeal cancer cells.Methods The cells were divided into normal group,test group and control group.Each group had 8 holes.The Hep-2 cells in test group were transfected with miR-483 inhibitor,while control cells were transfected with miR-483 inhibitor NC.The normal group was cultured without any treatment.The expression of miR-483 in transfected cells was detected by real-time polymerase chain reaction(Real-time PCR),the survi-val rate of Hep-2 cells was detected by CCK-8,the apoptosis was detected by annexin V-FITC/PI double staining flow cytometry,and the effect of miR-483 on the proliferation of laryngeal carcinoma cells was observed in vivo experiment of Hep-2 cell nude mice.Smad4 mRNA and protein expression in Hep-2 cells were detected by by Real-time PCR and Western Blot.Results Real-time PCR showed that the expression of miR-483 in normal group,test group and control group were 1.00±0.00,0.36±0.05 and 0.96±0.05;after 72 h intervention,the activity rates of Hep-2 cells in normal group,test group and control group were(99.32±0.21)%,(58.66±7.57)% and(99.67±0.42)%,the apoptosis rates were(2.36±0.15)%,(15.36±2.47)%and(2.06±0.12)%,compared with normal group and control group,there was significant in test group(all P<0.05).After Hep-2 cells were inoculated into nude mice,the tumor volume of normal group,test group and control group were(296.58±12.52),(125.46±23.69)and(305.24±15.26)mm^3 on the 10 th day after intervention,and the tumor volume were(1025.46±23.25),(563.48±22.56)and(996.52±24.51)mm^3 on the 30 th day after intervention.The tumor volume in test group was significantly smaller than that of normal group and control group(P<0.05).Real-time PCR assay showed that the relative expression of Smad4 mRNA in normal group,test group and control group were 0.16±0.02,0.69±0.11,0.18±0.03,and the relative expression of Smad4 protein were 0.56±0.13,1.24±0.32,0.33±0.05.Compared with test group,the relative expression of Smad4 mRNA and protein in normal and control groups decreased(all P<0.05).Conclusion miR-483 can inhibit the proliferation and promote the apoptosis of Hep-2 cells in vivo and in vitro,the mechanism may be related to the up-regulation of the expression of Smad4 gene transcriptional promoter protein.
作者
陈朝辉
吴宏林
徐奕莲
代丽丽
魏庆宇
CHEN Zhao-hui;WU Hong-lin;XU Yi-lian;DAI Li-li;WEI Qing-yu(Department of Otorhinolaryngology Head and Neck Surgery,Affiliated Hospital of Hangzhou Normal University,Hangzhou 310000,Zhejiang Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2020年第5期560-563,共4页
The Chinese Journal of Clinical Pharmacology
