微小RNA-125a-5p对人鼻咽癌细胞增殖的影响及机制

Effect and mechanism of microRNA-125a-5p on proliferation of nasopharyngeal carcinoma cells

目的探究微小RNA-125a-5p(miR-125a)对人鼻咽癌细胞(HK-1细胞)增殖的影响及机制。方法常规培养HK-1细胞及人胚肾细胞永生化细胞系(293T细胞),将HK-1细胞随机分为对照1组、过表达miR-125a组(转染miR-125a mimics)、沉默miR-125a组(转染miR-125a inhibitor);另取部分HK-1细胞随机分为对照2组、miR-125a过表达组(转染miR-125a mimics)及回补组[转染miR-125a mimics同时过表达含PDZ结合基序的转录共激活因子(TAZ)]。转染48 h后检测miR-125a表达验证转染效率。用RT-qPCR法检测miR-125a-5p、TAZ mRNA;用Western blotting法检测TAZ蛋白;CCK-8实验、集落形成实验检测细胞增殖能力;通过预测网站TargetScan(http://www.targetscan.org/)和miRDB(http://mirdb.org/)预测miR-125a与TAZ的结合位点。将293T细胞随机分为4组,WT+miR-ctrl组共转染TAZ野生型(TAZ-WT)和miR-125a空载对照(miR-ctrl)、Mut+miR-ctrl组共转染TAZ突变型(TAZ-MUT)和miR-ctrl、WT+miR-125a组共转染TAZ-WT和miR-125a模仿物(miR-125a)、Mut+miR-125a组共转染TAZ-MUT和miR-125a,用双荧光素酶报告基因实验进一步验证miR-125a的下游靶点。结果对照1组、过表达miR-125a组、沉默miR-125a组miR-125a的相对表达量比较差异有统计学意义(P均<0.05)。对照1组、过表达miR-125a组、沉默miR-125a组转染后第2、3、4、5、6天增殖活力(OD450值)比较差异均有统计学意义(P均<0.05)、集落形成数比较差异有统计学意义(P均<0.05)。对照1组、过表达miR-125a组、沉默miR-125a组TAZ蛋白相对表达量比较差异有统计学意义(P均<0.05)。网站预测显示miR-125a与TAZ存在结合位点,设计突变型序列(TAZ-3’UTR MT),miR-125a与TAZ的非编码区结合,miR-125a的直接下游靶点为TAZ。WT+miR-125a组相对荧光强度低于WT+miR-ctrl组、Mut+miR-ctrl组(P均<0.05),Mut+miR-125a组相对荧光强度与WT+miR-ctrl组、Mut+miR-ctrl组比较差异无统计学意义(P均>0.05)。对照2组、miR-125a mimics组、miR-125a mimics+TAZ组转染后第2、3、4、5、6天增殖活力比较差异有统计学意义(P均<0.05),集落形成数比较差异有统计学意义(P均<0.05)。结论miR-125a可抑制鼻咽癌细胞增殖,其作用机制与抑制其下游靶点TAZ的表达有关。

Objective To investigate the effect and mechanism of microRNA-125a-5p(miR-125a)on the proliferation of nasopharyngeal carcinoma cell line HK-1.Methods Human nasopharyngeal carcinoma cells(HK-1 cells)and human embryonic kidney immortalized cells(293T cells)were cultured routinely.HK-1 cells were randomly divided into the control group 1,miR-125a overexpression group(transfected with miR-125a mimics),and miR-125a silencing group(transfected with miR-125a inhibitor).Some cells were further divided into the control group 2,miR-125a overexpression group(transfected with miR-125a mimics),and rescue group(transfected with miR-125a mimics and TAZ overexpression simultaneously).The transfection efficiency was verified by detecting miR-125a expression at 48 h after transfection.RT-qPCR was used to detect the expression levels of miR-125a-5p and TAZ mRNA,while Western blotting was used to detect TAZ protein expression.Cell proliferation capacity was assessed using the CCK-8 assay and colony formation assay.TargetScan and miRDB prediction websites were used to predict the binding site of miR-125a and TAZ.The 293T cells were divided into four groups:WT+miR-ctrl group(co-transfected with TAZ wild-type and miR-ctrl),Mut+miR-ctrl group(cotransfected with TAZ mutant and miR-ctrl),WT+miR-125a group(co-transfected with TAZ-WT and miR-125a mimic),and Mut+miR-125a group(co-transfected with TAZ-MUT and miR-125a).Dual-luciferase reporter gene experiments were conducted to further validate the downstream target of miR-125a.Results There were significant differences in the relative expression levels of miR-125a among the control group 1,the miR-125a overexpression group,and the miR-125a silencing group(all P<0.05).Statistically significant differences were found in the proliferation activity(OD450 values)on the 2nd,3rd,4th,5th,and 6th days after transfection and colony formation numbers in the control group 1,miR-125a overexpression group,and miR-125a silencing group(all P<0.05).There was significant difference in the relative expression level of TAZ protein among the control group 1,miR-125a overexpression group,and miR-125a silencing group(all P<0.05).Website prediction indicated the existence of binding sites between miR-125a and TAZ,and mutated sequences(TAZ-3’UTR MT)were designed.MiR-125a directly targeted TAZ in the non-coding region.The relative fluorescence intensity of the WT+miR-125a group was lower than those of the WT+miR-ctrl group and the Mut+miR-ctrl group(both P<0.05),while there was no significant difference in relative fluorescence intensity among the WT+miR-125a group,WT+miR-ctrl group and the Mut+miR-ctrl group(all P>0.05).Statistically significant differences were found in proliferation activity on the 2nd,3rd,4th,5th,and 6th days after transfection and colony formation numbers between the control group 2,miR-125a mimics group,and miR-125a mimics+TAZ group(all P<0.05).Conclusion MiR-125a can inhibit the proliferation of nasopharyngeal carcinoma cells,and its mechanism of action is related to the inhibition of its downstream target TAZ expression.