ASB16反义RNA1在鼻咽癌组织中的表达及对鼻咽癌细胞增殖、凋亡的影响和作用机制

Expression of ASB16 antisense RNA 1 in nasopharyngeal carcinoma tissues and its effect on proliferation and apoptosis of nasopharyngeal carcinoma cells and the underlying mechanism

目的探讨ASB16反义RNA1(ASB16-AS1)在鼻咽癌组织中的表达及对鼻咽癌细胞增殖、凋亡的影响和作用机制。方法采用实时定量聚合酶链反应(qRT-PCR)检测33例鼻咽癌组织和对应癌旁组织中ASB16-AS1和miRNA-363-5p的表达水平。将鼻咽癌细胞C666-1分为si-NC组、si-ASB16-AS1组、si-ASB16-AS1+anti-miRNA-NC组和si-ASB16-AS1+anti-miRNA-363-5p组,分别采用CCK8法、克隆形成实验、流式细胞术检测细胞的增殖率、克隆形成数及凋亡率,采用蛋白质印迹法(Westernblot)检测细胞中Ki-67、cleaved caspase 3、pro-caspase 3蛋白表达水平。采用双荧光素酶报告基因实验验证ASB16-AS1和miRNA-363-5p的调控关系。结果鼻咽癌组织中ASB16-AS1相对表达量明显高于癌旁组织,miRNA-363-5p相对表达量明显低于癌旁组织,差异均有统计学意义(P﹤0.01),且鼻咽癌组织中ASB16-AS1与miRNA-363-5p的表达呈负相关(r=-0.992,P﹤0.05)。si-ASB16-AS1组C666-1细胞的光密度(OD)值(48、72 h)、克隆形成数、Ki-67、pro-caspase 3蛋白相对表达量均低于si-NC组,细胞凋亡率、cleaved caspase 3蛋白相对表达量均高于si-NC组,差异均有统计学意义(P﹤0.05)。ASB16-AS1在C666-1细胞中负调控miRNA-363-5p的表达。si-ASB16-AS1+anti-miRNA-363-5p组C666-1细胞凋亡率、cleaved caspase 3蛋白相对表达量均低于si-ASB16-AS1+anti-miRNA-NC组,OD值(48、72 h)、克隆形成数、Ki-67、pro-caspase 3蛋白相对表达量均高于si-ASB16-AS1+anti-miRNA-NC组,差异均有统计学意义(P﹤0.05)。结论ASB16-AS1可通过调控miRNA-363-5p促进鼻咽癌细胞增殖,抑制鼻咽癌细胞凋亡。

Objective To investigate the expression of ASB16 antisense RNA 1 in nasopharyngeal carcinoma tis-sues and its effect on the proliferation and apoptosis of nasopharyngeal carcinoma cells and the underlying mechanism.Method The expression levels of ASB16-AS1 and miRNA-363-5p in 33 nasopharyngeal carcinoma tissues and corre-sponding paracancerous tissues were detected by real-time quantitative polymerase chain reaction(qRT-PCR).Nasopha-ryngeal carcinoma cells C666-1 were divided into the si-NC group,si-ASB16-AS1 group,si-ASB16-AS1+anti-miRNA NC group,and si-ASB16-AS1+anti-miRNA-363-5p group.The proliferation rate,colony formation number,and apopto-sis rate of cells were detected by the CCK8 method,colony formation assay,and flow cytometry,respectively.The pro-tein expression levels of Ki-67,cleaved caspase 3 and pro-caspase 3 in cells were detected by Western blot.The regulato-ry relationship between ASB16-AS1 and miRNA 363-5p was verified by dual-luciferase reporter gene assay.Result The relative expression level of ASB16-AS1 in nasopharyngeal carcinoma tissues was significantly higher than that in paracancerous tissues,while the relative expression level of miRNA-363-5p was significantly lower than that in paracan-cerous tissues,and the differences were statistically significant(P<0.01).The expression of ASB16-AS1 and miRNA-363-5p was negatively correlated(r=-0.992,P<0.05).The optical density(OD)value(48,72 h),the number of clones,the rel-ative expression level of Ki-67,and pro-caspase 3 protein in C666-1 cells in the si-ASB16-AS1 group were lower than those in the si-NC group,while the apoptosis rate and the relative expression level of cleaved caspase 3 protein were high-er than those in the si-NC group,and the differences were statistically significant(P<0.05).ASB16-AS1 negatively regu-lated the expression of miRNA-363-5p in C666-1 cells.The apoptosis rate of C666-1 cells and the relative expression lev-el of cleaved caspase 3 protein in the si-ASB16-AS1+anti-miRNA-363-5p group were lower than those in the si-ASB16-AS1+anti-miRNA-NC group,while the OD value(48,72 h),the number of clones,and the relative expression level of Ki 67 and pro-caspase 3 proteins were higher than those in the si-ASB16-AS1+anti-miRNA-NC group,and the differences were statistically significant(P<0.05).Conclusion ASB16-AS1 could promote the proliferation of nasopharyngeal carci-noma cells and inhibit the apoptosis of these cells by regulating miRNA-363-5p.