miR-497靶向CDK6抑制喉鳞状细胞癌生长的实验研究

摘要

目的 研究miR-497和细胞周期蛋白依赖激酶6(cyclin-dependent kinase,CDK6)在喉鳞状细胞癌生长过程中的作用.方法 利用定量实时聚合酶链反应分析喉鳞状细胞癌及癌旁正常黏膜标本中CDK6 mRNA表达情况;评价CDK6mRNA的表达水平与喉鳞状细胞癌患者预后的关系;通过生物信息学预测miR-497的靶基因CDK6,并利用双荧光素酶报告基因验证;构建CDK6siRNA和质粒pcDNA3.1(+)CDK6,分别转染喉鳞状细胞癌细胞后,利用四甲基偶氮唑蓝法和克隆形成实验,观察喉鳞状细胞癌细胞生长的变化,并使用流式细胞术观察细胞周期的分布.统计学数据采用SPSS17.0进行分析.结果 CDK6在喉鳞状细胞癌中高表达(t=14.01,P=0.009),且CDK6高表达组患者的总生存率低于低表达组,差异有统计学意义(HR=3.236,P＜0.001);双荧光素酶报告基因显示,野生型CDK6荧光活性变化倍数与对照组相比差异有统计学意义(P＜0.01).而突变型与对照组比较,差异无统计学意义(P＞0.05).在细胞系Hep-2和TU-212中,CDK6 siRNA组A490值Hep-2细胞为(0.42±0.14,(x)±s),TU-212细胞为(0.51±0.13),较对照组Hep-2细胞(0.98±0.16)和TU-212细胞(1.17±0.20)明显减低,细胞的生长受到抑制;Hep-2细胞的克隆为(55±4)个,TU-212细胞为(51±3)个,也明显低于对照组Hep-2的(108±6)个和TU-212的(105±7)个;细胞阻滞在G0/G1期,Hep-2细胞G0/G1期百分比为(65.20± 10.12)％,TU-212为(63.42±8.97)％,明显高于对照组Hep-2的(45.31±7.55)％和TU-212的(42.37±7.28)％,S期细胞明显减少,Hep-2细胞中S期细胞数百分比为(25.39±5.51)％,TU-212为(27.21±5.43)％,明显低于对照组Hep-2的(42.87±6.85)％和TU-212的(44.76±7.02)％;miR-497/CDK6组与miR-497组相比,细胞的生长、克隆形成能力部分得到恢复.以上相应各组比较,差异均有统计学意义(P值均＜0.05).结论 CDK6在喉鳞状细胞癌中表达升高,具有癌基因的作用,且CDK6高表达提示患者预后不良.miR-497通过靶向CDK6抑制了喉鳞状细胞癌的生长,具有抑癌基因的作用.%Objective To study the effects of miR-497 and CDK6 on the growth of laryngeal squamous cell carcinoma (LSCC).Methods The expressions of CDK6 mRNA in fresh LSCC specimens,the adjacent normal mucosa of LSCC,and cell lines of LSCC were detected with quantitative real time polymerase chain reaction,pcDNA3.1(+) CDK6 plasmids were respectively transfected into the LSCC cells,and MTT assay and clone formation assay were performed to evaluate the growth of LSCC cells.Flow cytometry was employed for cell cycle analysis.SPSS17.0 software was used to analyze the data.Results CDK6 was highly expressed in LSCC(t=14.01,P=0.009) and the overall survival rate of the patients with high CDK6 expression was less than that with low CDK6 expression,with a significant difference (HR=3.236,P＜0.001).Double luciferase reporter gene analysis showed that fluorescence activity in wild type CDK6 group was significantly different from that in control group (P＜0.01),while there was no significant difference in the fluorescence activity between mutant CDK6 group and control group (P＞0.05).A490 values were respectively 0.42±0.14 (Mean±SD) in siRNA Hep-2 group,0.51±0.13 in siRNA TU-212 group;0.98± 0.16 in control Hep-2 group and 1.17±0.20 in control TU-212 group.Colonies were 55±4 in siRNA Hep-2 group,51±3 in siRNA TU-212 group,108±6 in control Hep-2 group and 105±7 in control TU-212 group,namely,cell growth and clone formation ability in CDK6 siRNA group were significantly lower than those in the control group.Cells cycle was blocked in G0/G1 phase (G0/G1:65.20％±10.12％ in siRNA Hep-2 group;63.42％±8.97％ in siRNA TU-212 group;45.31％±7.55％ in control Hep-2 group;and 42.37％±7.28％ in control TU-212 group),and cells decreased obviously in S phase (S:25.39％±5.51％ in siRNA Hep-2 group;27.21％±5.43％ in siRNA TU-212 group;42.87％±6.85％ in control Hep-2 group;and 44.76％±7.02％ in control TU-212 group).Compared with miR-497 group,cell growth and clone formation ability in miR-497/CDK6 group were partly restored (all P＜0.05).Conclusions CDK6 expression in LSCC is upregulated,functioning as an oncogene.High expression of CDK6 is a predictor for poor prognosis.miR-497,functioning as a tumor suppressor gene,inhibits the growth of LSCC by targeting CDK6.