miRNA-106b下调后抑制人喉鳞状细胞癌Hep-2和TU212细胞侵袭能力的机制研究  

Study on the mechanism of inhibiting invasion of human laryngeal squamous cell carcinoma Hep-2 and TU212 cells after the downregulation of miRNA-106b

作　　者：蔡克敏[1] 郭青 王菲[1] 杨波[1] 孔旭辉[1] Cai Kemin;Guo Qing;Wang Fei;Yang Bo;Kong Xuhui(Department of Otorhinolaryngology Head and Neck Surgery,Taizhou People's Hospital,Taizhou 225300,China)

机构地区：[1]江苏省泰州市人民医院耳鼻咽喉头颈外科,225300

出　　处：《肿瘤研究与临床》2020年第2期85-89,共5页Cancer Research and Clinic

基　　金：泰州市科技支撑计划(社会发展)项目(TS201530)。

摘　　要：目的探讨miRNA-106b(miR-106b)对人喉鳞状细胞癌Hep-2和TU212细胞的作用及其机制。方法将Hep-2和TU212细胞分为转染miR-106b抑制序列组(实验组)、转染miR-106b竞争性阴性序列组(阴性对照组)、未进行任何干预组(空白组)。采用反转录定量聚合酶链反应(qRT-PCR)验证miR-106b抑制序列对细胞miR-106b表达的抑制效应。应用生物信息学及荧光素酶报告载体分析磷酸酶和张力蛋白同源物(PTEN)是否为miR-106b的靶基因。利用PTEN小干扰RNA(siRNA)抑制Hep-2和TU212细胞中PTEN的表达。Transwell实验和蛋白质印迹法分别检测miR-106b沉默后和(或)PTEN干扰后Hep-2和TU212细胞侵袭能力的变化及PTEN、上皮性钙黏蛋白和波形蛋白表达变化。结果实验组Hep-2和TU212细胞miR-106b相对表达量分别为0.110±0.037、0.074±0.009,较阴性对照组(1.013±0.059、1.035±0.062)均降低(均P<0.05)。Transwell实验中,实验组Hep-2和TU212细胞较阴性对照组每个视野侵袭细胞数少[(37.09±4.02)个比(95.65±4.77)个,(29.16±2.49)个比(103.19±6.08)个,均P<0.05]。生物信息学分析显示PTEN mRNA的3'-UTR区与miR-106b的3'-UTR区域互补;双荧光素酶报告系统分析显示,转染miR-106b的野生型PTEN基因的荧光素酶报告子的活性降低至(22.84±2.68)%,转染miR-106b的突变型PTEN基因的荧光素酶报告子的活性几乎未改变[(92.08±3.44)%],二者差异有统计学意义(P<0.001)。实验组Hep-2和TU212细胞PTEN蛋白表达水平较阴性对照组高。Transwell实验检测显示,抑制PTEN表达的实验组Hep-2和TU212细胞比未抑制PTEN表达的实验组每个视野侵袭细胞数增加[(65.08±3.57)个比(26.72±2.58)个,(57.38±4.96)个比(31.81±2.97)个,均P<0.05]。蛋白质印迹实验显示,抑制PTEN表达的实验组Hep-2和TU212细胞上皮性钙黏蛋白表达水平上调,波形蛋白表达水平下调。结论人喉鳞状细胞癌Hep-2和TU212细胞中miR-106b可能通过靶向调节PTEN的表达,影响PTEN下游侵袭相关Objective To investigate the effect of miRNA-106b(miR-106b)on human laryngeal squamous cell carcinoma Hep-2 and TU212 cells and its mechanism.Methods Hep-2 and TU212 cells were divided into miR-106b inhibitory sequence transfected group(the experimental group),miR-106b competitive negative sequence transfected group(the negative control group)and non-intervention group(the blank group).The inhibitory effect of miR-106b inhibitory sequence on the expression of miR-106b was verified by using reverse transcription quantitative polymerase chain reaction(qRT-PCR).Whether phosphatase and tensin homolog(PTEN)was the target gene of miR-106b was analyzed by using bioinformatics and luciferase report vector.PTEN small interfering RNA(siRNA)was used to inhibit the expression of PTEN in Hep-2 and TU212 cells.Transwell method and Western blot were used to detect the change of invasion ability of Hep-2 and TU212 cells after miR-106b silencing or the PTEN intervening,and the expression change of PTEN,epithelial cadherin and vimentin.Results The relative expression levels of miR-106b in Hep-2 and TU212 cells in the experimental group were 0.110±0.037 and 0.074±0.009,respectively,which were lower than those in the negative control group(1.013±0.059 and 1.035±0.062,respectively;all P<0.05).In Transwell experiments,the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group was less than that in the negative control group[(37.09±4.02)vs.(95.65±4.77),(29.16±2.49)vs.(103.19±6.08),all P<0.05].The bioinformatics analysis results showed that 3'-UTR region of PTEN mRNA was complementary to 3'-UTR region of miR-106b.Dual-luciferase reporter system analysis showed that the luciferase reporter activity of wild-type PTEN gene transfected with miR-106b was decreased to(22.84±2.68)%,and that of mutant PTEN gene transfected with miR-106b was almost unchanged[(92.08±3.44)%],and the difference was statistically significant(P<0.001).The expression level of PTEN protein of Hep-2 and TU212 cells in the exp

关 键 词：喉肿瘤 癌 鳞状细胞 肿瘤侵润 RNA干扰 miRNA-106b 磷酸酶和张力蛋白同源物 

分 类 号：R73[医药卫生—肿瘤]