shRNA沉默β-catenin基因表达对人食管癌细胞生物学特性的影响

Effect of β-catenin gene silencing by shRNA on biologic characteristics of human esophageal carcinoma cells

目的 构建稳定抑制β-catenin表达的食管癌细胞克隆,观察shRNA介导的β-catenin基因沉默对人食管癌细胞生物学特性的影响,为以β-catenin为靶的食管癌基因治疗提供理论和实验依据.方法 通过细菌转化、酶切、测序鉴定和基因重组等方法构建针对β-catenin的RNA干扰质粒pGen-3-CTNNB1和阴性对照质粒pGen-3-con.利用脂质体介导转染技术转染人食管癌细胞系Eca-109,经G418筛选得到稳定抑制β-catenin表达的食管癌细胞模型（pGen-3-CTNNB1细胞）;逆转录聚合酶链反应（RT-PCR）、细胞免疫荧光和Western blot检测RNA干扰组（pGen-3-CTNNB1）、阴性对照组（pGen-3-con）及未转染组（Eca-109）3组细胞中β-catenin的表达;建立裸鼠皮下移植瘤模型,观察抑制β-catenin表达在活体内对肿瘤细胞生长能力的影响,免疫组织化学检测移植瘤组织中β-catenin的表达水平;体外浸润实验、迁移实验检测各组细胞的侵袭转移能力.结果 成功构建了针对β-catenin基因的RNA干扰载体pGen-3-CTNNB1,建立了稳定抑制β-catenin基因表达的食管癌细胞模型;与阴性对照组[（1.18±0.13）g]和未转染组[（1.38±0.21）g]比较,RNA干扰组[（0.42±0.09）g]移植瘤重量明显减轻（P＜0.05）;瘤组织中β-catenin的表达水平明显降低;抑制β-catenin基因表达后,食管癌细胞的浸润能力显著降低,阴性对照组浸润细胞数为（81±5）个/HPF、未转染组为（77±6）个/HPF、RNA干扰组为（41±4）个/HPF（P＜0.01）;迁移能力也显著下降,阴性对照组迁移细胞数为（73±5）个/HPF、未转染组为（69±5）个/HPF、RNA干扰组为（38±4）个/HPF（P＜0.05）.结论 在人食管癌细胞Eca-109中存在β-catenin表达异常和Wnt信号通路的异常激活,抑制β-catenin基因的表达可以在裸鼠体内显著抑制食管癌细胞的生长,并降低其侵袭转移的能力.

Objective To study the effects of short hairpin RNA （shRNA） mediated gene silencing of β-catenin on the biological characteristics of esophageal carcinoma cells, and to provide theoretical and experimental evidence for the gene therapy of esophageal carcinoma through target inhibition of β-catenin gene. Methods Single strand DNA was synthesized according to the hairpin RNA sequence, and then subcloned into eukaryotic expression vector pGenesil-3 to construct a shRNA-expression pDNAs driven by human U6 promoter of β-catenin （pGen-3-CTNNB1）. One additional construct of random siRNA （pGen-3-con） without homologous to any human genes was constructed in a similar fashion as control. Positive clones were identified and verified by restriction cleavage and DNA sequencing analyses. pGen-3-CTNNB1 and pGen-3-con were then transfected into esophageal carcinoma cell line Eca-109 with liposome, respectively.Positive colonies were selected with G418. Expression of β-catenin protein and mRNA in the transfected and nontransfected Eca-109 cells were examined by Western blotting, immunofluorescence and RT-PCR,respectively. Xenograft tumor model was used to compare the tumorigenesis of three different cells. Expressions of β-catenin in all tumor tissues were examined by immunohistochemistry staining The invasive abilities of three different cells were examined with transwell invasion filter and Matrigel. Results β-catenin expression levels were found markedly decreased in Eca-109 cells transfected with pGen-3-CTNNB1. In vivo, transfection with β-catenin shRNA greatly impeded the tumor growth, pGen-3-con （1.18±0.13） g, Eca-109 （1.38 ± 0.21） g, pGen-3-CTNNB1 （0.42 ± 0.09） g, P ＜ 0.05.Immunohistochemistry staining showed a significantly decreased expression of β-catenin in β-catenin shRNA transfected cells than in random shRNA transfected and nontransfected cells （P ＜ 0.05 ）. The infiltration abilities of esophageal carcinoma cells were significantly suppressed, pGen-3-con （81 ± 5） /HPF, Eca-109（77 ±6）/HPF, pGen-3-CTNNB1 （41 ±4） /HPF, P ＜0.01; along with significantly decreased migration abilities, pGen-3-con （73±5） /HPF, Eca-109 （69 ±5） /HPF, pGen-3-CTNNB1 （38 ±4）/HPF （P ＜0.05）. Conclusions There are abnormal expression of β-catenin and activation of Wnt signaling pathway in human esophageal carcinoma cell line Eca-109. RNA interference targeting β-catenin gene suppresses the growth of xenograft tumorigenesis in nude mouse and the invasiveness and metastatic capability of esophageal carcinoma cells.