shRNA干扰TTF-1基因对肺腺癌细胞EGFR-TKI敏感性及细胞上皮间质化的影响  

Effect of shRNA mediated TTF-1 gene silencing on sensitivity to EGFR-TKI and epithelial-mesenchymal transition in lung adenocarcinoma cells

作　　者：吕昕[1] 周林水[1] 陈瑞琳[1] 杨珺超[1] 王真[1] LYU Xin;ZHOU Linshui;CHEN Ruilin(Department of Respiratory Medicine,the First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310006,China)

机构地区：[1]浙江中医药大学附属第一医院呼吸内科

出　　处：《浙江医学》2019年第22期2374-2378,I0004,共6页Zhejiang Medical Journal

基　　金：浙江省自然科学基金(LQ16H270005);浙江省中医药管理局科研基金(2015ZB042)

摘　　要：目的探讨甲状腺转录因子(TTF-1)对肺腺癌细胞表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)药物敏感性及细胞上皮间质化的影响。方法将培养后的肺腺癌HCC827细胞分为空载体组、阴性质粒转染(NC-sh RNA)组和阳性质粒转染(TTF-1-sh RNA)组,通过sh RNA干扰技术抑制细胞中的TTF-1基因表达,用流式细胞仪测定转染前后细胞凋亡率和细胞周期变化。不同浓度的吉非替尼(10、20、40、60μmol/ml)作用各组细胞72h,CCK-8法检测细胞生长抑制率。5ng/ml转化生长因子-β1(TGF-β1)作用各组细胞48h,细胞出现上皮间质化,Western blot法测定TTF-1、α-平滑肌肌动蛋白(α-SMA)、E-钙黏蛋白(E-cadherin)表达。结果与NC-sh RNA组、空载体组比较,TTF-1-sh RNA组细胞凋亡率明显增加,G0/G1相百分比升高,S相和G2/M相百分比降低(均P<0.05);不同浓度吉非替尼作用后,每组细胞的生长抑制率均随药物浓度的增加而升高,不同浓度比较差异有统计学意义(P<0.05)。同一浓度时,3组细胞生长抑制率之间的差异有统计学意义(P<0.05);TTF-1-sh RNA组较NC-sh RNA组、空载体组均明显为高,差异均有统计学意义(均P<0.05);而空载体组与NC-sh RNA组比较,仅在吉非替尼20μmol/ml差异有统计学意义(P<0.05)。TGF-β1作用前后,与空载体组和NC-sh RNA组比较,TTF-1-sh RNA组细胞TTF-1、α-SMA蛋白表达水平均为低,E-cadherin蛋白表达水平均为高,差异均有统计学意义(均P<0.05)。与TGF-β1作用前比较,TGF-β1作用后NC-sh RNA组和空载体组TTF-1蛋白表达水平均下降,E-cadherin蛋白表达水平均下降,差异均有统计学意义(均P<0.05)。与NC-sh RNA组、空载体组比较,TTF-1-sh RNA组TGF-β1作用前后细胞E-cadherin、α-SMA蛋白变化不明显,差异无统计学意义(P>0.05)。结论sh RNA干扰TTF-1基因可增加肺腺癌HCC827细胞对EGFR-TKI药物敏感性,并部分抑制细胞上皮间质化。Objective To investigate the effect of thyroid transcription factor-1(TTF-1)on the drug sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)and epithelial-mesenchymal transition(EMT)in human lung adenocarcinoma HCC827 cells.Methods HCC827 cells were knocked down the expression of TTF-1 gene by sh RNA,then were divided into empty vector group,Sh RNA negative plasmid transfection group(NC-sh RNA group)and Sh RNA positive plasmid transfection group(TTF-1-sh RNA group).Flow cytometry was used to determine cell cycle.Cell Counting Kit-8(CCK-8)was used to detect the growth inhibition rate of gefitinib(10,20,40,60μmol/ml).EMT was induced by 5 ng/ml transforming growth factor-β1(TGF-β1)in each cell group.Expression of TTF-1,α-SMA and E-cadherin was determined by Western-blot.Results Compared with NC-sh RNA group and empty vector group,the apoptosis of TTF-1-sh RNA group was significantly increased,the percentage of G0/G1 phase in cell cycle was increased and the percentage of S phase and G2/M phase was decreased.After different concentrations of gefitinib treatment,the difference of cell growth inhibition rate between the three groups was statistically significant(P<0.05).After the same concentration of gefitinib treatment,compared with NC-sh RNA group and empty vector group,the cell growth inhibition rate of TTF-1-sh RNA group was increased,the difference was statistically significant(P<0.05).Compared with the NC-sh RNA group and the empty vector group,the growth inhibition rate of the TTF-1-sh RNA group was increased after adding different concentrations of gefitinib(10,20,40,60μmol/ml),and the difference was statistically significant(all P<0.05).Before and after TGF-β1 treatment,the expression of TTF-1 andα-SMA in TTF-1-sh RNA group was lower than those in empty vector group and NC-sh RNA group,and the expression of Eca was higher(all P<0.05).The expression of TTF-1 in NC-sh RNA group and empty vector group were decreased after TGF-β1 treatment,and the expression of Eca was decre

关 键 词：肺腺癌 TTF-1 上皮间质化 EGFR-TKI HCC827 

分 类 号：R73[医药卫生—肿瘤]