摘要
目的 构建稳定沉默Y盒结合蛋白1（YB-1）基因的人肺腺癌A549细胞株并观察其增殖能力.方法 以脂质体将两种沉默YB-1基因的shRNA真核表达载体转染人肺腺癌细胞株A549,G418筛选稳定转染细胞株.逆转录-聚合酶链反应（RT-PCR）、实时荧光定量聚合酶链反应（FQ-PCR）及Westem blot法检测YB-1基因表达;噻唑蓝（MTT）法绘制细胞生长曲线.结果 稳定转染细胞株稳定表达绿色荧光蛋白（GFP）;RT-PCR、FQ-PCR和Western blot法检测结果显示稳定转染组细胞株A549-746与A549-326 YB-1 mRNA和蛋白表达（0.41±0.01与0.39±0.06、0.43±0.10与0.20±0.24、0.49 ±0.06与0.24±0.02）较对照组明显下降（P＜0.05）;噻唑蓝（MTT）检测稳定转染组较对照组生长速度降低（P＜0.05）.结论 成功建立稳定表达GFP,并稳定沉默YB-1基因的人肺腺癌细胞株A549-746和A549-326,沉默YB-1可抑制A549细胞增殖能力.
Objective To establish a human lung adenocarcinoma A549 cell line with RNA interference for YB-1 gene silencing which can stably express green fluorescent protein （GFP） and to observe the effects of silencing Y-box binding protein （YB-1） gene on proliferation in lung adenocarcinoma A549 cells.Methods The human lung adenocarcinoma A549 cells were transfected with shRNA plasmid specific for the YB-1 gene,and were then selected through G418.G418-resistant clones were generated and screened using reverse transcription polymerase chain reaction （RT-PCR）,real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） and Western blotting analysis.Cell proliferation was examined by methyl thiazolyl tetrazolium.Results The G418-resistant clones （A549-746 and A549-326） showed much lower level of YB-1 mRNA （0.41 ± 0.01 and 0.39 ± 0.06） than A549-shNC （P 〈 0.05） under RT-PCR.A549-746 and A549-326 cell clones showed much lower level of YB-1 mRNA （0.43 ±0.10 and 0.20 ± 0.24） and protein expression （0.49 ± 0.06 and 0.24 ± 0.02） than A549-shNC （P 〈 0.05） under FQ-PCR and Western blotting analysis.Meanwhile,the G418-resistan clones （A549-746 and A549-326） stably expressed GFP in vitro.The cell growth rate in YBX1-homo-746 and YBX1-homo-326 groups was decreased significantly at the first 24 h （P 〈 0.05）.Conclusion The lung cancer cell lines A549-746 and A549-326 with stable YB-1 gene silencing and GFP expression were successfully established,and silencing YB-1 gene could inhibit the proliferation of lung adenocarcinoma A549 cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第11期2480-2482,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81173453）
大连市科技计划重大项目（2012E15SF141）
关键词
肺腺癌
Y盒结合蛋白-1
短发夹RNA
绿色荧光蛋白
Lung adenocarcinoma
Y-box binding protein-1
Short hairpin RNA
Green fluorescent protein
