CAJ | 학술논문

目的探讨短发夹RNA(short hairpin RNA,shRNA)表达载体稳定沉默叉头框M1(forkhead box M1,FOXM1)基因对人肝癌细胞体外生长的影响.方法构建针对人类FOXM1mRNA的不同干扰靶点的4个shRNA表达载体,选择干扰效果最佳的表达载体和阴性对照质粒转染人肝癌细胞QGY-7703,用新霉素(G418)筛选稳定转染的克隆.用四甲基偶氮唑盐(MTT)比色法和平板克隆形成实验检测FOXM1基因沉默前后肝癌细胞体外生长能力的变化.用Annexin V-APC/PI双染法检测细胞凋亡.结果不同人肝癌细胞株中普遍表达FOXM1蛋白.4个shRNA表达载体中,shRNA-1026表达载体的干扰效果最佳,对FOXM1 mRNA和蛋白表达水平的抑制率分别为38.5%和53.2%.用shRNA-1026稳定沉默FOXM1基因后,QGY-7703细胞增殖受到抑制,培养48、72和96 h后,沉默组的吸光值均显著低于对照组(分别t=10.830,3.578,5.734,均P＜0.05);沉默组的克隆形成能力较对照组显著降低(t=5.336,P＜0.05),而细胞凋亡较对照组显著增加(t=6.827.P＜0.05).结论 shRNA表达载体稳定沉默FOXM1基因抑制肝癌细胞的生长.
목적 탐토단발협RNA(short hairpin RNA,shRNA)표체재체은정침묵차두광M1(forkhead box M1,FOXM1)기인대인간암세포체외생장적영향.방법 구건침대인류FOXM1mRNA적불동간우파점적4개shRNA표체재체,선택간우효과최가적표체재체화음성대조질립전염인간암세포QGY-7703,용신매소(G418)사선은정전염적극륭.용사갑기우담서염(MTT)비색법화평판극륭형성실험검측FOXM1기인침묵전후간암세포체외생장능력적변화.용Annexin V-APC/PI쌍염법검측세포조망.결과 불동인간암세포주중보편표체FOXM1단백.4개shRNA표체재체중,shRNA-1026표체재체적간우효과최가,대FOXM1 mRNA화단백표체수평적억제솔분별위38.5%화53.2%.용shRNA-1026은정침묵FOXM1기인후,QGY-7703세포증식수도억제,배양48、72화96 h후,침묵조적흡광치균현저저우대조조(분별t=10.830,3.578,5.734,균P＜0.05);침묵조적극륭형성능력교대조조현저강저(t=5.336,P＜0.05),이세포조망교대조조현저증가(t=6.827.P＜0.05).결론 shRNA표체재체은정침묵FOXM1기인억제간암세포적생장.
Objective To evaluate the effect of sustained silencing Forkhead box Ml (F0XM1) gene by short-hairpin RNA (shRNA) expression vector on cell growth of hepatocelluar carcinoma (HCC) in vitro.Methods Four shRNA expression vectors targeting different sequences of human F0XM1 mRNA were constructed.The expression vector with the best interfering effect and the negative control plasmid were used to transfect HCC cell line QGY-7703, stably transfected cell clones were selected by neomycin (G418).Cell growth was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation was assessed by clonogenic assay.Cell apoptosis was detected by double staining with APC conjugated Annexin V and PI.Results F0XM1 protein was detected with different levels in all these studied human cell lines.The expression vector shRNA-1026 exhibited excellent interference effect after transient transfection, which showed 38.5% and 53.2% reduction of FOXM1 mRNA and protein level respectively.The growth of QGY-7703 cells was inhibited after stable inhibition of FOXM1 expression by shRNA-1026, which was indicated by decreased absorbance value of the test group after culture for 48, 72 and 96 h compared to control group (t = 10.830,3.578 and 5.734 respectively, P ＜ 0.05).Stable inhibition of F0XM1 also led to reduced colony formation ( t = 5.336, P ＜ 0.05 ) and increased apoptosis of QGY-7703 cells in comparison to control cells (t = 6.827, P ＜ 0.05 ).Conclusions Stable silencing F0XM1 gene by shRNA suppresses the growth of HCC cells in vitro.