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郭启帅,黄曦,吴永忠,李少林.靶向EGFR基因RNA干扰对人卵巢癌SKOV3细胞增殖的抑制作用[J].陆军军医大学学报(原第三军医大学学报),2009,31(23):2338-2341.
GUO Qi-shuai,HUANG Xi,WU Yong-zhong,LI Shao-lin.RNA interference targeting EGFR on proliferation of human ovarian carcinoma cell line SKOV3[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(23):2338-2341.
郭启帅,黄曦,吴永忠,李少林.靶向EGFR基因RNA干扰对人卵巢癌SKOV3细胞增殖的抑制作用[J].陆军军医大学学报(原第三军医大学学报),2009,31(23):2338-2341. DOI:
GUO Qi-shuai,HUANG Xi,WU Yong-zhong,LI Shao-lin.RNA interference targeting EGFR on proliferation of human ovarian carcinoma cell line SKOV3[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(23):2338-2341. DOI:
目的 利用RNA干扰技术下调表皮生长因子受体(epidermal growth factor receptor,EGFR)基因的表达,观察其对卵巢癌SKOV
3
细胞增殖的影响。 方法 构建针对EGFR基因的siRNA真核表达载体,转染入卵巢癌SKOV
3
细胞中,并筛选稳定表达siRNA的转化克隆,RT-PCR及Western blot分别检测EGFR mRNA和蛋白表达,流式细胞仪检测细胞周期和凋亡
克隆形成实验及MTT法检测细胞克隆形成率及细胞体外的增殖情况。 结果 成功构建表达质粒pGenSil-HK、pGenSil-EGFR1和pGenSil-EGFR2,并转染SKOV
3
细胞,通过G418筛选后获得稳定转染克隆,RT-PCR及Western blot分析表明转染pGenSil-EGFR1、pGenSil-EGFR2后的细胞EGFR mRNA和蛋白表达均受到了明显抑制,EGFR mRNA抑制率分别为41.87%和68.07%,蛋白表达抑制率分别为45.21%和70.25%。与未转染组和pGenSil-HK组相比,pGenSil-EGFR2组细胞凋亡比例显著增加,G
1
期细胞增多,而S期细胞减少(P0.05);转染pGenSil-HK组克隆形成率为(0.82±0.03),转染pGenSil-EGFR2组克隆形成率为(0.61±0.04),二者比较差异显著(P0.05),MTT显示细胞培养36 h后,pGenSil-EGFR-2组的细胞生长均显著低于未转染组和转染pGenSil-HK组(P0.05)。 结论 靶向EGFR基因RNA干扰能够阻抑卵巢癌SKOV
3
细胞中的EGFR表达,诱导细胞凋亡、调控细胞周期再分布、抑制细胞增殖。
Objective To explore the effect of downregulating epidermal growth factor receptor (EGFR) expression by RNA interference on the proliferation of ovarian carcinoma SKOV
3
cells. Methods The recombinant eukaryotic expression plasmids pGenSi
l-HK
pGenSil-EGFR1 and pGenSil-EGFR2 were constructed and transfected into SKOV
3
cells respectively. The mRNA and protein expressions of EGFR in SKOV
3
cells were detected by RT-PCR and Western blotting
respectively. The cell cycle and apoptosis were evaluated by flow cytometry. The proliferation of SKOV
3
cells was determined by clone formation assay and MTT assay. Results We successfully constructed the recombinant eukaryotic expression plasmids pGenSil-HK
pGenSil-EGFR1 and pGenSil-EGFR2 and transfected into SKOV
3
cells. Three cell clones were screened by G418. Compared with untransfected SKOV
3
cells and SKOV
3
cells transfected with pGenSil-HK
the expressions of EGFR in SKOV
3
cells transfected with pGenSil-EGFR1
pGenSil-EGFR2 were inhibited significantly at both mRNA and protein levels
with an inhibitory rate of 41.87% and 68.07% for EGFR mRNA and of 45.21% and 70.25% for EGFR protein respectively. Compared with untransfected SKOV
3
cells and SKOV
3
cells transfected with pGenSil-HK
the cell apoptotic rate was significantly increased significantly
the cell cycle was arrested in G
1
phase and S phase decreased significantly in pGenSil-EGFR2 SKOV
3
cells (P0.05). The clone formation efficiency was significantly lower in pGenSil-EGFR2 SKOV
3
cells than in untransfected SKOV
3
cells and SKOV
3
cells transfected with pGenSil-HK [(0.61±0.04) vs (0.82±0.03)
(P0.05)
]
. The proliferative rate was markedly lower in pGenSilEGFR2 SKOV
3
cells than in untransfected SKOV
3
cells and SKOV
3
cells transfected with pGenSilHK 36 h after transfection (P0.05). Conclusion RNA interference targeting EGFR gene down-regulates the EGFR expression
induces cell apoptosis
regulates cell phase redistribution and inhibits cell proliferation in ovarian carcinoma SKOV
3
cells.
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