摘要
目的探讨RNA干扰沉默信号转导及转录活化因子3(signal transducer and activator of transcription 3,STAT3)基因对人喉癌Hep-2细胞放射敏感性的影响。方法构建STAT3短发夹RNA(short hairpin RNA,shRNA)真核表达质粒pshSTAT3,将pshSTAT3和阴性对照质粒(pshNeg)转染喉癌Hep-2细胞,24小时后以60Coγ射线0、2、4、6、8、10Gy照射,培养48小时后,MTT法检测细胞增殖状态,流式细胞术检测细胞凋亡率、6Gy射线照射后的STAT3、p-STAT3、B淋巴细胞2(bcl-2)蛋白的表达。结果成功构建STAT3重组质粒pshSTAT3,质粒转染人喉癌Hep-2细胞后,pshSTAT3与pshNeg组和空白对照组相比,细胞增殖明显受抑制,凋亡率明显升高(P<0.05)。pshSTAT3+照射组STAT3、p-STAT3、bcl-2的蛋白表达量均明显低于空白对照组、单纯照射组、pshNeg组、pshNeg+照射组和pshSTAT3组(P<0.05),p-STAT3蛋白表达与bcl-2表达呈正相关(r=0.974,P<0.05)。结论RNA干扰抑制STAT3基因表达能提高Hep-2细胞对放射线的敏感性,STAT3基因可能是联合放疗治疗头颈肿瘤的理想靶点。
OBJECTIVE To explore the radiosensitivity of human laryngeal carcinoma cell line Hep-2 after inhibition of STAT3（signal transducer and activator transcription 3）gene by transfecting plasmid carrying short hairpin RNA（shRNA）. METHODS The vector expressing short hairpin RNA targeting STAT3 gene（pshSTAT3）was constructed. pshSTAT3 and negative control plasmid were transfected into Hep-2 cells by Lipofectamine 2000, and transfection efficiency was observed by fluorescence microscopy and detected by FCM（flow cytometry）at 24h after transfection. Subsequently the cells were radiated by 60Co γ ray at 0, 2, 4, 6, 8,10 Gy. Cell survival after irradiation was evaluated by MTT. FCM was used to detect cell apoptosis rate and STAT3,p-STAT3, bcl-2 protein levels after radiation with 6 Gy. RESULTS The vector expressing short hairpin RNA targeting STAT3 was constructed successfully. MTT assay showed that pshSTAT3 combined with ^60Coγ ray significantly inhibited proliferation of Hep-2 cells compared with that in pshNeg and control groups（P 〈0.05）. Flow cytometry showed that apoptosis rate of the pshSTAT3 group was significantly higher than that of pshNeg and untransfected group at the same radiation dosage 0, 2, 4, 6, 8 and 10 Gy（P 〈0.05）. After 6 Gy radiation, STAT3, p-STAT3 and bcl-2 protein levels ofpshSTAT3＋radiation group decreased significantly than that of control, radiation, pshNeg, pshNeg＋radiation, and pshSTAT3 groups（P 〈0.05）. Meanwhile, FI （fluorescence index）of p-STAT3 showed a positive correlation with levels of bcl-2（r =0.974,P 〈0.05）. CONCLUSION The shRNA targeting STAT3 gene can significantly enhance the sensitivity of human laryngeal carcinoma cells to radiation. The combination of RNA interference targeting STAT3 gene with radiotherapy may be more effective in the treatment of head and neck cancers.
出处
《中国耳鼻咽喉头颈外科》
北大核心
2009年第11期623-627,共5页
Chinese Archives of Otolaryngology-Head and Neck Surgery
基金
军队医药卫生"十一五"科研资助项目(200606MA076)
