摘要
目的探讨抑制人类生殖器形成抑制基因（hSMG-1）对人非小细胞肺癌H1299细胞化疗敏感性的影响。方法靶向hSMG．1基因的小干扰RNA（siRNA）转染H1299细胞，实时定量逆转录PCR和免疫荧光验证敲降效果；CCK-8法了解抑制hSMG-1基因后H1299细胞对抗癌药物敏感性；流式Annexin V—FITCPI双染法检测各组细胞的凋亡率；比色法测定各组细胞活性半胱天冬酶（caspase）3和caspase9的量。结果hSMG-1小干扰RNA（siRNA）显著抑制hSMG．1基因mRNA的表达，抑制率达（73．8±10．3）％（P〈0．01）；免疫荧光检测也证实hSMG-1蛋白的表达被明显抑制。抑制hSMG-1后，H1299细胞对吉西他滨和顺铂的敏感性增加，10．0mg／L的吉西他滨和10．0mg／L的顺铂分别作用48h后，转染hSMG-1siRNA组细胞存活率与转染对照siRNA组比均显著降低（0．51±0．02比0．69±0．01，0．34±0．03比0．48±0．01，均P〈0．01）。流式Annexin V—FITCPI双染检测显示抑制hSMG-1后再给予抗癌药物作用时能诱导更多的H1299细胞凋亡（P〈0．05）。抑制hSMG-I的H1299细胞给予抗癌药物作用时线粒体凋亡通路相关活性caspase3和caspase9的量均显著升高（P〈0．05）。结论抑制hSMG-1基因能通过诱导更多的肿瘤细胞凋亡，提高人肺癌H1299细胞对化疗药物的敏感性，其机制可能与增强肿瘤细胞线粒体凋亡信号通路活性有关。
Objective To investigate the effect of inhibiting the expression of human suppressor of morphogenesis in genitalia-1 （ hSMG-1 ） on chemosensitivity in human lung cancer H1299 cells. Methods Specialized small interference RNAs （siRNAs） of hSMG-1 were transfected into H1299 cells. The knockdown effect was evaluated by quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR） and immunoflnorescenee. After the administration of anti-cancer drugs, the cell viable rate was determined by cell counting kit （CCK-8） and apoptotie rate was measured by Annexin V-FITC PI double staining on flow cytometry. The apoptotic related activated caspase 3 and easpase 9 were determined by eolorimetric assay. Results The expression of hSMG-1 mRNA was significantly inhibited by hSMG-1 siRNA. And the inhibition rate of （73.8 ± 10. 3） % was obtained （ P 〈 0. 01 ）. The knockdown effect was further confirmed by immunofluorescence. The inhibition of hSMG-1 enhanced the sensitivity of H1299 cells to gemcitabine and cisplatin. The survival rates significantly decreased when the hSMG-1 siRNA transfected cells were treated with 10. 0 mg/L gemcitabine and 10. 0 mg/L cisplatin for 48 h respectively （0. 51 ±0. 02 vs 0. 69 ± 0. 01, P 〈 0. 01 and 0. 34 ±0. 03 vs 0. 48 ± 0. 01, P 〈 0. 01 ; all compared with control siRNA group）. Annexin V-FITC PI double staining showed that, under the treatment of anti-cancer drugs, the apoptoic rate of H1299 cells was significantly increased by hSMG-1 knockdown [ gemcitabine, （ 20. 9 ±3.4）% vs （12.0±2.7）%, P〈0.05; cisplatin, （10.2±1.8）% vs （4.5±2.0）%, P〈0.05; all compared with control siRNA group ]. Further study showed the inhibition of hSMG-1 up-regulated the activated caspase 3 and caspase 9 in the treated H1299 cells （gemcitabine, 0. 1 mg/L, 48 h, caspase 3 ： 14.4 ±3.8 vs 2. 3 ±0. 4, P 〈0. 01 ; caspase 9：15.5 ±2. 4 vs 4.2 ±0. 9, P 〈0. 01 ; cisplatin, 1.0 mg/L, 48 h, caspase3： 18.8±3.0vs6.5±1.5, P〈0.01; caspase9： 20.3±4.2vs8.1±2.0, P〈0.05; all compared with control siRNA group）. Conclusion The inhibition of hSMG-1 significantly enhances the sensitivity of human lung cancer H1299 ceils to gemcitabine and cisplatin through an induction of more apoptotic cells. And the activation of mitochondrial apoptotic pathway is involved.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第8期554-559,共6页
National Medical Journal of China
基金
基金项目：国家自然科学基金（30672406）
关键词
癌
非小细胞肺
肿瘤细胞
培养的
细胞凋亡
化学疗法
肿瘤
Carcinoma, non-small cell lung
Tumor cells, cultured
Apoptosis
Chemotherapy, cancer
