CAJ | 학술논문

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-FITC PI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caspase 9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,10.0 mg/L的吉西他滨和10.0 mg/L的顺铂分别作用48 h后,转染hSMG-1 siRNA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-FITC PI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-1的H1299细胞给予抗癌药物作用时线粒体凋亡通路相关活性caspase 3和caspase 9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.
목적 탐토억제인류생식기형성억제기인(hSMG-1)대인비소세포폐암H1299세포화료민감성적영향.방법 파향hSMG-1기인적소간우RNA(siRNA)전염H1299세포,실시정량역전록PCR화면역형광험증고강효과;CCK-8법료해억제hSMG-1기인후H1299세포대항암약물민감성;류식Annexin V-FITC PI쌍염법검측각조세포적조망솔;비색법측정각조세포활성반광천동매(caspase)3화caspase 9적량.결과 hSMG-1소간우RNA(siRNA)현저억제hSMG-1기인mRNA적표체,억제솔체(73.8±10.3)%(P＜0.01);면역형광검측야증실hSMG-1단백적표체피명현억제.억제hSMG-1후,H1299세포대길서타빈화순박적민감성증가,10.0 mg/L적길서타빈화10.0 mg/L적순박분별작용48 h후,전염hSMG-1 siRNA조세포존활솔여전염대조siRNA조비균현저강저(0.51±0.02비0.69±0.01,0.34±0.03비0.48±0.01,균P＜0.01).류식Annexin V-FITC PI쌍염검측현시억제hSMG-1후재급여항암약물작용시능유도경다적H1299세포조망(P＜0.05).억제hSMG-1적H1299세포급여항암약물작용시선립체조망통로상관활성caspase 3화caspase 9적량균현저승고(P＜0.05).결론 억제hSMG-1기인능통과유도경다적종류세포조망,제고인폐암H1299세포대화료약물적민감성,기궤제가능여증강종류세포선립체조망신호통로활성유관.
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 immunofluorescence. After the administration of anti-cancer drugs, the cell viable rate was determined by cell counting kit (CCK-8) and apoptotic rate was measured by Annexin V-FITC PI double staining on flow cytometry. The apoptotic related activated caspase 3 and caspase 9 were determined by colorimetric 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. 02; all compared with control siRNA group). Annexin V-FITC PI double staining showed that, under the treatment of anti-cancer drugs, the apoptotic 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, caspase 3:18.8±3.0 vs 6.5 ±1.5, P＜0. 01; caspase 9:20.3±4.2 vs 8. 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 cells to gemcitabine and cisplatin through an induction of more apoptotic cells. And the activation of mitochondrial apoptotic pathway is involved.