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目的 观察靶向缺氧诱导因子 (HIF)- 1α的短发夹状小干扰RNA基因转染对骨肉瘤生长的影响。方法 针对HIF- 1α构建短发夹状siRNA真核表达载体 (pSilencer) HIF并转染骨肉瘤细胞系(SaOS) 2。骨肉瘤细胞置于缺氧诱导培养基中培养。逆转录 聚合酶链反应 (RT -PCR)和免疫印迹沉淀观察HIF -1α的基因沉默效果。四甲基偶氮唑盐 (MTT)比色分析检测细胞体外增殖活力,透射电镜,原位末端标记TUNEL法,AnnexinV/PI双标记法检测细胞的凋亡。同时,通过裸鼠移植瘤模型观察HIF- 1α基因沉默后对骨肉瘤体内治疗效果。结果 测序证实成功构建HIF- 1α的短发夹状siRNA真核表达载体pSilencer HIF,转染骨肉瘤细胞后,HIF -1α基因表达显著抑制 ( 90% )。与对照组比较,pSilencer HIF转染组骨肉瘤细胞缺氧状态下的增殖活性显著降低。缺氧培养 72h后,透射电镜、TUNEL法显示细胞典型凋亡特征,AnnexinV/PI双标检测转染组细胞凋亡率为 18. 71% ±0. 98%,明显高于pSilencer neo空载体转染组和未转染组 (P< 0 .01 )。裸鼠移植瘤实验显示pSilencer HIF转染组肿瘤生长减慢,成瘤率下降,肿瘤组织中可见大量坏死组织。结论 靶向HIF- 1α基因的短发夹状siRNA可以有效阻断骨肉瘤缺氧耐受转导通路,抑制骨肉瘤细胞的生长。
Objective To observe the effect of short hairpin RNA interference (shRNA) targeting hypoxia inducible factor (HIF) -1α on the growth of osteosarcoma. Methods A short hairpin siRNA eukaryotic expression vector (pSilencer) HIF was constructed and transfected into osteosarcoma cell line (SaOS) 2 against HIF-1α. Osteosarcoma cells were cultured in hypoxia-induced medium. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to observe the gene silencing effect of HIF-1α. MTT assay was used to detect cell proliferation in vitro. Transmission electron microscopy, TUNEL method and Annexin V / PI double staining were used to detect cell apoptosis. At the same time, the in vivo therapeutic effect of HIF-1α gene silencing on osteosarcoma was observed by transplanted tumor model in nude mice. Results Sequencing confirmed that the short hairpin siRNA eukaryotic expression vector pSilencer HIF was successfully constructed and the expression of HIF - 1α was significantly inhibited (90%) after transfection of osteosarcoma cells. Compared with the control group, the proliferative activity of osteosarcoma cell in hypoxia in pSilencer HIF transfected group was significantly reduced. After hypoxia for 72 hours, the typical apoptotic features of cells were observed by transmission electron microscope and TUNEL method. The apoptotic rate of the transfected group was 18. 71% ± 0. 98%, which was significantly higher than that of pSilencer neo vector Dye group and untransfected group (P <0.01). Nude mice transplantation tumor experiments showed that pSilencer HIF transfection group tumor growth slowed down, tumor formation rate decreased, a large number of tumor tissue visible necrotic tissue. Conclusion Short hairpin siRNA targeting HIF-1α gene can effectively block the transduction pathway of hypoxia tolerance of osteosarcoma and inhibit the growth of osteosarcoma cells.