靶向survivin的siRNA对肝癌细胞生物学行为的影响

Effect of siRNA targeting survivin gene on biological behavior of hepatocellular carcinoma

目的:探讨转入靶向survivin的siRNA对肝癌细胞生物学行为的影响. 方法:设计、合成一对survivin编码基因的反向重复序列, 中间间隔9个核苷酸序列,通过定向克隆至载体PSilencer2.1, 构建siRNA真核表达载体,经稳定转染HepG2细胞后对转基因后肿瘤细胞的生物学行为进行观察. 结果:测序证实siRNA真核表达载体构建成功.通过RT- PCR及流式细胞术检测,siRNA在mRNA及蛋白质水平抑制survivin基因表达分别达73%和75%.转染细胞生长速度明显减慢,凋亡率增加15倍.裸鼠体内成瘤率下降75%,9-30 d肿瘤体积分别为0.10±0.01 cm3,0.30±0.03 cm3,0.39±0.11 cm3,0.45±0.13 cm3,0.49±0.07 cm3,0.58±0.01 cm3,0.60±0.10 cm3,0.65±0.07 cm3,与对照组相比差异显著(P<0.01). 结论:靶向survivin的siRNA能有效降低目的基因的表达并能在体内体外抑制肝癌细胞株HepG2的生长.为进一步逆转肿瘤细胞的耐药性提供了理论指导.

AIM: To study the influence of siRNA targeting survivin on the biological behavior of hepatocellular carcinoma (HCC). METHODS: One pair of 21 bp reverse repeated motifs of survivin target sequence with 9 spacer were synthesized and inserted into plasmid psilencer 2.1 to generate siRNA eukaryotic expression vector. After stable transfection into HepG2 cells, the biological behaviors of the survivin siRNA transfected HCC cells were observed. RESULTS: The recombinant plasmid Psilence(+)-survivin was successfully constructed. survivin mRNA and protein expression inhibitory ratio reached 73% and 75% respectively. It was demonstrated that transfected cells with survivin siRNA were inhibited on growth and increased on apoptosis. Subsequent study in nude mouse model demonstrated lower succeeding rate in cells transfected with survivin siRNA and the tumor size from 9-30 day was 0.10±0.01 cm3, 0.30±0.03 cm3, 0.39±0.11 cm3, 0.45±0.13 cm3, 0.49±0.07 cm3, 0.58±0.01 cm3 0.60±0.10 cm3, and 0.65±0.07 cm3 respectively. The difference was obvious (P<0.01). CONCLUSION: siRNA targeting survivin gene can specifically suppress survivin expression in HepG2 cells and inhibit tumor cells growth both in vivo and in vitro. This provides a theory basis to reverse the drug resistance in tumor cells.