Construction and Identification of a siRNA Expression Plasmid Targeting Sox9 and Its Effect on the Growth and Apoptosis of Human Chondrosarcoma Cells
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摘要: 目的: 利用小干扰阻断目的基因的原理将构建的目的基因Sox9的pSilencer3,1-H1 neo siRNA(small interfering RNA)表达质粒转染人体软骨肉瘤细胞HTB-94,观察目的基因被阻断后肿瘤细胞目的基因的表达、肿瘤细胞生长和凋亡所受到的影响。 方法: 设计并合成Sox9(siRNA),鉴定后将其转染HTB-94肿瘤细胞,观察肿瘤细胞的Sox9基因的mRNA和蛋白表达的变化,以及被转染肿瘤细胞的生长曲线和肿瘤细胞凋亡的情况。 结果: Sox9(siRNA)的插入片段测序结果与合成的siRNA结果一致,被转染的HTB-94Sox9基因的mRNA的表达量下降了35.4%,蛋白的表达量下降了31.3%;Sox9(siRNA)转染HTB-94细胞24h和96h的光吸收值分别为0.146±0.037和0.412±0.036,而正常对照细胞组两个同样时间段的光吸收值分别为0.152±0.0367和0.607±0.029。其中细胞生长增殖速度的差异为32.0%,肿瘤细胞的生长明显受到抑制;Sox9(siRNA)转染HTB-94细胞的干扰组细胞凋亡率为39.2%;而未经干扰的HTB-94肿瘤细胞的细胞凋亡率为0.1%。 结论: 经过设计合成的Sox9(siRNA)表达质粒可以稳定转染人体软骨肉瘤细胞HTB-94肿瘤细胞,被Sox9(siRNA)表达质粒转染的人体软骨肉瘤细胞HTB-94肿瘤细胞Sox9基因的mRNA和蛋白的表达都受到抑制,同时肿瘤细胞的生长繁殖也受到明显抑制,肿瘤细胞的凋亡明显增加。
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关键词:
- siRNA(Sox9) /
- RNA干扰 /
- Sox9蛋白和mRNA表达 /
- 细胞生长曲线 /
- 细胞凋亡
Abstract: Objective: To construct a siRNA expression plasmid targeting Sox9 and to investigate its effect on thegrowth and apoptosis of human chondrosarcoma cells. Methods: After designing and synthesizing the siRNA expressionplasmid, it was transfected into HTB-94 human chondrosarcoma cells and confirmed by enzyme digestion analysis andDNA sequencing. The expression of Sox9 mRNA and protein was assessed and cell growth and apoptosis were observed. Results: Compared with that of untransfected cells, the expression of Sox9 mRNA in transfected HTB-94 cells was re-duced by 35.4%, and the protein expression was reduced by 31.3%. The light absorbance values in transfected cells after24h and 96h were 0.146± 0.037 and 0.412± 0.036, but were 0.152± 0.0367 and 0.607± 0.029 in untransfected cells. Thecell growth in transfected cells was 32% lower than that in untransfected cells. The apoptosis rate was 39.2% in transfect-ed cells and 0.1% in untransfected cells. Conclusion: After transfection into HTB-94 human chondrosarcoma cells, asiRNA expression plasmid targeting Sox9 reduced mRNA and protein expression, inhibiting growth and causing apoptosisof the tumor cells.-
Keywords:
- siRNA /
- Sox9 /
- RNA interference /
- Tumor cell growth /
- Tumor cell apoptosis
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