增强PIAS3表达对CHG-5细胞生物学效应的影响

Effects of the overexpression of PIAS3 in CHG-5 glioma cells in vitro

目的探讨增强人胶质瘤CHG-5细胞内的活化STAT3抑制蛋白(protein inhibitor of activated STAT3,PIAS3)表达对CHG-5细胞增殖及凋亡等生物学效应的影响。方法构建PIAS3真核表达载体pEGFP-N1-PIAS3,使用脂质体法瞬时转染人CHG-5胶质瘤细胞,同时设置空白对照组和阴性对照组,转染48h后用RT-PCR、蛋白质印迹法和免疫组化法分别在核酸和蛋白水平检测目的蛋白表达,采用流式细胞技术检测转染后的胶质瘤细胞凋亡和细胞增殖变化。结果转染后的CHG-5细胞可见PIAS3绿色荧光蛋白表达,显微镜观察发现细胞形态改变,坏死细胞增多。转染组PIAS3mRNA的积分密度值为523 414.50±34 502.89,与空白对照组的135 668.00±6 693.66和阴性对照组的154 511.67±8 266.89相比,转染组PIAS3mRNA表达增强,χ2=13.053,P=0.01。转染组PIAS3蛋白的积分密度值为30.13±3.76,与空白对照组的13.83±0.77和阴性对照组的15.02±0.87比较,CHG-5细胞转染PIAS3基因后,PIAS3蛋白表达增强,χ2=14.193,P=0.001。提示转染后PIAS3mRNA和蛋白表达增加。转染组的早期细胞凋亡率为(12.5±1.9)%,较空白对照组的(6.4±1.1)%和阴性对照组的(5.4±1.8)%升高,χ2=7.407,P=0.005;转染组的活细胞率为(86.9±2.2)%,较空白对照组的(92.1±1.2)%和阴性对照组的(93.1±2.0)%降低,χ2=4.775,P=0.019。转染组S期细胞百分率为(35.2±4.2)%,高于空白对照组的(24.5±5.1)%和阴性对照组的(23.0±3.7)%,χ2=8.179,P=0.003;转染组G2期细胞百分率为(10.7±5.4)%,高于空白对照组的(21.3±4.0)%和阴性对照组的(27.8±5.2)%,χ2=17.121,P<0.01。结论外源性增强PIAS3表达,引起CHG-5胶质瘤细胞生长缓慢,出现S期阻滞,并促进细胞凋亡发生。

OBJECTIVE To observe the effects of overexpression of protein inhibitor of activated STAT3（PIAS3） on the proliferation and apoptosis in CHG-5 glioma cells. METHODS The constructed eukaryotic expression vectors of PIAS3 was constructed named as pEGFP-N1-PIAS3. In vitro the CHG-5 glioma cells were transfected with pEGFP-N1-PIAS3 with O1- igofectamine. At the same time, the blank control and negative control groups were set. After 48 hours,the expression of PIAS3 was analyzed by RT-PCR,western blots and immunohistochemistry. The apoptosis and proliferation of the trans- fected cells were analyzed by flow cytometry. RESULTS The expression of PIAS3 in the transfected cells was increased. The change of cell shape and increased necrotic cells were observed by microscope. The IDV （Integrated density value） of PIAS3 mRNA （523 414.5±34 502.89） in the PIAS3-transfected group was significantly different from that of the blank control group （135 668.0±6 693.66） and the negative control group （154 511.67±8 266.89;x^2 =13. 053,P=0.01）.The IDV of PIAS3 protein （30. 13 ±3.76） in the PIAS3-transfected group was significantly different from that of the blank control group （13.83±0. 77） and the negative control group （15.02 ± 0.87;x^2 = 14. 193, P= 0. 001）. The results suggested that the expression of PIAS3 was increased in the PIAS3-transfected cells. The percentage of early apoptotic cells （12. 5 ±1.9）% in the PIAS3-transfeeted group was significantly higher than that of the blank control group （6.4 ± 1.1）% and the negative control group （5.4±1.8）% （x^2=7. 407,P=0. 005）. The rate of living cells （86.9±2.2）G was significantly lower than that of the blank control group （92.1 ± 1.2） % and the negative control group （93.1 ± 2.0） （x^2 =4. 775,P=0.019）. The percentage of the cells in S phase （35.2±4.2）% in the PIAS3-transfected group was sig- nificantly higher than that of the blank control group （24.5 ±5.1 ） % and the negative control group （23.0± 3.7） % （x^2 = 8. 179,P=0. 003）. The percentage of the cells in Gz phase （10.7±5.4）% in the PIAS3-transfected group was signifi- cantly lower than that of the blank control group （21.3±4.0）% and the negative control group （27.8±5.2）% （ x^2 = 17. 121,P〈0. 01）. CONCLUSION The overexpression of PIAS3 in CHG-5 glioma cells significantly suppresses cells proliferation, induces S-phase arrest and cell apoptosis.