RNA干扰组蛋白去乙酰化酶1对人胰腺癌细胞增殖、凋亡的调控机制研究

Mechanism of RNA interference on proliferation and apoptosis regulation of pancreatic cancer cell line PaTu8988 by HDAC1 siRNA

目的观察沉默组蛋白去乙酰化酶1(HDAC1)基因对人胰腺癌PaTu8988细胞增殖、凋亡的影响及可能机制。方法胰腺癌PaTu8988细胞株分为空白对照组(不予任何处理)、阴性对照siRNA组(予以30nmol/L阴性siRNA)、siRNA1组(予以15nmol/LHDAC1siRNA)和siRNA2组(予以30nmol/LHDAC1siRNA)。siRNA转染48h后,用相对实时定量RT-PCR法和West-ernblotting检测HDAC1基因表达情况;相对实时定量RT-PCR法检测细胞增殖凋亡相关基因p21、Bcl-2、Bax的表达;WST-8法检测细胞增殖情况;流式细胞术检测细胞凋亡变化。结果转染HDAC1siRNA48h后人胰腺癌PaTu8988细胞中HDAC1mRNA表达量在siRNA1组和siRNA2组分别为46.1%±6.1%和32.3%±1.4%,均显著低于空白对照组(100.0%±3.4%)和阴性对照siRNA组(87.4%±28.3%,P<0.05)。Westernblotting显示HDAC1蛋白表达量亦明显下降,以siRNA2组表达量最低(P<0.05)。WST-8法检测显示4组细胞存活率分别为100%±17.1%、87.1%±5.0%、68.7%±4.7%、61.6%±2.0%,siRNA1组和siRNA2组与其他各组相比差异显著(P<0.05)。流式细胞术检测显示上述4组胰腺癌细胞凋亡率分别为4.20%±0.95%,4.59%±1.26%,10.09%±1.36%,11.19%±6.07%,与空白对照组和阴性对照siRNA组相比,siRNA1组和siRNA2组凋亡率显著增高(P<0.05)。siRNA1组和siRNA2组p21、BaxmRNA相对表达量明显高于其他各组(P<0.05),而Bcl-2mRNA相对表达量明显低于其他各组(P<0.05)。结论HDAClsiRNA能特异、有效地抑制人胰腺癌PaTu8988细胞HDACl的表达,同时抑制细胞增殖、诱导细胞凋亡,其机制可能与上调p21、Bax表达,下调Bcl-2表达有关。

Objective To investigate the effects of histone deacetylase 1 （HDAC1） gene silencing on cell proliferation and apoptosis of pancreatic cancer cell line PaTu8988.Methods PaTu8988 cells were cultured and divided into four groups：control group （untreated）,negative siRNA group （treated with 30nmol/L negative control siRNA）,siRNA1 group （treated with 15nmol/L HDACl siRNA） and siRNA2 group （treated with 30nmol/L HDACl siRNA）.RT-PCR and Western blotting were used to detect the gene expression of HDAC1 48 hours after HDAC1 siRNA transfection.The expression of p21,Bax and bcl-2 mRNA was detected by RT-PCR.Cell proliferation and apoptosis were evaluated using cell counting kit and flow cytometry,respectively.Results After HDAC1 siRNA trasnfection,the expression of HDAC1 mRNA in PaTu8988 cells was 46.1%±6.1% in siRNA1 group and 32.3%±1.4% in siRNA2 group,respectively,and they were significantly lower than that in control group （100.0%±3.4%,P0.05） and in negative siRNA group （87.4%±28.3%,P0.05）.Western blotting showed that the expression of HDAC1 protein decreased,especially in siRNA2 group （P0.05）.Cell survival rate was 100%±17.1% in control group,87.1%±5.0% in negative control siRNA group,68.7%±4.7% in siRNA1 group and 61.6%±2.0% in siRNA2 group,and significant difference existed between the latter two groups （P0.05）.Cell apoptosis rate was 4.20%±0.95%,4.59%±1.26%,10.09%±1.36% and 11.19%±6.07% respectively,in the four groups,and significantly increased in siRNA1 and siRNA2 group （P0.05）.The mRNA expression of p21 and Bax was higher than that in other two groups （P0.05）,while that of Bcl-2 was lower （P0.05）.Conclusions HDACl siRNA can efficiently and specifically inhibit the expression of HDAC1 and the proliferation of PaTu8988 cells,and meanwhile induce cell apoptosis,which may be attributable to up-regulation of p21 and Bax,and down-regulation of bcl-2 mRNA expression.