杆状病毒介导的DNA甲基转移酶1 siRNA对人胰腺癌细胞凋亡的影响

Effects of baculovirus-mediated siRNA of DNA methyltransferase 1 on pancreatic cancer cell apoptosis

目的:观察杆状病毒介导的DNA甲基转移酶1(DNA-methyltransferase 1,DNMT1)siRNA(小干扰RNA)对人胰腺癌PaTu8988细胞凋亡的影响。方法:人胰腺癌细胞PaTu8988用6孔板培养并随机分成5组:空白组、阴性组、病毒r-Bac-si-DNMT1-D1组(D1组)、r-Bac-si-DNMT1-D2组(D2组)、r-Bac-si-DNMT1-D3组(D3组)。空白组仅加10%胎牛血清和DMEM培养基培养,阴性组、D1组、D2组、D3组加血清和DMEM培养基培养24 h后,分别加入空白载体r-Bac-CMV-EGFP和重组杆状病毒r-Bac-si-DNMT1-D1、r-Bac-si-DNMT1-D2、r-Bac-si-DNMT1-D3,150μL/孔。72 h后收集5组细胞,用流式细胞术测肿瘤细胞凋亡,蛋白质印迹法检测抗凋亡蛋白Bcl-2和促凋亡蛋白Bax的表达情况。结果:D1组、D2组和D3组的细胞凋亡率明显高于空白组和阴性组(P<0.05)。与空白组和阴性组相比,D1组、D2组和D3组Bcl-2蛋白表达明显降低(P<0.05),Bax蛋白表达显著增加(P<0.05)。结论:杆状病毒介导的DNMT1siRNA通过抑制Bcl-2蛋白表达,增加Bax蛋白表达,诱导胰腺癌细胞的凋亡。

Objective： To observe the effects of baculovirus-mediated RNA small interference（siRNA） of DNA methyltransferase 1（DNMT1） on the apoptosis of pancreatic cancer cell line PaTu8988 cells.Methods： Human pancreatic cancer cell line PaTu8988 cells were cultured in 6-well plates.Pancreatic cancer cells were randomly divided into five groups： control group（C1 group）,negative control group（C2 group）,r-Bac-si-DNMT1-D1 group（D1 group）,r-Bac-si-DNMT1-D2 group（D2 group）,r-Bac-si-DNMT1-D3 group（D3 group）.C1 group cells were cultured only with 10% fetal bovine serum（FBS） and DMEM.At 24 h after plating with FBS and DMEM,C2 group,D1 group,D2 group and D3 group were transfected with carrier control r-Bac-CMV-EGFP,and recombinant baculovirus r-Bac-si-DNMT1-D1,r-Bac-si-DNMT1-D2,r-Bac-si-DNMT1-D3,150 μL each well.At 72 h after transfecting,flow cytometry was performed to analyze cell apoptosis.Western blotting was used to detect the expression of antiapoptosis protein Bcl-2 and proapoptosis protein Bax.Results： Flow cytometry analysis showed that cell apoptosis rate in D1 group,D2 group,and D3 group were significant higher than that in C1 group and C2 group（P＆lt;0.05）.Compared with C1 group and C2 group,the protein expression of Bcl-2 in D1 group,D2 group and D3 group were deeply decreased（P＆lt;0.05）,but the protein expression of Bax in D1 group,D2 group and D3 group were obviously higher（P＆lt;0.05）.Conclusion： Baculovirus-mediated siRNA of DNMT1 could induce apoptosis of human pancreatic cancer cells by decreasing Bcl-2 expression and increasing Bax expression.