CYP3A5通过影响cyclin D1蛋白表达水平促进胰腺癌细胞增殖  

CYP3A5 promotes proliferation of pancreatic cancer cells by regulating cyclin D1 protein expression

作　　者：朱建伟[1] 孔祥毓[1] 孔凡扬[1] 朱惠云[1] 安薇[1] 吴洪玉[1] 李兆申[1] 金震东[1] Zhu Jianwei;Kong Xiangyu;Kong Fanyang;Zhu Huiyun;An Wei;Wu Hongyu;Li Zhaoshen;Jin Zhendong(Department of Gastroenterology,Changhai Hospital,Second Military Medical University,Shanghai 200433,China)

机构地区：[1]第二军医大学长海医院消化内科,上海200433

出　　处：《中华胰腺病杂志》2018年第3期175-179,共5页Chinese Journal of Pancreatology

基　　金：国家自然科学青年基金(81400669);上海市卫计委课题青年项目(20144Y0255).

摘　　要：目的探讨CYP3A5对胰腺癌细胞增殖的影响及其可能机制。方法采用蛋白质印迹法检测BxPC-3、FG、MDA28、8902、PANC15株胰腺癌细胞株CYP3A5蛋白表达,选取蛋白表达量最低的PANC1细胞和表达量最高的BxPC-3细胞分别进行CYP3A5过表达质粒转染和靶向CYP3A5的siRNA(siRNA-CYP3A5)转染,分别以空载质粒转染和非特异性siRNA(siRNA-NC)转染作为对照。采用CCK-8法和克隆形成实验检测CYP3A5过表达及表达抑制后对胰腺癌细胞增殖能力的影响。采用蛋白质印迹法及定量PCR法检测各组细胞周期调控基因cyclinE、cyclinD1及凋亡相关基因Bcl-2的蛋白和mRNA表达量。结果CYP3A5过表达质粒转染PANC1后细胞CYP3A5蛋白表达显著增加(1.66±0.14比1.00,P=0.0021)。siRNA-CYP3A5转染BxPC-3细胞后CYP3A5蛋白表达显著下降(0.18±0.02比1.00,P<0.0001)。CYP3A5过表达组与空载质粒组PANC1细胞培养48、72h的A450值分别为1.36±0.05比1.15±0.03、2.10±0.09比1.42±0.03,过表达组显著高于空载质粒组,差异均有统计学意义(P值<0.005或0.001);siRNA-CYP3A5转染组与siRNA-NC转染组BxPC-3细胞培养48、72h的A450值分别为0.62±0.01比0.77±0.03、0.83±0.01比1.18±0.02,siRNA-CYP3A5转染组显著低于siRNA-NC转染组,差异均有统计学意义(P值<0.05或<0.001)。过表达组PANC1细胞克隆形成率为(19.33±0.58)%,显著高于空载质粒组的(9.67±0.63)%;siRNA-CYP3A5组克隆形成率为(8.50±0.80)%,显著低于siRNA-NC组的(16.00±0.60)%,差异均有统计学意义(P值均<0.01)。过表达组PANC1细胞cyclinD1蛋白表达量为2.00±0.11,显著高于空载质粒组的1.00;siRNA-CYP3A5组BxPC-3细胞cyclinD1蛋白表达量为0.45±0.04,显著低于siRNA-NC组的1.00,差异均有统计学意义(P值均<0.01)。但CYP3A5过表达或抑制对cyclinD1mRNA表达量以及cyclinE、Bcl-2蛋白和mRNA的表达均无影响。结论CYP3A5通过上调cyclinD1蛋白表达促进胰腺癌细胞增殖。ObjectiveTo investigate the effect of CYP3A5 on the proliferation of pancreatic cancer cells and its underlying mechanisms.MethodsThe protein expression of CYP3A5 in five pancreatic cancer cell lines BxPC-3, FG, MDA28, 8902 and PANC1 was detected by Western blotting. The PANC1 cells with the lowest protein expression of CYP3A5 and the BxPC-3 cells with highest expression of CYP3A5 were transfected with CYP3A5 overexpression plasmid and CYP3A5 targeted-siRNA(siRNA-CYP3A5), respectively. CCK-8 and cloning formation assay were used to investigate the role of CYP3A5 overexpression and knockdown in the proliferation of pancreatic cancer cells. The changes of the protein and mRNA expression of cell cycle regulating gene cyclin E, cyclin D1 and apoptosis related gene Bcl-2 were detected by Western blotting and PCR, respectively.ResultsCYP3A5 protein expression in PANC1 cells increased significantly after the transfection of CYP3A5 overexpression plasmid (1.66±0.14 to 1, P=0.0021), which greatly decreased in BxPC-3 cells transfected with siRNA CYP3A5 (0.18±0.02 to 1, P<0.0001). A450 values of the CYP3A5 overexpression group and the empty plasmid group in PANC1 cells cultured for 48 and 72 h were 1.36±0.05 vs 1.15±0.03, 2.1±0.09 vs 1.42±0.03, respectively, which were significantly higher in CYP3A5 overexpression group than empty plasmid group, and the differences were statistically significant (P value <0.005 or 0.001). The A450 values of BxPC-3 cells in CYP3A5-siRNA transfected group and siRNA-NC transfected group were 0.62±0.01 vs 0.77±0.03、0.83±0.01 vs 1.18±0.02, respectively, which in The CYP3A5-siRNA transfection group was significantly lower than that of siRNA-NC transfection group, and the difference was statistically significant (P<0.05 or <0.001). The clone formation rate of PANC1 cells in the overexpression group was (19.33±0.58)%, which was significantly higher than that in the empty plasmid group (9.67±0.63)%, and the clone formation rate in CYP3A5-siRNA group was (8.5±0.8)%, which was significa

关 键 词：胰腺肿瘤 细胞色素类 细胞增殖 CYP3A5 CYCLIN D1 

分 类 号：R735.9[医药卫生—肿瘤]