DNMT3B对甲状腺乳头状癌细胞增殖和侵袭的影响

Effect of DNMT3B on proliferation and invasion of papillary thyroid carcinoma cells

目的探讨敲减DNA甲基化转移酶3B(DNA methyltransferase 3 beta,DNMT3B)对甲状腺乳头状癌细胞BCPAP增殖和侵袭的影响及其作用机制。方法采用Western blot方法检测正常人甲状腺细胞Nthy-ori 3-1和甲状腺癌细胞株TPC-1、K1和BCPAP中DNMT3B的表达,以DNMT3B高表达的BCPAP细胞作为后续研究对象,实验分为空白对照组(control组)、阴性对照组(si-NC组,转染阴性siRNA)和敲减DNMT3B组(si-DNMT3B组,转染干扰DNMT3B表达的siRNA)。应用RNA敲减(RNAi)技术合成针对DNMT3B的siRNA并转染BCPAP细胞,qRT-PCR和Western blot检测各组中DNMT3B、死亡相关蛋白激酶(death-associated protein kinase,DAPK)、细胞凋亡调控因子(Bcl-2-associated X protein,Bax)和Ras相关区域家族1A(Ras-associated domain family 1A,RASSF1A)这4个基因的mRNA和蛋白水平。CCK-8法检测细胞增殖能力。Transwell实验检测细胞侵袭和迁移能力。流式细胞术检测敲减DNMT3B对细胞凋亡的影响。甲基化特异性PCR检测敲减DNMT3B对DAPK,Bax和RASSF1A基因甲基化的影响。结果与Nthy-ori 3-1细胞比较,DNMT3B在TPC-1细胞和K1细胞中的mRNA和蛋白水平升高,在BCPAP细胞中的mRNA和蛋白水平最高(P<0.01)。与control组和si-NC组相比,敲减DNMT3B组中DNMT3B基因的mRNA和蛋白水平显著降低(P<0.01)。与control组和si-NC组相比,敲减DNMT3B组BCPAP细胞增殖、迁移和侵袭能力显著降低(P<0.01)。与control组和si-NC组相比,敲减DNMT3B组BCPAP细胞凋亡增多(P<0.01)。与control组和si-NC组相比,敲减DNMT3B组DAPK、Bax和RASSF1A启动子甲基化水平下降,mRNA水平和蛋白水平上升(P<0.01)。结论敲减DNMT3B可能通过调控凋亡相关基因启动子区域的甲基化水平来影响基因的表达,从而抑制BCPAP细胞的增殖、迁移及侵袭。

Objective To investigate the effect of DNMT3B on the proliferation and invasion of thyroid papillary carcinoma cell line BCPAP and its mechanism.Methods Western blot was used to detect the expression of DNMT3B in normal thyroid Nthy-ori 3-1 cells and thyroid cancer cell lines TPC-1,K1 and BCPAP.BCPAP cells with high expression of DNMT3B were divided into blank control group(control group),negative control group(NC group,transfected with DNMT3B intervention null siRNA)and DNMT3B interference group(transfected with DNMT3B effective intervention siRNA).The siRNA targeting DNMT3B was synthesized by RNA interference(RNAi)technique and transfected into BCPAP cells.The effects of interfering DNMT3B on the mRNA and protein expression of DAPK,Bax and RASSF1A were detected by qRT-PCR and Western blot.The ability of cell proliferation was detected by CCK-8 assay.The invasion and migration abilities were detected by Transwell assay.The effect of interfering DNMT3B on apoptosis was detected by flow cytometry.Methylation-specific PCR was used to detect the effect of interfering DNMT3B on cell growth and methylation of apoptosis-related genes.Results Compared with Nthy-ori 3-1 cells,the expression level of DNMT3B in TPC1 and K1 cells was significantly increased(P<0.01),and it was the highest in BCPAP cells.Compared with control group and si-NC group,the mRNA and protein levels of DNMT3B were decreased significantly in DNMT3B interference group(P<0.01),the proliferation,migration and invasion abilities of BCPAP cells were significantly inhibited(P<0.01),and the apoptosis of BCPAP cells was promoted(P<0.01).Compared with control group and si-NC group,the methylation levels of DAPK,Bax and RASSF1A promoters were decreased,while their expression was increased in DNMT3B interference group(P<0.01).Conclusion Interfering with DNMT3B may affect gene expression by regulating the level of methylation in the promoter region of apoptosis-related genes,thus inhibiting the proliferation,migration and invasion of BCPAP cells.