CAJ | 학술논문

背景与目的：DNA甲基转移酶(DNA methyltransferase，DNMT)3B有近40种异构体，它们的表达有组织和疾病的特异性，细胞中这些异构体的功能尚不清楚。本研究旨在探讨外源性DNMT3B4基因过表达对人胚肾细胞株293A增殖的影响及其机制。方法：将携带DNMT3B4基因的质粒pCMV-DNMT3B4及空质粒pCMV-2B转染293A细胞(293A-vector)并形成稳定表达细胞系，培养300 d后用MTT法检测细胞的增殖；流式细胞仪检测细胞的周期分布；real-time PCR蛋白质印迹法(Western blot)检测细胞中p21表达情况；甲基化特异PCR(MS-PCR)检测p21基因启动子区甲基化状态。结果：在接种于96孔板的第4天，2株过表达DNMT3B4的293A细胞(293A-DNMT3B4-1和293A-DNMT3B4-2)吸光度(A)值分别是293A-vector细胞的(58.92±3.47)%和(68.82±5.64)%，过表达DNMT3B4抑制细胞增殖，差异有统计学意义(P<0.05)；293A-DNMT3B4-1和293A-DNMT3B4-2的S期细胞比例分别为(35.88±2.00)%和(37.00±1.79)%，293A-vector细胞比例为(40.44±0.91)%，过表达DNMT3B4降低S期细胞比例，差异有统计学意义(P<0.05)。过表达DNMT3B4增加p21的表达，但不改变p21基因启动子区甲基化状态。结论：DNMT3B4基因过表达可抑制293A细胞增殖并增加p21表达。
배경여목적：DNA갑기전이매(DNA methyltransferase，DNMT)3B유근40충이구체，타문적표체유조직화질병적특이성，세포중저사이구체적공능상불청초。본연구지재탐토외원성DNMT3B4기인과표체대인배신세포주293A증식적영향급기궤제。방법：장휴대DNMT3B4기인적질립pCMV-DNMT3B4급공질립pCMV-2B전염293A세포(293A-vector)병형성은정표체세포계，배양300 d후용MTT법검측세포적증식；류식세포의검측세포적주기분포；real-time PCR단백질인적법(Western blot)검측세포중p21표체정황；갑기화특이PCR(MS-PCR)검측p21기인계동자구갑기화상태。결과：재접충우96공판적제4천，2주과표체DNMT3B4적293A세포(293A-DNMT3B4-1화293A-DNMT3B4-2)흡광도(A)치분별시293A-vector세포적(58.92±3.47)%화(68.82±5.64)%，과표체DNMT3B4억제세포증식，차이유통계학의의(P<0.05)；293A-DNMT3B4-1화293A-DNMT3B4-2적S기세포비례분별위(35.88±2.00)%화(37.00±1.79)%，293A-vector세포비례위(40.44±0.91)%，과표체DNMT3B4강저S기세포비례，차이유통계학의의(P<0.05)。과표체DNMT3B4증가p21적표체，단불개변p21기인계동자구갑기화상태。결론：DNMT3B4기인과표체가억제293A세포증식병증가p21표체。
Background and purpose:DNMT3B has nearly 40 known splice variants expressed in a tissue-and disease-speciifc manner, but the roles of these splice variants in the cell are still unclear. The aim of this study was to investigate the effects of overexpression of DNA methyltransferase 3B4 (DNMT3B4) gene on proliferation of human embryo kidney 293A cells. Methods:293A cells were transfected with plasmid pCMV-DNMT3B4 or pCMV-2B and then treated with G418 to get the stable cell line. The stable cell lines were determined for proliferation level by MTT method, and for cell cycle distribution by lfow cytometry. The expression of p21 was detected by real-time PCR and Western blot. The methylation status of p21 gene promoter was detected by methylation-speciifc PCR (MS-PCR). Results:The absorbance value in DNMT3B4-1 and DNMT3B4-2 clone were (58.92±3.47)%and (68.82±5.64)%as compared to 293A-vector cells using MTT method. DNMT3B4 overexpression signiifcantly decreased cell proliferation (P<0.05). S phase fraction of 293A-vector cells was (40.44±0.91)%. While in DNMT3B4-1 and DNMT3B4-2 clone cells, the S phase fraction was (35.88±2.00)%and (37.00±1.79)%respectively. Overexpression of DNMT3B4 could significantly decrease S phase fraction (P<0.05). The expression of p21 in DNMT3B4 overexpressed cells was increased, but the methylation status of p21 gene promoter was unchanged.Conclusion:Overexpression of DNMT3B4 can inhibit 293A cell proliferation and can facilitate p21 expression.