CAJ | 학술논문

目的研究miR-34b在白血病细胞株的表达及其启动子区CpG岛甲基化状态,探讨miR-34b甲基化对白血病细胞增殖的影响.方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测8种白血病细胞株(U937、HL-60、MV4-11、M2R、K562、Raji、CCRF、DAMI) miR-34b的相对表达量,选择同期20例非血液病患儿骨髓细胞作为对照并与其比较.采用甲基化特异PCR检测miR-34b在8种白血病细胞株miR-34b的甲基化程度,选择同期23例非血液病患儿骨髓细胞作为对照并与其比较.地西他滨(5-aza-2-dC)处理HL60及K562细胞,检测其甲基化状态及miR-34b的相对表达量.采用脂质体转染hsa-miR-34b到K562细胞、通过流式细胞仪检测转染效率、CCK-8法检测各时期hsa-miR-34b转染组细胞增殖情况并与未转染组、阴性对照转染组比较.结果 miR-34b在非血液病患儿骨髓细胞、白血病细胞株组的相对表达量分别为5.22±1.15、0.03±0.03,白血病细胞株组与非血液病患儿骨髓细胞相比,差异有统计学意义(t=4.538,P＜0.叭).8种白血病细胞株均出现甲基化现象,其甲基化阳性率为100％;23例非血液病患儿骨髓细胞,无一例出现甲基化现象.5-aza-2-dC处理白血病细胞株HL-60及K562后,其甲基化条带均明显减弱,miR-34b的相对表达量均明显升高,分别为处理前的49.5倍和18.8倍.脂质体转染hsa-miR-34b到K562细胞其转染效率为61％,CCK-8法检测hsa-miR-34b转染组各时期均出现细胞增殖抑制作用,与阴性对照转染组相比,差异有统计学意义(48 h:t=9.303,P＜0.01;72 h:t=65.617,P＜0.01;96 h:t =36.878,P ＜0.01;120 h:t=18.748,P＜0.01).抑制率分别为12.2％、45.7％、32.5％、22.9％,其中72 h抑制作用最明显.结论启动子区CpG岛甲基化使miR-34b在白血病细胞株表达降低,导致细胞增殖抑制作用减弱,可能是白血病发生或发展的原因之一.
목적 연구miR-34b재백혈병세포주적표체급기계동자구CpG도갑기화상태,탐토miR-34b갑기화대백혈병세포증식적영향.방법 채용실시형광정량취합매련반응(qRT-PCR)검측8충백혈병세포주(U937、HL-60、MV4-11、M2R、K562、Raji、CCRF、DAMI) miR-34b적상대표체량,선택동기20례비혈액병환인골수세포작위대조병여기비교.채용갑기화특이PCR검측miR-34b재8충백혈병세포주miR-34b적갑기화정도,선택동기23례비혈액병환인골수세포작위대조병여기비교.지서타빈(5-aza-2-dC)처리HL60급K562세포,검측기갑기화상태급miR-34b적상대표체량.채용지질체전염hsa-miR-34b도K562세포、통과류식세포의검측전염효솔、CCK-8법검측각시기hsa-miR-34b전염조세포증식정황병여미전염조、음성대조전염조비교.결과 miR-34b재비혈액병환인골수세포、백혈병세포주조적상대표체량분별위5.22±1.15、0.03±0.03,백혈병세포주조여비혈액병환인골수세포상비,차이유통계학의의(t=4.538,P＜0.팔).8충백혈병세포주균출현갑기화현상,기갑기화양성솔위100％;23례비혈액병환인골수세포,무일례출현갑기화현상.5-aza-2-dC처리백혈병세포주HL-60급K562후,기갑기화조대균명현감약,miR-34b적상대표체량균명현승고,분별위처리전적49.5배화18.8배.지질체전염hsa-miR-34b도K562세포기전염효솔위61％,CCK-8법검측hsa-miR-34b전염조각시기균출현세포증식억제작용,여음성대조전염조상비,차이유통계학의의(48 h:t=9.303,P＜0.01;72 h:t=65.617,P＜0.01;96 h:t =36.878,P ＜0.01;120 h:t=18.748,P＜0.01).억제솔분별위12.2％、45.7％、32.5％、22.9％,기중72 h억제작용최명현.결론 계동자구CpG도갑기화사miR-34b재백혈병세포주표체강저,도치세포증식억제작용감약,가능시백혈병발생혹발전적원인지일.
Objective To study the expression level and CpG island methylation status of miR-34b in leukemia cell lines and to research the effect of DNA methylation on the proliferation of leukemia cells regulated by miR-34b.Method Taqman real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was carried out to detect the relative expression of miR-34b in control group (bone marrow cells of 20 children without blood disease) and 8 leukemia cell lines (U937,HL-60,MV4-11,M2R,K562,Raji,CCRF,DAMI).Methylation-specific polymerase chain reaction (MSP) was carried out to detect the methylation differences of miR-34b in control group (Bone marrow cells of 23 children without blood disease),8 leukemia cell lines.HL-60 and K562 were treated with methyltransferase inhibitor (5-aza-2-dC) for further detection of its methylation status and expression of miR-34b.Hsa-miR-34b mimics was transfected into K562 cell by liposome,the transfection efficiency was detected by flow cytometry.The cell proliferation of hsa-miR-34b transfected group in each stage was measured with CCK-8 assay,and then compared with non-transfected group and negative control group.Result The relative expression level of miR-34b in the group of children without blood disease and the group of leukemia cell lines were 5.22 ± 1.15,0.03 ±0.03.The results showed that,the group of leukemia cell lines was significantly different from the control group(t =4.538,P ＜ 0.01).Eight leukemia cell lines showed methylation,the positive rate of the methyl was 100％.There was no methylation in the 23 cases of control group.After leukemia cell lines HL-60 and K562 were treated with 5-aza-2-dC,the methylated bands became obviously weakened,and the relative expression levels of miR-34b substantially increased 49.5 times and 18.8 times respectively.After hsa-miR-34b mimics was transfected into K562 cell by liposome,its transfection efficiency detected by flow cytometry was 61％ and the cell proliferation was measured with CCK-8 assay from which it was found that the cell proliferation was significantly suppressed compared with the control group at 48 h (t =9.303,P ＜ 0.01),72 h (t=65.617,P＜0.01),96 h (t=36.878,P＜0.01) and 120 h (t=18.748,P＜0.01) in hsa-miR-34b transfected group,with the inhibition rate of 12.2％ (48 h),45.7％ (72 h),32.5％ (96 h) and 22.9％ (120 h).Conclusion The hypermethylation of promoter leads to decrease in the expression levels of miR-34b in leukemia cell lines,which attenuate mechanism of proliferative inhibition may be one of the reasons of occurrence or development of childhood leukemia.