[1]王双琳,王慧睿,肖蓬莉,等.多发性骨髓瘤细胞中ZNF382甲基化状态及其意义[J].新乡医学院学报,2020,37(7):611-616.[doi:10.7683/xxyxyxb.2020.07.003]
　WANG Shuanglin,WANG Huirui,XIAO Pengli,et al.Significance of ZNF382 methylation in multiple myeloma cells[J].Journal of Xinxiang Medical University,2020,37(7):611-616.[doi:10.7683/xxyxyxb.2020.07.003]
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多发性骨髓瘤细胞中ZNF382甲基化状态及其意义
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
37
期数:
2020年7
页码:
611-616
栏目:
基础研究
出版日期:
2020-07-05

文章信息/Info

Title:
Significance of ZNF382 methylation in multiple myeloma cells
作者:
王双琳1; 2; 王慧睿2; 肖蓬莉2; 郭淑利2; 王万里2; 王松云2; 李　治2
（1.新乡医学院，河南　新乡　453003；2.郑州大学附属洛阳中心医院血液内科，河南　洛阳　471009）
Author(s):
WANG Shuanglin1; 2; WANG Huirui2; XIAO Pengli2; GUO Shuli2; WANG Wanli2; WANG Songyun2; LI Zhi2
（1.Xinxiang Medical University,Xinxiang 453003,Henan Province,China；2.Department of Hematology,Luoyang Central Hospital Affiliated to Zhengzhou University,Luoyang 471009,Henan Province,China）
关键词:
多发性骨髓瘤; ZNF382; 甲基化
Keywords:
multiple myeloma; ZNF382; methylation.
分类号:
R733.3
DOI:
10.7683/xxyxyxb.2020.07.003
文献标志码:
A
摘要:
目的　研究多发性骨髓瘤（MM）患者骨髓细胞和U266细胞株中ZNF382的表达及其在启动子区的甲基化状态。方法　选择2018年1～6月洛阳市中心医院收治的6例MM患者（MM组）和6例缺铁性贫血患者（对照组），抽取2组患者骨髓细胞并选择生长状态良好的MM细胞系U266细胞，分别采用反转录聚合酶链式反应（RT-PCR）和Western blot法检测MM组、对照组患者骨髓细胞及U266细胞中ZNF382 mRNA和蛋白表达，采用甲基化特异性PCR（MSP）检测MM组、对照组患者骨髓细胞及U266细胞中ZNF382基因启动子区甲基化状态。将U266细胞分为空白对照组、二甲基亚砜（DMSO）组、5 μmol·L-1地西他滨（DAC）组和10 μmol·L-1DAC组，应用RT-PCR检测4组细胞中ZNF382 mRNA表达水平，采用MSP检测空白对照组、5 μmol·L-1DAC组和10 μmol·L-1DAC组细胞中ZNF382基因启动子区甲基化状态。取生长状态良好的U266细胞分为空白对照组、空载质粒转染组和ZNF382转染组，空白对照组细胞正常培养，不添加任何类型慢病毒，空载质粒转染组细胞转染空载质粒慢病毒，ZNF382转染组细胞转染过表达ZNF382慢病毒，筛选稳定表达ZNF382的U266细胞，并应用细胞计数试剂盒-8和流式细胞术检测U266细胞增殖和凋亡情况。结果　MM组患者骨髓细胞和U266细胞中ZNF382 mRNA及蛋白的表达低于对照组患者骨髓细胞（P<0.05）。MM组患者骨髓细胞及U266细胞中ZNF382基因启动子区甲基化水平明显高于对照组患者骨髓细胞（P<0.05）。空白对照组和DMSO组U266细胞中ZNF382 mRNA表达比较差异无统计学意义（P>0.05）；与空白对照组和DMSO组比较，5、10 μmol·L-1DAC组U266细胞中ZNF382 mRNA表达水平增加（P<0.05）；10 μmol·L-1DAC组U266细胞中ZNF382 mRNA表达水平高于5 μmol·L-1DAC组（P<0.05）。与空白对照组比较，5、10 μmol·L-1DAC组细胞中ZNF382启动子区甲基化水平降低（P<0.05）；5 μmol·L-1DAC组和10 μmol·L-1DAC组细胞中ZNF382启动子区甲基化水平比较差异无统计学意义（P>0.05）。转染24、48、72 h后，空载质粒转染组与空白对照组U266细胞增殖能力比较差异无统计学意义（P>0.05）；ZNF382转染组U266细胞增殖能力低于空白对照组与空载质粒转染组（P<0.05）。空白对照组与空载质粒转染组U266细胞凋亡率比较差异无统计学意义（P>0.05），ZNF382转染组U266细胞凋亡率高于空载质粒转染组和空白对照组（P<0.05）。结论　ZNF382基因启动子区高甲基化可能是引起MM患者骨髓细胞及细胞株U266中ZNF382表达降低的重要原因，ZNF382能够有效抑制U266细胞的增殖并促进其凋亡。
Abstract:
Objective　To investigate the expression of ZNF382 in bone marrow cells of multiple myeloma(MM) patients and MM cell line U266 and its methylation status in promoter region.Methods　Six patients with MM（MM group）and six patients with iron-deficiency anemia（control group） in the Luoyang Central Hospital from January to June 2018 were selected,the bone marrow cells of patients in the above two groups were extracted，and the U266 cells in good growth condition were selected.The expression of ZNF382 mRNA and protein in the control group,MM group and U266 cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The methylation level of ZNF382 gene promoter region in bone marrow cells of the control group,MM group and U266 cells was detected by methylation specific PCR (MSP).The U266 cells were taken and divided into blank control group,dimethyl sulfoxide（DMSO) group,5 μmol·L-1 dicithabine (DAC) group and 10 μmol·L-1DAC group；the expression of ZNF382 mRNA in the blank control group,DMSO group,5 μmol·L-1DAC group and 10 μmol·L-1DAC group was detected by RT-PCR;the methylation level of ZNF382 gene promoter region in the blank control group,5 μmol·L-1DAC group and 10 μmol·L-1DAC group was detected by MSP.The U266 cells in good growth condition were divided into blank control group,empty plasmid transfection group and ZNF382 transfection group;the cells in the blank control group were cultured normally without adding any type of lentivirus,the cells in the empty plasmid transfection group were transfected with empty plasmid lentivirus,the cells in the ZNF382 transfection group were transfected with over expressed ZNF382 lentivirus.Then the U266 cells which stably expressing ZNF382 gene were screened and the cell proliferation and apoptosis were detected by cell counting kit-8 and flow cytometry,respectively.Results　The expressions of ZNF382 mRNA and protein in bone marrow cells of the MM group and U266 cells were significantly lower than those in the control group(P<0.05).The methylation level of ZNF382 gene promoter region in bone marrow cells of the MM group and U266 cells were significantly higher than that in the control group(P<0.05).There was no significant difference in ZNF382 mRNA expression between the control group and DMSO group (P>0.05).Compared with the control group and DMSO group,the ZNF382 mRNA expression level in U266 cells of 5,10 μmol·L-1DAC groups increased(P<0.05).The expression of ZNF382 mRNA in U266 cells of 10 μmol·L-1DAC group was higher than that of 5 μmol·L-1DAC group(P<0.05).The methylation level of ZNF382 gene promoter region in the 5,10 μmol·L-1DAC group was lower than that in the blank control group(P<0.05);there was no statistic difference in the methylation level of ZNF382 gene promoter region between 5 μmol·L-1DAC group and 10 μmol·L-1DAC group(P>0.05).After 24,48 and 72 hours of transfection,there was no significant difference in the proliferation ability of U266 cells between the empty plasmid transfection group and the blank control group (P>0.05),the proliferation ability in the of U266 cells in the ZNF382 transfection group was lower than that in the blank control group and the empty plasmid transfection group (P<0.05).There was no significant difference in apoptosis rate of U266 cells between the blank control group and the empty plasmid transfection group(P>0.05),the apoptosis rate of U266 cells in the ZNF382 transfection group was higher than that in the empty plasmid transfection group and the blank control group (P<0.05).Conclusion　Hypermethylation in the promoter region of ZNF382 gene may contribute to the decreased expression of ZNF382 in MM cells and MM cell line U266.ZNF382 can effectively inhibit the proliferation and promote the apoptosis of the MM cell line U266.

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更新日期/Last Update: 2020-07-05