吉林大学学报(医学版) ›› 2022, Vol. 48 ›› Issue (2): 284-290.doi: 10.13481/j.1671-587X.20220203

• 基础研究 • 上一篇    下一篇

急性淋巴细胞白血病移植瘤模型小鼠髓外浸润过程中血清Wnt5A和LINE-1启动子甲基化水平检测及其意义

王一涵,王奕丹,惠赫童,范馨元,王添琦,夏薇(),刘丽梅()   

  1. 北华大学医学技术学院实验中心,吉林 吉林 132013
  • 收稿日期:2021-06-11 出版日期:2022-03-28 发布日期:2022-05-10
  • 通讯作者: 夏薇,刘丽梅 E-mail:xiawei4016@126.com;liulm74@163.com
  • 作者简介:王一涵(1994-),女,吉林省长春市人,在读硕士研究生,主要从事疾病的生物化学和分子诊断指标及方法方面的研究。
  • 基金资助:
    国家自然科学基金项目(81201354);吉林省科技厅科技发展计划项目(20200403118SF);吉林省教育厅科学技术研究“十二五”规划项目(吉教科验字〔2017〕196);吉林省吉林市科技局项目中科院苏州医工所合作专项(E055PY03);北华大学研究生创新计划项目(北华研创合字〔2019〕062)

Detection of promoter methylation levels of Wnt5A and LINE-1 in serum during extramedullary infiltration in acute lymphoblastic leukemia transplanted tumor model mice and its significance

Yihan WANG,Yidan WANG,Hetong HUI,Xinyuan FAN,Tianqi WANG,Wei XIA(),Limei LIU()   

  1. Experiment Center,College of Medical Technology,Beihua University,Jilin 132013,China
  • Received:2021-06-11 Online:2022-03-28 Published:2022-05-10
  • Contact: Wei XIA,Limei LIU E-mail:xiawei4016@126.com;liulm74@163.com

摘要: 目的

建立绿色荧光蛋白(GFP)标记的急性淋巴细胞白血病(ALL)移植瘤小鼠模型,研究ALL髓外浸润过程中血清游离DNA(cfDNA)特异基因甲基化的变化,为ALL髓外浸润监测提供理论和实验依据。

方法

60只ICR小鼠随机分为对照组、实验15 d组和实验30 d组,每组20只。实验组小鼠通过尾静脉注射构建GFP标记ALL移植小鼠模型,流式细胞术检测白血病细胞髓外浸润情况。分别在建模后第15和30天时采用实时荧光定量PCR(RT-qPCR)法检测各组小鼠血清中双加氧酶2(TET2)、DNA甲基转移酶3α(DNMT3a)、DNA甲基转移酶3β(DNMT3b)、Wnt家族成员5A(Wnt5A)及逆转座子长散布核元件-1(LINE-1) mRNA表达水平,重亚硫酸盐测序法(BSP)检测各组血清中Wnt5A和LINE-1启动子区甲基化水平。

结果

实验15 d组和实验30 d组小鼠脾脏组织中均可见明显绿色荧光,且实验30 d组较实验15 d组荧光强度增强;实验30 d组小鼠脾脏质量高于对照组(P<0.05),脾肿大较为明显。流式细胞术检测,与对照组比较,实验15 d组和实验30 d组小鼠外周血细胞荧光强度均明显增强,且实验30 d组小鼠外周细胞血荧光强度较实验15 d组增强(P<0.05)。RT-qPCR法检测,与对照组比较,实验15 d组和实验30 d组小鼠血清中Wnt5A和TET2 mRNA表达水平均明显降低(P<0.05),LINE-1、DNMT3a和DNMT3b mRNA表达水平明显升高(P<0.05)。BSP法检测,与对照组比较,实验15 d组和实验30 d组小鼠血清 LINE-1启动子区甲基化水平明显降低(P<0.05),在浸润过程中逐步呈现低甲基化;抑癌基因Wnt5A启动子区甲基化水平明显升高(P<0.05)。

结论

ALL移植瘤小鼠模型于建模早期即出现髓外浸润,血清cfDNA中Wnt5A和LINE-1启动子区的异常甲基化直接影响其异常表达。cfDNA特异位点甲基化水平检测可为ALL早期诊断与治疗监测提供新的方法和策略。

关键词: 急性淋巴细胞白血病, 髓外浸润, 循环游离DNA, 甲基化, 表观遗传

Abstract: Objective

To establish the acute lymphoblastic leukemia(ALL) transplanted tumor mouse models labled with green fluorescent protein(GFP)and to study the methylation changes in the serum circulating free DNA (cfDNA) during the extramedullary infiltration, and to provide the theroretical and experimental basis for monitoring extramedullary infiltration of ALL.

Methods

Sixty ICR mices were randonly divided into control group,treatment 15 d group and treatment 30 d group,with 20 mice in each group,and the ALL transplanted tumor mouse models labled with GFP were established by tail vein injection, the extramedullary infiltration of leukemia cells was derected by flow cytometry.The expression levels of ten-eleven-translocation2(TET2), DNA methyltransferase 3 alpha(DNMT3a), DNA methyltransferase 3 beta(DNMT3b), Wnt family member 5A(Wnt5A) and long interspersed nuclear elements(LINE-1) mRNA in the blood of the mice in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR) and the methylation levels in the promoter region of Wnt5A and LINE-1 in the blood of the mice in various groups were determined by bisulfite sequencing PCR (BSP).

Results

Significant green fluorescence was found in the spleen tissue of the mice in treatment 15 d group and treatment 30 d group, and the fluorescence intensity in treatment 30 d group was higher than that in treatment 15 d group. The mass of spleen of the mice in treatment 30 d group was higher than that in control group(P<0.05), and the splenomegaly was significant.The flow cytometry results showed that compared with control group, the fluorescence intensity of peripheral blood cells in treatment 15 d group and treatment 30 d group were increased significantly, and the fluorescence intensity in treatment 30 d group was higher than that in treatment 15 d group(P<0.05). Compared with control group, the expression levels of Wnt5A and TET2 mRNA in treatment 15 d group and treatment 30 d group were decreased significantly(P<0.05). The expression levels of LINE-1, DNMT3a and DNMT3b mRNA were increased significantly(P<0.05). Compared with control group, the methylation levels of LINE-1 promoter in serum of the mice in treatment 15 d group and treatment 30 d group were decreased significantly(P<0.05),and hypomethylation was gradually showed during infiltration; the methylation level of Wnt5aA promoter of tumor suppressor gene was increased significantly(P<0.05).

Conclusion

Extramedullary infiltration occurres early in the establishment of ALL fransplanted tumor mouse models.the abnormal methylation of Wnt5A and LINE-1 promoter in serum cfDNA affects their abnormal expressions. The methylation detection of specific ectopic sites in cfDNA can provide a new method and strategy for early diagnosis and treatment monitoring of ALL.

Key words: Acute lymphoblastic leukemia, Extramedullary involvement, Circulating free DNA, Methylation, Epigenetics

中图分类号: 

  • R733.71