METTL3通过IGF2BP2依赖性方式参与m6A修饰调控HBV复制的机制

余玲; 张自力; 曾蓉; 张节; 王鹏; 潘万龙

doi:10.16016/j.2097-0927.202307062

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METTL3通过IGF2BP2依赖性方式参与m6A修饰调控HBV复制的机制

基础医学 | 更新时间：2026-01-09

- METTL3通过IGF2BP2依赖性方式参与m6A修饰调控HBV复制的机制
- METTL3 participates in m6A modification to regulate HBV replication through an IGF2BP2-dependent manner
- 陆军军医大学学报 2024年46卷第5期页码：442-449
- 作者机构：
  
  1.637100 四川南充，川北医学院基础医学与法医学院
  2.637100 四川南充，川北医学院附属医院老年科
- 作者简介：
  
  [潘万龙，E-mail: panwl@aliyun.com PAN Wanlong, E-mail: panwl@aliyun.com
- 基金信息：
  
  南充市市校科技战略合作专项(22SXQT0318)
- DOI：10.16016/j.2097-0927.202307062
  中图分类号： R341;R373.21;R394.3
- 收稿：2023-07-12，
  
  修回：2023-08-28，
  
  纸质出版：2024-03-15
- 稿件说明：
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余玲, 张自力, 曾蓉, 等. METTL3通过IGF2BP2依赖性方式参与m6A修饰调控HBV复制的机制[J]. 陆军军医大学学报, 2024,46(5):442-449.

Ling YU, Zili ZHANG, Rong ZENG, et al. METTL3 participates in m6A modification to regulate HBV replication through an IGF2BP2-dependent manner[J]. Journal of Army Medical University, 2024, 46(5): 442-449.
余玲, 张自力, 曾蓉, 等. METTL3通过IGF2BP2依赖性方式参与m6A修饰调控HBV复制的机制[J]. 陆军军医大学学报, 2024,46(5):442-449. DOI： 10.16016/j.2097-0927.202307062.

Ling YU, Zili ZHANG, Rong ZENG, et al. METTL3 participates in m6A modification to regulate HBV replication through an IGF2BP2-dependent manner[J]. Journal of Army Medical University, 2024, 46(5): 442-449. DOI： 10.16016/j.2097-0927.202307062.

摘要

目的

探究胰岛素样生长因子2 mRNA结合蛋白2(insulin like growth factor 2 mRNA binding protein 2，IGF2BP2)协助甲基转移酶样3(methytransferase like 3，METTL3)通过N6-甲基腺苷(N6-methyladenosine，m6A)修饰调控HBV复制的分子机制。

方法

以HBV稳定复制细胞系HepG2.2.15及其亲本细胞HepG2为模型，斑点杂交分析m6A修饰水平，RT-qPCR和Western blot检测METTL3、IGF2BP2表达。生物信息学及免疫共沉淀分析METTL3与m6A阅读蛋白及其相互作用。将METTL3质粒、METTL3 siRNA和(或)IGF2BP2 siRNA转染至

HepG2.2.15细胞，分别记为OE-METTL3、si-METTL3、si-IGF2BP2、OE-METTL3+si-IGF2BP2、si-METTL3+si-IGF2BP2、Control组；qPCR检测

HBV DNA、HBV rcDNA、HBV cccDNA

拷贝数，RT-qPCR或Western blot检测

HBV pgRNA

、METTL3、IGF2BP2表达。

结果

与HepG2细胞相比，在HepG2.2.15细胞中m6A修饰富集，METTL3、IGF2BP2表达水平升高(

＜0.05)。与Control组相比，在OE-METTL3组中m6A修饰富集增强，IGF2BP2表达水平升高(

＜0.05)，HBV复制相关指标(

HBV DNA、HBV rcDNA、HBV cccDNA、HBV pgRNA

)升高(

＜0.01)；在si-METTL3组中则相反。生物信息学分析及免疫共沉淀显示METTL3与IGF2BP2为互作蛋白。与Control组相比，在si-IGF2BP2组中m6A修饰富集减弱，METTL3表达水平降低(

＜0.01)，HBV复制相关指标降低(

＜0.01)。在OE-METTL3+si-IGF2BP2组中，HBV复制相关指标较OE-METTL3组降低(

＜0.001)。在si-METTL3+si-IGF2BP2组中，HBV复制相关指标较si-METTL3组、si-IGF2BP2组降低(

＜0.05)。

结论

METTL3依赖IGF2BP2富集m6A通过增强

HBV rcDNA

转化为

cccDNA

，进而增强

pgRNA

逆转录复制病毒。

Abstract

Objective

To investigate the molecular mechanism by which methytransferase like3 (METTL3) cooperates with insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) to regulate HBV replication through N6-methyladenosine (m6A) modifications.

Methods

HBV stably replicating cell line HepG2.2.15 and its source cells HepG2 were used as models. Spot hybridization was used to analyze m6A modification level

and RT-qPCR and Western blotting were utilized to detect the expression of METTL3 and IGF2BP2 at mRNA and protein levels. Bioinformatics analysis and co-immunoprecipitation assay were applied to analyze METTL3 and m6A reading proteins and their interactions. After METTL3 plasmid

METTL3 siRNA and/or IGF2BP2 siRNA were transfected into HepG2.2.15 cells

respectively

the cells were assigned into OE-METTL3 group

si-METTL3 group

si-IGF2BP2 group

OE-METTL3+si-IGF2BP2 group

si-METTL3+si-IGF2BP2 group

and the cells untreated were subjected as control group. Copy numbers of HBV DNA

HBV rcDNA

and HBV cccDNA were detected by qPCR

and the expression of HBV pgRNA

METTL3

and IGF2BP2 were measured with RT-qPCR or Western blotting.

Results

Compared with HepG2 cells

m6A modification was enriched

and the expression levels of METTL3 and IGF2BP2 were elevated in HepG2.2.15 cells (

0.05). Compared with the control group

m6A modification enrichment was enhanced and IGF2BP2 expression level was elevated in the OE-METTL3 group (

0.05)

and HBV replication-related indexes (HBV DNA

HBV rcDNA

HBV cccDNA

and HBV pgRNA) were increased (

0.01); and opposite phenomena were observed in the si-METTL3 group. Bioinformatics analysis and co-immunoprecipitation assay showed that METTL3 and IGF2BP2 were interacting proteins. Compared with the control group

m6A modification enrichment was attenuated and METTL3 expression level was reduced (

0.01)

and HBV replication-related indexes were decreased in the si-IGF2BP2 group (

0.01). In the OE-METTL3+si-IGF2BP2 group

HBV replication-related indexes were reduced when compared with the OE-METTL3 group (

0.01). In the si-METTL3+si-IGF2BP2 group

HBV replication-related indexes were reduced when compared with the si-METTL3 group and si-IGF2BP2 group (

0.05).

Conclusion

METTL3 enriches m6A by depending on IGF2BP2

replicates the virus by enhancing the conversion of HBV rcDNA to cccDNA

and in turn enhances pgRNA reverse transcription.

关键词

Keywords

references

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