METTL3 participates in m6A modification to regulate HBV replication through an IGF2BP2-dependent manner

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 (P < 0.05). Compared with the control group, m6A modification enrichment was enhanced and IGF2BP2 expression level was elevated in the OE-METTL3 group (P < 0.05), and HBV replication-related indexes (HBV DNA, HBV rcDNA, HBV cccDNA, and HBV pgRNA) were increased (P < 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 (P < 0.01), and HBV replication-related indexes were decreased in the si-IGF2BP2 group (P < 0.01). In the OE-METTL3+si-IGF2BP2 group, HBV replication-related indexes were reduced when compared with the OE-METTL3 group (P < 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 (P < 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.

Keywords

• methytransferase like 3
• insulin like growth factor 2 mrna binding protein 2
• m6a methylation
• hepatitis b virus