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. 2025 Jul 28;21(11):4942–4960. doi: 10.7150/ijbs.112133

Figure 3.

Figure 3

The m6A level of MALAT1 is upregulated by the recruitment of the m6A methyltransferase complex. A Heatmap of 20 m6A-related genes in the livers of HBV-HCC patients (n = 21) and peritumor (n = 21) tissues. B Pearson's correlation analysis was used to determine the relationships between the mRNA levels of 20 m6A-related genes. C The overall m6A content in HepG2.2.15 and HepG2 cells was analyzed by m6A quantitation analysis. D Relative expression of the 7 m6A methyltransferase genes was detected by qRT-PCR in HepG2.2.15 and HepG2 cells. E The protein levels of METTL3, METTL14, and WTAP in HepG2.2.15 and HepG2 cells were detected by WB. F Venn diagram showing the overlap of METTL3-, METTL14-, and WTAP-bound transcripts revealed by CLIP-seq and the TCGA-LIHC cohort DEGs. G MeRIP of MALAT1 transcripts in HepG2.2.15 and HepG2 cells upon indicated knockdown. The abundance of MALAT1 among MeRIP with anti-m6A antibodies was measured via qPCR and normalized to that of IgG. H MeRIP of MALAT1 transcripts in HepG2.2.15 cells. I METTL3, METTL14, and WTAP were pulled down by biotin-labeled sense MALAT1 (S) but not MALAT1 antisense (AS) RNA in HepG2.2.15 and HepG2 cells. J METTL3, METTL14, and WTAP were pulled down in cytoplasmic and nuclear fractions of HepG2.2.15 and HepG2 cells. K MALAT1 secondary structure was predicted by lnCAR. L Scheme of full-length biotinylated-MALAT1 (S#2), antisense MALAT1 (S#1), and 8 truncated biotinylated-MALAT1 fragments based on the sub-structures: S#3(1-1097 nt), S#4 (1098-2195 nt), S#5 (2196-3293 nt), S#6 (3294-4391 nt), S#7 (4392-5489 nt), S#8 (5490-6587 nt), S#9 (6588-7685 nt) and S#10 (7686-8779 nt). The full-length biotinylated-MALAT1 and its truncations were separately transfected into HepG2.2.15 cells. M Dual-luciferase reporter assays were used to confirm the interaction between METTL3, METTL14, WTAP and 2 mutations (6021C and 7265C) of the MALAT1 m6A sites. N MeRIP of MALAT1 transcripts in HepG2.2.15 cells transfected with MALAT1 mutant plasmids. *P < 0.05; **P < 0.01; ***P < 0.001. n.s., not significant.