Fig. 6

YTHDF2 mediates the mRNA degradation of LHPP and NKX3–1 in m⁶A-dependent way. a-c MeRIP-seq data in two groups (shNC and shYTHDF2, three triplicates respectively). a Scatter plot of differentially methylated genes. The values of X and Y axes in the scatter plot are the averaged RPM (reads per million) values of each group (log₂ scaled). Genes above the top line (2949 red dots, upregulation in shNC group) or below the bottom line (3567 blue dots, upregulation in shYTHDF2 group) indicate more than 2-fold change between two compared groups. Brown dots indicate methylation level without differentially expression. b Volcano plot of differentially methylated genes. The values of X and Y axes in the volcano plot are the fold change (log₂ transformed) and P value (−log₁₀ transformed) between two groups, respectively. Red/Blue dots indicate 2-fold change differentially methylated genes with statistical significance (3567 blue dots, upregulated in shYTHDF2 group, 2949 red dots, upregulated in shNC group). Brown circles indicate non-differentially methylated gene. c Heatmap analyzed from MeRIP-seq data listed the representative upregulated genes in m⁶A levels after knocking down YTHDF2, black arrow referred to the candidate target genes discussed below. d Venn diagram was plotted to show the intersected genes from MeRIP-seq, RIP-seq, mRNA-seq and YTHDF2/METTL3 negatively-correlated genes. Fourteen common genes were screened out. e The correlation between LHPP and YTHDF2 or METTL3 was plotted with GraphPad prism 6.0 by analyzing the negatively-correlated genes downloaded from LinkedOmics (TCGA data). And r = − 0.2078 (P = 2.993e-6) or − 0.2123 (P = 1.802e-6) separately. f The prostate cancer pathway involved in KEGG analysis was obtained from DAVID tool by inputting the 3567 upregulated genes in shYTHDF2 group in MeRIP-seq results as gene list. The graph was downloaded from KEGG database. g and h The mRNA and protein levels of LHPP and NKX3–1were detected with RT-qPCR and western blot after knocking down YTHDF2 or METTL3. GAPDH was the internal reference. Data were analyzed with student’s t test. i RIP-RT-qPCR was utilized to confirm the LHPP and NKX3–1 mRNA enrichment by YTHDF2 in DU-145 and PC-3 cell lines. Data were analyzed with student’s t test. j The mapped reads represent enriched RNA fragments by MeRIP experiment. RNA methylation profiles were loaded in IGV and m⁶A modification peak alterations in LHPP and NKX3–1 mRNA full length were visualized. k The potential m⁶A sites of LHPP and NKX3–1 predicted by SRAMP were combined and co-localized with m⁶A MeRIP-seq results. Different color lines indicated different confidences (red, purple, blue and green respectively represents very high, high, moderate and low confidence). The sequence beside is the fragments captured in MeRIP-seq, which was co-localized with predicted sites. l MeRIP-RT-qPCR was used to detect the m⁶A level alterations of LHPP and NKX3–1 after knocking down METTL3 in PC-3 cell line. Data was analyzed with student’s t test. m The total RNA m⁶A level after treatment of DAA was detected with m⁶A RNA dot-blot assay and compared with DMSO treatment. Methylene blue staining was loading control. n RT-qPCR was utilized to detect the mRNA level of LHPP and NKX3–1 after DAA treatment. Data was analyzed with student’s t test. Error bars represent the SD obtained from at least three independent experiments; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001