N6-methyladenosine methylation analysis of long noncoding RNAs and mRNAs in 5-FU-resistant colon cancer cells

Figures & data

Figure 1. General characteristics of RNA m6A methylation. (a) Venn diagram showing the number of shared and unique m6A peaks in the two groups. (b) Chromosomal distribution of m6A peaks in both groups. (c) Number distribution of m6A peaks spanning different exon numbers in the two groups. (d) The number of single-base sites and the average log2 fold enrichment of single-base site peaks in the 5-fu group and the control group. (e) The positional distribution of m6A peaks on functional elements of genes in the two groups.

Figure 2. Conserved sequence motifs of m6A peaks between 5fu and control groups.

Figure 3. Overview of the differentially methylated m6A peaks landscape in the 5fu and control samples. (a) The differentially m6A modification volcano plot showing distinct m6A peaks and their statistical significance. (b) Pie charts displaying the positional distribution of the differentially methylated m6A peaks on functional elements of genes. (c) Distribution of the differentially methylated m6A peaks on chromosomes. (d) Number distribution of the differentially methylated m6A peaks spanning different exon numbers. (e) The number of single-base sites and m6A peaks in upregulated and downregulated m6A peaks. (f) The average log2 fold enrichment of the differentially methylated m6A peaks. (g) The distribution of p-value of the differentially methylated m6A peaks.

Table 1. The top ten significantly up- regulated or down-regulated m6A modified peaks.

Chr	Gene Name	Peak Start	Peak End	Log2-Fold Change	Peak Region	P-value	Regulation
5	MAP3K1	56865932	56871985	5.98	CDS	0.002	Hypermethylated
9	KIF24	34255982	34256222	5.38	CDS	0.000	Hypermethylated
2	NDUFS1	206115476	206115717	4.89	3’UTR	0.000	Hypermethylated
3	B4GALT4	119213465	119213645	4.79	3’UTR	0.000	Hypermethylated
1	CDKN2C	50974004	50974155	4.66	CDS	0.001	Hypermethylated
X	TAB3	30854256	30854437	4.66	CDS	0.000	Hypermethylated
4	SLC26A1	990056	990295	4.64	CDS	0.000	Hypermethylated
4	ANTXR2	79905855	79906066	4.64	3’UTR	0.000	Hypermethylated
1	DNAJC11	6678403	6701745	4.63	CDS	0.000	Hypermethylated
19	NUP62	49928744	49928925	4.45	5’UTR	0.000	Hypermethylated
18	VPS4B	63390537	63390688	−6.44	3’UTR	0.000	Hypomethylated
18	ANKRD12	9239536	9254370	−6.06	CDS	0.000	Hypomethylated
1	CCDC18	93205545	93207291	−5.97	CDS	0.000	Hypomethylated
10	STAMBPL1	88880452	88882462	−5.63	5’UTR	0.000	Hypomethylated
2	SP140L	230402924	230403134	−5.52	exon	0.000	Hypomethylated
10	KNDC1	133186321	133186622	−5.41	CDS	0.000	Hypomethylated
3	PLCH1	155564997	155568289	−5.29	CDS	0.000	Hypomethylated
8	VPS28	144425992	144426173	−5.25	exon	0.000	Hypomethylated
5	AC093297.2	44826674	44826855	−5.2	exon	0.000	Hypomethylated
10	UBE2D1	58368756	58368936	−5.06	3’UTR	0.002	Hypomethylated

Figure 4. GO function and KEGG pathway enrichment of hyper-methylated and hypo-methylated mRnas. (a) The top 20 GO terms of genes with upregulated m6A peaks. (b) The top 20 GO terms of genes with downregulated m6A peaks. (c) The top 20 KEGG pathways of genes with upregulated m6A peaks. (d) The top 20 KEGG pathways of genes with downregulated m6A peaks.

Figure 5. The differential expression of lncRnas and mRnas and functional enrichment analysis of mRnas. (a) The differentially expressed lncRnas in the 5FU group and control group. (b) The differentially expression of mRnas in the 5FU group and control group. (c) Hierarchical cluster analysis of differentially expressed mRnas genes. (d) Hierarchical cluster analysis of differentially expressed lncRnas genes. (e) GO enrichment analysis of differentially expressed mRnas. (f) KEGG pathways enrichment analysis of differentially expressed mRnas.

Figure 6. Integrated analysis of m6A methylation and mRNA expression. (a) Four quadrant diagram showing differentially methylated and differentially expressed RNAs. (b) The RNA expression level was positively correlated with the methylation level (R² = 0.11, p < 0.0001). (c) The top 10 statistically significant GO enrichment analysis terms. (d) GO enrichment analysis of mRnas with both differential methylation and expression. (e) KEGG pathways enrichment analysis of mRnas with both differential methylation and expression.

Figure 7. Enrichment analysis of target genes with differentially m6A methylated lncRnas. (a) GO biological processes enrichment analysis. (b) GO cellular component enrichment analysis. (c) GO molecular function enrichment analysis. (d) KEGG enrichment analysis.

Figure 8. Integrated analysis of lncRnas in the RNA-seq and MeRIP-seq data. (a) The network of lncRNA-mRNA regulation, the circle represents mRNA and the diamond represents lncRnas with both differential methylation and expression, red means high expression and blue means low expression, purple means high methylation and green means low methylation. (b) GO biological processes enrichment analysis. (c) GO cellular component enrichment analysis. (d) GO molecular function enrichment analysis. (e) KEGG enrichment analysis.

Figure 9. Validation of MeRIP-seq and RNA-seq. (a) RT-qPCR validation of four lncRnas. (b) SELECT-PCR validation of four lncRnas. (c) MeRIP-qPCR validation of three m6A-modified lncRnas and mRnas.

Figure 10. Knockdown of lncRNA ADIRF-AS1 and AL139035.1 promoted CRC proliferation and invasive metastasis in vitro. (a) Validation of siRNA knockdown inefficiency. (b) Knockdown of ADIRF-AS1 and AL139035.1 promotes CRC proliferation in vitro (c) knockdown of ADIRF-AS1 and AL139035.1 promotes CRC invasion and migration.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article. The sequencing data can be accessed in the NCBI SRA (Sequence Read Achieve) database at https://www.ncbi.nlm.nih.gov/sra/PRJNA923388.