Distinct 5-methylcytosine profiles of LncRNA in breast cancer brain metastasis

Abstract

Background: Recent studies have identified a complex relationship between methylation patterns and the development of various cancers. Breast cancer (BC) is the second leading cause of cancer mortality among women. Approximately 5-20% of BC patients are at risk of BC brain metastases (BCBM). Although 5-methylcytosine (m5C) has been identified as an important regulatory modifier, its distribution in BCBM is not well understood. This study aimed to investigate the distribution of m5C in BCBM.

Materials and methods: Samples from BCBM (231-BR cells) and BC (MDA-MB-231 cells) groups were subjected to a comprehensive analysis of the m5C methylation in long non-coding RNA (lncRNA) using methylated RNA immunoprecipitation next-generation sequencing (MeRIP-seq). The expression levels of methylated genes in BC and adjacent tissues were verified through quantitative real-time polymerase chain reaction (RT-qPCR). Enrichment pathway analyses were through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to predict the potential functions of m5C in BCBM.

Results: The MeRIP-seq analysis identified 23,934 m5C peaks in BCBM and 21,236 m5C in BC. A total of 9,480 annotated genes (BCBM) and 8,481 annotated genes (BC) were mapped. Notably, 1,819 methylation sites in lncRNA were upregulated in BCBM, whereas 2,415 methylation sites were upregulated in BC. Significant m5C hypermethylated lncRNAs included ENST00000477316, ENST00000478098 and uc002gtt.1, whereas hypomethylated lncRNAs included ENST00000600912, ENST00000493668, ENST00000544651 and ENST00000464989. These results were verified by qPCR and MeRIP-qPCR in BC and BCBM. Considering the strong association between m5C RNA methylation regulators and lncRNA, we examined the expression levels of 13 m5C RNA methylation regulators and observed significant differences between BC tissues and adjacent normal tissues. In addition, the interaction between regulators of altered expression and the differentially expressed genes in vitro was analyzed. The GO and KEGG pathways analyses revealed that genes significantly associated with m5C sites in lncRNA were linked to the BCBM signaling pathways.

Conclusion: This uncovered significant variations in the levels and distribution of m5C in BCBM compared to BC. The findings provide a new theoretical understanding of the mechanisms of BCBM.

Keywords: 5-methylcytosine (m5C); Breast Cancer Brain Metastases (BCBM); Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG); Long non-coding RNA (lncRNA); Methylated RNA Immunoprecipitation next-generation sequencing (MeRIP-seq); Methylation regulators.

Fig. 1

RNA MeRIP-seq library construction and sequencing

Fig. 2

RNA MeRIP-seq was performed to determine the transcriptome-wide m5C methylation and the overall characteristics of lncRNA in BCBM. (A) A Venn diagram illustrating the m5C methylation sites in lncRNA from BCBM and BC. (B) A Venn diagram displaying the m5C genes in both BCBM and BC. (C) The proportion of lncRNAs with varying m5C methylation peaks between BCBM and BC cells, with the majority having only a single m5C peak. (D) The histogram showing the distribution of m5C methylation sites in various chromosomes

Fig. 3

Motif analysis of methylation sites. (A-B) The motif of m5C in BCBM and BC. (C) Heat map displaying the methylation patterns between BCBM and BC cells. (D) Differential expression of lncRNAs between BCBM and BC cells. Up-regulated methylated genes are shown in red, while down-regulated methylated genes are represented by purple

Fig. 4

Gene Ontology (GO) enrichment analysis of the differentially methylated lncRNA-associated genes in BCBM cells. The top 10 GO terms in the (A) biological processes (BP), (B) cellular components (CC), and (C) molecular functions (MF), illustrating the enrichment of the up-methylated m5C genes in BCBM. The top 10 GO terms in the (D) BP, (E) CC, and (F) MF, showing enrichment of the down-methylated m5C genes in BCBM

Fig. 5

KEGG pathway analysis for the m5C genes among BCBM lncRNAs. (A) The bar chart illustrating the top ten enrichment scores for significant pathways associated with up-methylated m5C genes in BCBM. (B) The bar chart showing the top ten enrichment scores of the significant pathways associated with down-methylated m5C genes in BCBM. (C) The dot plot indicating the gene ratio values for the ten most significantly enriched pathways associated with the up-methylated m5C genes in BCBM. (D) The dot plot showing the gene ratio values of the ten most significantly enriched pathways associated with the down-methylated m5C genes in BCBM

Fig. 6

The expression profile of differential methylation genes in BCBM and BC. (A-G) The relative expression levels of 7 different methylated genes in BCBM and BC as determined by qPCR (n = 3; Data are presented as the mean ± SD; P < 0.05)

Fig. 7

(A-G) MeRIP-qPCR analysis of the three up-regulated genes and four down-regulated genes following methylation in the BCBM and BC. (n = 3; data are presented as the mean ± SD; P < 0.05). (H) Comparison of mean fold change (log2 conversion) between MeRIP-qPCR and RNA MeRIP-seq. (I) The correlation of the mean fold changes (log2 transformed) between MeRIP-qPCR and the RNA MeRIP-seq data

Fig. 8

Integration analysis of m5C lncRNA methylation and lncRNA transcript expression. (A) Cluster analysis of the lncRNA levels of BCBM and BC. (B) Scatter Plot analysis of differential lncRNA expression in BC and BCBM. Up-regulated lncRNA are shown in red, while down-regulated lncRNA are indicated in green. (C) A volcano plot showing significant differences in lncRNA expression between the BCBM and BC (Fold change > 2.0 and P < 0.05). (D) Nine-quadrant diagram for m5C methylation and lncRNA expression. (E) Up-set graph for different m5C methylation and lncRNA expression profiles

Fig. 9

Differential expression of methylation genes in BC and adjacent tissues. (A-G) Relative expression levels of 8 different methylated genes in BC and adjacent tissues by qPCR. adjacent tissues: Control (CTR) (n = 8; data are presented as the mean ± SD; P < 0.05)

Fig. 10

Analysis of m5C RNA methylation regulators. (A) Analysis of the relative expression of eight RNA methylation regulators in BC and adjacent tissues by qPCR. The expression of NSUN2 in BC and BCBM was quantified by qPCR (B) and western-blot (C-D, Supplementary Original western blots). NSUN2 knockdown efficiency in BCBM cells measured by immunofluorescent (E, scale bars: 100 μm), qPCR (F), and Western-blot (G-H, Original blots/gels are shown in Supplementary Original western blots). (I) The relative expression levels of 7 different methylation genes in BCBM following NSUN2 knock-down. (n = 3; Data are presented as the mean ± SD; P < 0.05)