LINE-1 hypomethylation, increased retrotransposition and tumor-specific insertion in upper gastrointestinal cancer

Abstract

The long interspersed nuclear element-1 (LINE-1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE-1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE-1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE-1 hypomethylation, retrotransposition, and tumor-specific insertion in upper GI cancers remain unknown. We used bisulfite-pyrosequencing and quantitative real-time PCR to verify LINE-1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE-1 retrotransposition profile with an originally developed L1Hs-seq. In tumor samples, LINE-1 methylation levels were significantly lower than non-tumor controls, while LINE-1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE-1 methylation level and copy number in tumor tissues, with lower LINE-1 methylation levels corresponding to higher LINE-1 copy numbers. Of particular importance is that somatic LINE-1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE-1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor-suppressive genes. LINE-1 hypomethylation in upper GI cancers is related to increased LINE-1 retrotransposition and tumor-specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor-suppressive genes.

Keywords: LINE-1; esophageal cancer; gastric cancer; methylation; retrotransposition.

FIGURE 1

Long interspersed nuclear element‐1 (LINE‐1) methylation level and copy number. (A) The LINE‐1 structure and methylation site, together with copy number estimation sites. (B) LINE‐1 methylation levels in the cancerous tissue were significantly lower than those in the normal tissue (left, total; middle, esophageal cancer; right, gastric cancer). (C) LINE‐1 copy numbers in the cancerous tissue were significantly higher than those in the normal tissue (left, total; middle, esophageal cancer; right, gastric cancer). (D) Relationship between LINE‐1 methylation level and copy numbers in upper GI cancers (left, total; middle, esophageal cancer; right, gastric cancer). The LINE‐1 methylation level exhibited a significant degree of inverse correlation with copy number, with lower LINE‐1 methylation levels corresponding to higher LINE‐1 copy numbers.

FIGURE 2

The number of long interspersed nuclear element‐1 (LINE‐1) insertions in upper gastrointestinal (GI) cancers. (A) Number of LINE‐1 insertions from 20 samples of patients with upper GI cancer. Deep blue indicates esophageal cancer and orange indicates gastric cancer. Each diagonal bar indicates the number of insertions in matched normal tissues. (B) The number of somatic LINE‐1 insertions was higher in tumor compared to matched normal tissues (left, total; middle, esophageal cancer; right, gastric cancer). (C) Correlation between LINE‐1 methylation level and number of LINE‐1 insertions. The number of LINE‐1 insertions was significantly higher in instances of LINE‐1 hypomethylation in tumors (left). Such a relationship was not observed in normal tissues (right).

FIGURE 3

Locations of long interspersed nuclear element‐1 (LINE‐1) insertions. (A) The plot of chromosomes where somatic LINE‐1 insertion was found in 20 tumors. LINE‐1 inserted randomly and evenly throughout autosomal and sex chromosomes. Deep blue indicates esophageal cancer and orange indicates gastric cancer. The bar on the upper right shows the gene density of the human genome. (B) Genomic locations of LINE‐1 insertion sites. LINE‐1 insertions were predominantly distributed among intergenic (shown as black) and intron (shown as gray) regions, which accounted for 80%–85% of all LINE‐1 insertions identified throughout the genome.