The relationship between DNA methylation andReprimogene expression in gastric cancer cells

Abstract

Reprimo (RPRM) is a tumor suppressor involved in the development of a number of malignant tumors including gastric cancer which is highly related to its gene hypermethylation. However, the regulation of RPRM gene expression by DNA methylation in gastric cancer is not well understood. We examined the RPRM gene methylation in gastric cancer tissues or plasma samples by bisulfite sequencing, and investigated the relationship between DNA methylation and the RPRM gene expression by quantitative reverse transcription-PCR and Western blotting. We found that the RPRM gene promoter region is hypermethylated in gastric cancer tissues (75%, 45/60), plasma samples (86.3%, 44/51) and various cancer cell lines (75%, 3/4), which is correlated with the decrease of RPRM gene expression. The hypermethylation-induced RPRM reduction can be recovered by treating with zebularine, a demethylating agent, and by inhibition of the DNA methyltransferases via RNA interference and CRISPR/Cas9-mediated gene knockout. In addition, we generated RPRM gene-knockout cells and studied the effects of the RPRM deficiency on tumor formation by inoculating these cells in mice. The data show that the loss of RPRM can promote tumorigenesis. These data suggest that the RPRM expression is inhibited by DNA methyltransferases and the RPRM normal function can be restored by treating with DNA methylation inhibitors. The study provides important information regarding the role of RPRM and its methylation related to gastric cancer development.

The Reprimo Gene Family: A Novel Gene Lineage in Gastric Cancer with Tumor Suppressive Properties

“…In gastric cancer cells, restoring the expression of RPRM by transfecting exogenous cDNA results in reduced colony formation and anchorage-independent growth [ 3 , 31 ]. Correspondingly, mouse xenografts models of gastric cancer cells deficient in RPRM expression have demonstrated enhanced tumor formation and volume [ 31 , 32 ]. In other tumors, such as breast cancer, pituitary tumors and renal cell carcinoma cell lines, overexpression of RPRM suppresses cell proliferation, cell migration, clonogenic capacity and invasiveness [ 33 , 34 , 35 ].…”

“…Clinical studies have shown that the loss of RPRM expression is as common event in gastric cancer [ 3 , 31 , 32 , 36 ] as in other tumors of the gastrointestinal tract including Barrett’s-associated esophageal adenocarcinoma, pancreatic and colorectal carcinoma [ 37 , 38 , 39 , 40 , 41 ]. Loss of expression of RPRM has been also reported in non-digestive tumors including breast, renal cell carcinoma, adrenocortical and pituitary tumors [ 33 , 34 , 35 , 42 , 43 ].…”

“…In the case of RPRM , bisulfite sequence experiments to evaluate the density of methylated CpG sites of the promoter region have correlated positively with the levels of the transcriptional expression of the gene [ 3 ]. Consequently, restoring the expression of RPRM by the use of demethylating agents, such as 5-aza-cytidine has confirmed that the expression of RPRM gene is regulated by DNA methylation in gastric cancer cells [ 3 , 32 , 48 ]. In other neoplasm similar results have been obtain confirming the role of DNA methylation as the main mechanisms of regulation of RPRM gene expression [ 35 , 49 ].…”

Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment

“…Up to the present, studies using CRISPR/Cas9 in relation to tumor development and research into treatment involving tumor suppressor genes have yielded some positive results. The main anti‐tumor research results of tumor suppressor genes are listed in Table (Part A‐D) …”

DNA Methyltransferases and Gastric Cancer: Insight Into Targeted Therapy

“…Guo et al have shown that exposure with 5-Aza-dC could restitute IRX1 protein level in gastric cancer cells [47]. Reprimo (RPRM) has been reported to be inhibited by DNMTs and its function was restored by treatment with zebularine, a DNMT inhibitor [48]. Moreover, a possible association between DNMT1 amplification, triggering hypermethylation of CpG islands and down-regulation of TSGs including human MutL homolog 1 (hMLH1), thrombospondin 1 (THBS1), cadherin 1 (CDH1) has been suggested in gastric cancer [49].…”