Methylation of mitochondrial DNA displacement loop region regulates mitochondrial copy number in colorectal cancer

Abstract

It is not established whether de-methylation of the displacement loop (D-loop) region if mitochondrial DNA (mtDNA) directly influences mtDNA copy number and further alters the cell cycle, apoptosis and cell proliferation in colorectal cancer. The current study employed cell viability assays, cell cycle analysis, and mtDNA methylation analysis using 5 colorectal cancer cell lines. The present results demonstrated that 5-aza-2'-deoxycytidine (5-AZA), a DNA hypomethylating agent, significantly increased proliferation of Lovo and Colo-205 colorectal cancer cell lines. In Colo-205 cells, the proportion of G0/G1 phase cells was increased following 5-AZA treatment. Additionally, the apoptosis rate in Colo-205 cells was decreased by 5-AZA treatment. Compared with their controls, a significantly higher mtDNA copy number was observed in Colo-205 and Lovo cells following 5-AZA treatment. Notably, the Colo-205 and Lovo cells had relatively higher methylation levels at the 4 and 6th/7th CpG sites of D-loop region, respectively, compared with the levels at the corresponding sites following 5-AZA treatment. However, in HCT116, SW480, LS-174T, and HT-29 cells, 5-AZA treatment did not induce a significant change in proliferation, cell cycle, apoptosis and mtDNA copy number. Demethylation at the 4 and 6th/7th CpG sites of the D-loop region of HCT116, SW480, LS-174T and HT-29 cells was not observed following 5-AZA treatment. In conclusion, de-methylation of specific sites on CpG islands of D-loop promoter may lead to the elevation of mtDNA copy number in colorectal cancer, triggering alterations in biological behaviors, including increased cell proliferation, reduced apoptosis and a relative cell cycle arrest in G0/G1 phase.