DNA methylation is involved in the aberrant expression of miR-133b in colorectal cancer cells

Abstract

The dysregulation of microRNA (miRNA) expression is highly involved in cancer. Recently, a number of studies have demonstrated that the silencing of specific miRNAs is associated with DNA methylation. The muscle-specific miRNA-113b (miR-133b) is markedly downregulated in human colorectal cancer (CRC) compared with healthy colon cells, and is critical in the regulation of CRC cell proliferation and apoptosis. However, the mechanism of miR-133b downregulation in CRC has yet to be elucidated. Therefore, the aim of the present study was to determine the existence of an association between DNA methylation and miR-133b expression in CRC cells. It was identified that miR-133b promoter hypermethylation is upregulated in CRC tissues. To investigate the role of miR-133b methylation in CRC cells, the survival, cell cycle and invasion were analyzed in HT-29 and SW620 CRC cells treated with 5-aza-2'-deoxycytidine (5-Aza-CdR), 4-phenylbutyric acid (PBA) and 5-Aza-CdR/PBA. Functional analysis demonstrated that demethylation increased the expression of miR-133b, which restored migration and apoptosis in CRC cells. Thus, these results indicate that the regulation of miR-133b methylation may provide a novel therapeutic strategy for CRC treatment.

Keywords: DNA methylation; colorectal carcinoma; microRNA-133b.

Figure 1.

miR-133b expression is downregulated in CRC tissues and cell lines. (A) Reverse transcription-quantitative polymerase chain reaction identified significant downregulation in the expression levels of miR-133b in CRC tissues but not in healthy or matched adjacent non-tumor tissues. U6 small nuclear RNA was used as the endogenous control. (B) Relative expression level of miR-133b in one healthy colon cell line and five CRC cell lines. CRC, colorectal cancer; miR, microRNA.

Figure 2.

miR-133b methylation in CRC tissue samples and cell lines, and reactivation of miR-133b by demethylation. (A and B) Methylation-specific PCR, revealing frequent methylation of miR-133b in CRC tissue samples and the CRC HT-29 and SW60 cell lines, and healthy human colonic HCoEpiC cell lines. A1-A2, Healthy colon tissue; A3-A4, adjacent non-tumor tissue; C1-C6, human CRC tissue. (C) Reverse transcription-quantitative PCR and (D) combined bisulfite restriction analysis demonstrating that pharmacological demethylation can restore miR-133b expression in CRC cells. (E) Survival curve of CRC cells following treatment with 5-Aza/PBA, determined by using CCK-8. CRC, colorectal cancer; PCR, polymerase chain reaction; M, methylated; U, unmethylated; miR, microRNA; con, control; 5-Aza, 5-aza-2′-deoxycytidine; PBA, 4-phenylbutyric acid; OD, optical density; CCK-8, cell counting kit-8.

Figure 3.

Effect of miR-133b demethylation on the cell cycle distribution of CRC HT-29 and SW620 cells. Representative cell cycle analysis graphs of (A) control HT-29 cells, (B) HT-29 5-Aza/PBA-treated cells, (C) control SW620 cells and (D) SW620 5-Aza/PBA-treated cells. (E) Summarized flow cytometry data. Results are presented as the mean ± standard deviation and are based on three independent experiments. *P<0.05 vs. untreated control cells, determined using a Student's t-test. miR, microRNA; 5-Aza, 5-aza-2′-deoxycytidine; PBA, 4-phenylbutyric acid.

Figure 4.

miR-133b demethylation inhibits tumor cell invasion. Colorectal cancer HT-29 and SW620 cells were treated with 5-Aza, PBA and 5-Aza/PBA. The number of invading tumor cells was calculated by analyzing Transwell invasion assays, and the miR-133b demethylation and control groups were compared. *P<0.05 vs. untreated control cells. miR, microRNA; 5-Aza, 5-aza-2′-deoxycytidine; PBA, 4-phenylbutyric acid; OD, optical density.