Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer

Abstract

Background: MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker.

Methods: The 5-aza-2'-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a.

Results: The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561.

Conclusions: These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker.

Figure 1

Methylation status of miR-34b/c and miR-148a in a panel of CRC cell lines assessed by RT–qPCR and methylation-specific PCR (MSP). Fold change expression of miR-34b/c (A, upper panel) and miR-148a (B, upper panel) following 10 μM demethylating agent 5-aza-2′-deoxycytidine over 5 days of incubation in HCT116, HT29, LoVo and HCT15 assessed by RT-qPCR and by means of a 2^{^−ΔΔ}C_q method. Corresponding methylation-specific PCR for miR-34b/c (A, lower panel) and miR-148a (B, lower panel) showing the decrease in methylation pattern following 10 μM demethylating agent 5-aza-2′-deoxycytidine. Lane UM and M corresponded to unmethylated and methylated reaction respectively. Qiagen methylated and unmethlated control (ctrl) DNAs served as a reaction control for PCR. NTC=negative template control; POS=positive template control; AZA=5-aza-2′-deoxycytidine.

Figure 2

Methylation-specific PCR (MSP) reactions for the miR-34b/c and miR-148a promoter region in tumour and faecal specimens derived from CRC and/or normal individual. (A and B) MSP analysis for miR-34b/c in matched CRC samples. (C) MSP analysis for miR-34b/c in faeces of CRC (upper panel), colonoscopy negative individuals (middle panel) and high-grade dysplasia (lower pane). (D) MSP analysis for miR-148a in matched CRC tissues. Lane UM and M corresponded to unmethylated and methylated reaction, respectively. Qiagen methylated and unmethylated control (CTRL) DNAs served as a reaction control for polymerase chain reaction. NTC=negative template control; POS=positive template control; P=patients; F-P1=samples derived from faeces of CRC and normal individuals.

Figure 3

Box and Whisker plot and Kaplan–Meier disease-free survival analysis for CRC patients according to miR-148a hypermethylation pattern. (A) The 10-year survival rate (60%) of CRC patients was calculated using the Kaplan–Meier method. (B) Survival rate of CRC patients according to depth of invasion. The difference is statistically significant (log-rank test: P<0.05). (C) The prognostic value of miR-148a methylation status was with a trend towards lower survival rate in patients with methylated alleles (10-year survival probability: 48%) compared with unmethylated cases (10-year survival probability: 65%), log-rank test: P=0.561.