Circulating exosomal microRNAs as biomarkers of colon cancer

Abstract

Purpose: Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined.

Experimental design: Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients.

Results: The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis.

Conclusion: Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.

Figure 1. Strategy for the identification of CRC-specific exosomal miRNAs.

Figure 2. Serum exosomal expression levels of 16 miRNAs that were up-regulated in colon cancer.

Serum exosomal miRNA levels in 11 HCs (blue) and 88 CRC patients (red) at different TNM stages (I to IV). The signal intensities were normalized to the total signal intensity of the microarray. The horizontal lines indicate the mean normalized signal intensity for each group. Statistically significant differences were determined by Welch's t-test.

Figure 3. Exosomal miRNA levels in matched serum samples from CRC patients before and after tumor resection.

(A) Scatter plot of the 16 commonly up-regulated serum exosomal miRNAs in CRC patients (n = 29) before (Pre) and after (Post) surgical removal of tumors. The sample set included stage I (n = 6), stage II (n = 6), stage IIIa (n = 5), stage IIIb (n = 9), and stage IV (n = 4) patients. The data represent the mean normalized signal intensities (%). The red dots indicate the eight miRNAs that were significantly down-regulated after tumor resection: let-7a, miR-1224-5p, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a. (B) Individual changes in the serum exosome levels of the eight down-regulated miRNAs in CRC patients (n = 29) before (Pre) and after (Post) surgical removal of tumors. Statistically significant differences between the mean Pre values and the mean Post values were determined by paired Student's t-tests.

Figure 4. ROC curve analysis of eight miRNAs in serum exosomes of HCs and CRC patients.

The signal intensities of the miRNAs are shown as percentages of the total signal intensity. The cut-off values of the eight miRNAs that were up-regulated in colon cancer and down-regulated after tumor resection were analyzed using a ROC curve. Black boxes indicate patients over the cut-off value of the biomarkers or miRNA levels. The normalized intensities of undetectable miRNAs in serum exosomes were calculated as 0.

Figure 5. Validation of CRC-associated increases in the expression of eight miRNA in serum exosomes by qRT-PCR.

Box-and-whisker plots of the expression levels of the eight selected miRNAs in an independent set of HCs (n = 8) and CRC patients with primary tumor (n = 13). Statistically significant differences between the HC and CRC datasets were determined by Welch's t-tests. The comparative cycle threshold (Ct) method was used to quantify the levels of exosomal miRNAs in HC and CRC patients. The relative ratio was calculated using the 2^-ΔΔCt method. The Ct value of miR-451 was used as an internal standard. Each data point was normalized to a representative HC sample.