Signature mRNA markers in extracellular vesicles for the accurate diagnosis of colorectal cancer

Abstract

Background: With the increasing incidence of colorectal cancer (CRC), its accurate diagnosis is critical and in high demand. However, conventional methods are not ideal due to invasiveness and low accuracy. Herein, we aimed to identify efficient CRC mRNA markers in a non-invasive manner using CRC-derived extracellular vesicles (EVs). The expression levels of EV mRNAs from cancer cell lines were compared with those of a normal cell line using quantitative polymerase chain reaction. Eight markers were evaluated in plasma EVs from CRC patients and healthy controls. The diagnostic value of each marker, individually or in combination, was then determined using recessive operating characteristics analyses and the Mann-Whitney U test.

Results: Eight mRNA markers (MYC, VEGF, CDX2, CD133, CEA, CK19, EpCAM, and CD24) were found to be more abundant in EVs derived from cancer cell lines compared to control cell lines. A combination of VEGF and CD133 showed the highest sensitivity (100%), specificity (80%), and accuracy (93%) and an area under the curve of 0.96; hence, these markers were deemed to be the CRC signature. Moreover, this signature was found to be highly expressed in CRC-derived EVs compared to healthy controls.

Conclusions: VEGF and CD133 mRNAs comprise a unique CRC signature in EVs that has the potential to act as a novel, non-invasive, and accurate biomarker that would improve the current diagnostic platform for CRC, while also serving to strengthen the value of EV mRNA as diagnostic markers for myriad of diseases.

Keywords: CD133; Colorectal cancer; Extracellular vesicle; Non-invasive biomarker; VEGF; mRNA.

Fig. 1

EV mRNA analysis in cell lines. The relative changes in gene expression of each marker from four CRC cell lines (SW620, Wi-Dr, LS174T, and HCT116) were calculated and compared with that in a normal cell line (CCD-18Co) as the control group. EV mRNA markers were selected for further investigation if they were highly expressed in at least one CRC cell line with a relative change in gene expression of ca. 100 (ca, circa); mRNA level (a.u., arbitrary unit) running from bottom to top

Fig. 2

Analysis of clinical samples for expression of specific extracellular vesicle mRNA markers. The relative changes in MYC, VEGF, CDX2, CD133, CEA, CK19, EpCAM and CD24 gene expression from ten CRC patients and five healthy controls were calculated and compared with a healthy control (C2) group (P, CRC patient; HC, Healthy control; C, control; Signature, combined marker of VEGF and CD133); mRNA level (a.u.) running from bottom to top

Fig. 3

Receiver Operating Characteristic (ROC) curve and Area Under the Curve (AUC). a Individual EV mRNA markers (only ROC curves with AUC > 0.5). b-d Combinations of EV mRNA markers

Fig. 4

Differentiation of CRC patients from healthy controls using the CRC signature. a Relative changes in gene expression of the CRC signature between CRC patients and a healthy control (C2) group. Data are shown as mean + standard deviation. The two-tailed P value was determined by Mann-Whitney U test. b Bar graph representation for mRNA level of CRC signature in clinical samples. Cut-off value for the CRC signature is shown as a dotted line