Circulating miR-182 is a biomarker of colorectal adenocarcinoma progression

Abstract

MiR-182 expression was evaluated by qRT-PCR and in situ hybridization in 20 tubular adenomas, 50 colorectal carcinoma (CRC), and 40 CRC liver metastases. Control samples obtained from patients with irritable bowel syndrome, or tumor-matched normal colon mucosa were analyzed (n=50). MiR-182 expression increased progressively and significantly along with the colorectal carcinogenesis cascade, and in CRC liver metastases. The inverse relation between miR-182 and the expression of its target gene ENTPD5 was investigated by immunohistochemical analysis. We observed that normal colocytes featured a strong ENTPD5 cytoplasmic expression whereas a significantly and progressively lower expression was present along with dedifferentiation of the histologic phenotype. Plasma samples from 51 CRC patients and controls were tested for miR-182 expression. Plasma miR-182 concentrations were significantly higher in CRC patients than in healthy controls or patients with colon polyps at endoscopy. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy compared to preoperative samples. Our results strengthen the hypothesis of a central role of miR-182 dysregulation in colon mucosa transformation, demonstrate the concomitant progressive down-regulation of ENTPD5 levels during colon carcinogenesis, and indicate the potential of circulating miR-182 as blood based biomarker for screening and monitoring CRC during the follow-up.

Figure 1. miR-182 is up-regulated during colon carcinogenesis

(A) miR-182 expression was evaluated by qRT-PCR after RNA extraction from FFPE samples of colon normal mucosa, LG-IEN, HG-IEN lesions and CRCs. (B) miR-182 expression was evaluated by qRT-PCR in matched surgical samples of normal colon mucosa, primary CRC and liver metastatis. (C) Representative ISH evaluation of miR-182 in matched tissue sections of normal colon, primary tumor and metastatic CRC (N= normal colon mucosa; K= primary CRC). The presence of miR-182 is shown by a grainy blue cytoplasmic stain; slides counterstained in fast red. (Original magnifications 10x and 20x). Significance (Student's t test); *p<0.05; **p<0.01. nRQ, normalized Relative Quantity. Data were expressed as mean values ± SD.

Figure 2. miR-182 targets ENTPD5 during colon carcinogenesis

(A) Luciferase reporter assay of miR-182 and 3′UTR ENTPD5 region. Relative light units (RLU) of biological replicates are shown as means ± standard deviation (SD) of three experiments performed in triplicate. Mutation of the binding site completely restored luciferase expression. (B) ENTPD5 IHC scores distribution (expressed as %) was significantly down-regulated during colorectal carcinogenesis (p<0.001). (C) ENTPD5 expression, as assessed by both qRT-PCR and IHC, is significantly lower in primary and metastatic CRCs in comparison to normal colic mucosa. (D) Representative examples of ENTPD5 expression during colon carcinogenesis. (Original magnifications 10x and 20x ; * liver parenchyma). Significance (Student's t test); *p<0.05; **p<0.01. Immunohistochemical scores: black = score 3 (% positive cases), dark gray = score 2, light gray = score 1, white = score 0 (% negative cases). nRQ, normalized Relative Quantity. Data were expressed as mean values ± SD.

Figure 3. miR-182 plasma levels are significantly elevated in CRC patients

(A) miR-182 plasma levels were analyzed in 10 healthy volunteers, 10 patients with colic adenomas at endoscopy, 10 early stages (stages I and II) and 10 late stages (stages III and IV) CRC patients. (B) miR-182 plasma expression in advanced CRC patients and in early CRC patients. (C) Plasma miR-182 concentration before and after curative liver metastasectomy (p=0.020). Significance (Student's t test); *p<0.05; **p<0.01. nRQ, normalized Relative Quantity. Data were expressed as mean values ± SD.