Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer

Abstract

Objective: To evaluate the ability of epithelial-to-mesenchymal transition-related microRNAs (miRNAs) as serum biomarkers for prognosis and prediction of metastasis in patients with colorectal cancer (CRC).

Background: Epithelial-to-mesenchymal transition-related miRNAs drive CRC progression and metastasis. However, their potential as serum biomarkers in CRC has not been studied.

Methods: This was a 3-phase study using 446 colorectal specimens. In the first phase, we selected candidate miRNAs associated with metastasis by analyzing the expression of 4 miR-200 family members (miR-200b, -200c, -141, and -429) in serum samples from 12 patients with stage I and IV CRC. The second phase involved independent validation of candidate miRNAs in serum from 182 patients with CRC and 24 controls. Finally, we analyzed expression in matched 156 tumor tissues from 182 patients with CRC and an independent set of 20 matched primary CRC and corresponding liver metastases to identify the source of circulating miRNAs.

Results: After initial screening, miR-200c was selected as the candidate serum miRNA best associated with metastasis. Validation analysis revealed that serum miR-200c levels were significantly higher in stage IV than in stage I-III CRCs. High serum miR-200c demonstrated a significant positive correlation with lymph node metastasis, distant metastasis, and prognosis (P = 0.0026, P = 0.0023, and P = 0.0064, respectively). More importantly, serum miR-200c was an independent predictor for lymph node metastasis (odds ratio: 4.81, 95% confidence interval: 1.98-11.7, P = 0.0005) and tumor recurrence (hazard ratio: 4.51, 95% confidence interval: 1.56-13.01, P = 0.005) and emerged as an independent prognostic marker for CRC (hazard ratio: 2.67, 95% confidence interval: 1.28-5.67, P = 0.01).

Conclusions: Serum miR-200c has strong potential to serve as a noninvasive biomarker for CRC prognosis and predicting metastasis.

Conflict of interest statement

Conflict of interest: The authors have no conflict of interests to disclose

Figures

Figure 1. Expression analysis of miR-200 family members in the serum of stage I and stage IV CRC patients

Box plots of serum levels of miR-200b (A), miR-200c (B), miR-429 (C) and miR-141 (D) in stage I (n=12) and stage IV (n=12) CRC patients. MiR-200c levels in serum from stage IV patients were significantly higher than that of stage I patients. The boxes represent the interquartile range, and the lines across the boxes indicate the median values. Expression levels of these miRNAs (log10 scale on the y-axis) were normalized to cel-miR-39. Statistical analysis was performed using Mann-Whitney test.

Figure 2. Validation of miR-200c expression in a validation cohort of CRC patients

(A) Dot plots of serum miR-200c levels in healthy normal controls (NC) (n=24) and patients with CRC (n=182). (B) Dot plots of serum miR-200c levels across various stages of CRCs. MiR-200c levels in serum from CRC patients were significantly elevated compared with those of normal controls, and the expression levels in stage IV CRC patients were significantly higher than those in stage I–III patients. (C) Dot plots of serum miR-200c levels subdivided by H Stage. (D) Dot plots of serum miR-200c levels subdivided by N Stage. Serum miR-200c increased depending on the higher N and H stages. The lines indicate the mean values. Expression levels of miR-200c (log10 scale on the y-axis) were normalized to cel-miR-39. Statistically significant differences were determined using Mann-Whitney tests and Kruskal-Wallis test.

Figure 3. Kaplan-Meier survival curves of CRC patients subdivided by miR-200c levels in serum and matched primary tumors from CRC patients

Overall survival rates of stage I–IV (A) and stage II–IV (B) CRC patients with high serum miR-200c levels were significantly lower than for those with low miR-200c expression (stage I–IV, P=0.0064; stage II–IV, P=0.003; Log-rank test). Overall survival rates of stage I–IV (C) and stage II–IV (D) CRC patients with low miR-200c expression in primary CRC were lower than for those with high miR-200c expression (stage I–IV, P=0.0935; stage II–IV, P=0.154; Log-rank test). The miRNA expression cut-off thresholds for miR-200c expression in serum were deduced from the ROC curves with Youden’s Index.

Figure 4. Expression of miR-200c in tissues from CRC patients

(A) Dot plots of miR-200c levels in adjacent normal mucosae (NC) (n=20) and CRC tissues (n=156). (B) Dot plots of miR-200c tissue levels across various CRC stages. (C) Dot plots of miR-200c tissue levels for comparisons between matched primary CRCs (CRC) (n=20) and their corresponding liver metastasis (LM) (n=20). (D)In situ hybridization analysis of miR-200c in matched primary CRCs and their corresponding LM. Representative photomicrographs are shown from 2 primary CRCs (left panels) and matched LM (right panels). MiR-200c expressions in LMs were higher than that in primary CRCs expressed. In contrast, miR-200c expression in normal liver tissues was either very low or absent. Line indicates the mean value. Expression levels of miR-200c were normalized to has-miR-16. Statistically significant differences were determined using Mann-Whitney tests and Kruskal-Wallis test.