Identification of a metastasis-specific MicroRNA signature in human colorectal cancer

Abstract

Background: Distant metastasis is the major cause of mortality in colorectal cancer (CRC). We performed a systemic, comprehensive discovery for expression patterns of metastasis-specific microRNAs (miRNAs) by directly comparing primary CRCs (pCRCs) and matched liver metastases (LMs) and evaluated the feasibility of their clinical application as metastasis-specific biomarkers.

Methods: CRC metastasis-specific miRNA profiles were generated by analyzing nine pairs of pCRC and LM tissues, followed by quantitative validation in an independent cohort of 58 pairs of matched pCRC and LM tissues. We evaluated associations between miRNA expression and patient survival and ability to predict metastasis in another 84 patients with CRC. Subsequently, associations were quantitatively validated in 175 CRC tissues and 169 serum samples. Kaplan-Meier, Cox regression, and logistic regression analyses were used. All statistical tests were two-sided.

Results: Twenty-three miRNAs were identified that were differentially expressed between pCRC and LM (P < .001; FDR < .5). Four miRNAs downregulated in LM (let-7i, miR-10b, miR-221, and miR-320a) and one upregulated miR (miR-885-5p) were quantitatively validated in pCRC (P < .0001). Low let-7i expression in pCRC tissue predicted worsened prognosis (hazard ratio [HR] = 5.0, 95% confidence interval [CI] = 1.0 to 24.4, P = .0479) as well as distant metastasis (odds ratio [OR] = 5.5, 95% CI = 1.1 to 26.8, P = .0334). High miR-10b expression in pCRC tissue independently predicted distant metastasis (OR = 4.9, 95% CI = 1.2 to 19.7, P = .0248). High serum miR-885-5p expression independently predicted prognosis (HR = 2.9, 95% CI = 1.1 to 7.5, P = .0323), LN metastasis (OR = 3.0, 95% CI = 1.3 to 7.2, P = .0116), and distant metastasis (OR = 3.1, 95% CI = 1.0 to 10.0, P = .0456), whereas tissue miR-885-5p expression did not. Expression patterns of miRNAs were confirmed by in situ hybridization.

Conclusions: We discovered a metastasis-specific miRNA signature in pCRCs and discovered novel tissue- and serum-based CRC metastasis-specific miRNA biomarkers through intensive validation. These unique miRNAs may be clinically applicable to predict prognosis and distant metastasis in CRC.

Figure 1.

Expression status of candidate microRNAs (miRNAs) as colorectal cancer (CRC) metastasis-specific biomarkers. Quantitative validation of selected four downregulated miRNAs (let-7i, miR-320a, miR-10b, and miR-221) and two upregulated miRNAs (miR-30b and miR-885-5p) in liver metastasis (LM) compared with primary CRC (pCRC) in an independent validation cohort of 58 pairs of matching pCRC and LM tissues. The gray horizontal bar represents mean expression levels; ***P < .0001, paired t test; ns = no significance; P < .0001, multiple variables assessment of six miRNAs by O’Brien test. LM = liver metastasis; miRNA = microRNA; pCRC = primary colorectal cancer.

Figure 2.

Kaplan-Meier Survival Analysis based on microRNA (miRNA) microarray cohort (frozen tissue). Overall survival analysis based on four miRNAs (let-7i, miR-10b, miR-221, and miR-320a) in miRNA microarray cohort. The P values were determined by log-rank test. All statistical tests were two-sided.

Figure 3.

Kaplan-Meier Survival Analysis based on different types of specimen cohorts (FFPE tissue and serum). A) Overall survival (left panel) and disease-free survival (right panel) analyses based on let-7i in colorectal cancer (CRC) tissue cohort. B) Overall survival (left panel) and disease-free survival (right panel) analyses based on miR-10b in CRC tissue cohort. C) Overall survival (left panel) and disease-free survival (right panel) analyses based on miR-885-5p in CRC serum cohort. The P values were determined by log-rank test. Disease recurrence was observed in 19 patients, which included three patients with stage II disease and 16 with stage III cancers. All statistical tests were two-sided.

Figure 4.

In situ expression of colorectal cancer (CRC) metastasis-specific microRNAs (miRNAs; let-7i, miR-10b, and miR-885-5p). Pathologic expression patterns of three validated CRC metastasis-specific miRNAs (let-7i, miR-10b, and miR-885-5p) were determined by hybridization with LNA-modified and 5`- and 3`-DIG-labeled oligonucleotide probes. In situ hybridization analysis of let-7i, miR-10b, and miR-885-5p in (A) positive and negative controls (primary colorectal cancer [pCRC]) and (B) matched primary pCRC with and without metastases and expression in the matched liver metastasis (positive control, U6 snRNA; negative control, scrambled miRNA control). LM = liver metastasis; pCRC = primary colorectal cancer.