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. 2013 Jun 19;105(12):849-59.
doi: 10.1093/jnci/djt101. Epub 2013 May 23.

Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer

Yuji Toiyama  1 , Masanobu Takahashi, Keun Hur, Takeshi Nagasaka, Koji Tanaka, Yasuhiro Inoue, Masato Kusunoki, C Richard Boland, Ajay Goel
Affiliations

Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer

Yuji Toiyama et al. J Natl Cancer Inst. .

Abstract

Background: The oncogenic microRNAs (miRNAs) miR-21 and miR-31 negatively regulate tumor-suppressor genes. Their potential as serum biomarkers has not been determined in human colorectal cancer (CRC).

Methods: To determine whether miR-21 and miR-31 are secretory miRNAs, we screened expression in medium from 2 CRC cell lines, which was followed by serum analysis from 12 CRC patients and 12 control subjects. We validated expression of candidate miRNAs in serum samples from an independent cohort of 186 CRC patients, 60 postoperative patients, 43 advanced adenoma patients, and 53 control subjects. We analyzed miR-21 expression in 166 matched primary CRC tissues to determine whether serum miRNAs reflect expression in CRC. Patient survival analyses were performed by Kaplan-Meier analyses and Cox regression models. All statistical tests were two-sided.

Results: Although miR-21 was secreted from CRC cell lines and upregulated in serum of CRC patients, no statistically significant differences were observed in serum miR-31 expression between CRC patients and control subjects. In the validation cohort, miR-21 levels were statistically significantly elevated in preoperative serum from patients with adenomas (P < .001) and CRCs (P < .001). Importantly, miR-21 expression dropped in postoperative serum from patients who underwent curative surgery (P < .001). Serum miR-21 levels robustly distinguished adenoma (area under the curve [AUC] = 0.813; 95% confidence interval [CI] = 0.691 to 0.910) and CRC (AUC = 0.919; 95% CI = 0.867 to 0.958) patients from control subjects. High miR-21 expression in serum and tissue was statistically significantly associated with tumor size, distant metastasis, and poor survival. Moreover, serum miR-21 was an independent prognostic marker for CRC (hazard ratio = 4.12; 95% CI = 1.10 to 15.4; P = .03).

Conclusions: Serum miR-21 is a promising biomarker for the early detection and prognosis of CRC.

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Figures

Figure 1.
Figure 1.
Expression of miR-21 and miR-31 in culture media of colorectal cancer (CRC) cell lines (HCT116 and SW620). MiR-21 levels in media of both HCT116 A) and SW620 B) increased with increased cell counts (0.5–2×106 cells/well) and longer incubation intervals, whereas miR-31 levels did not change in either cell line C, D). The y-axis represents relative expression of miR-21 and miR-31 normalized to cel-miR-39. Initial screening for miR-21 and miR-31 expression in the screening phase, using a small subset of tissue and serum specimens from CRC patients, was done. Box plots are shown for miR-21 expression E) and miR-31 expression F) levels in primary tumor tissues (CRC) and adjacent normal mucosa (N) from eight CRC patients. Box plots are shown for serum levels of miR-21 G) and miR-31 H) in mucosa from normal control subjects (N; n = 12) and CRC patients (n = 12). Boxes represent interquartile range, and the horizontal line across each box indicates median value. The y-axis represents relative expression of miR-21 and miR-31, and data were normalized to cel-miR-39 and miR-16 expression in sera and tissue, respectively. Statistical analysis was performed using two-sided Wilcoxon and Mann–Whitney U tests. *P < .05; **P < .01; ***P < .001; ns, not statistically significant.
Figure 2.
Figure 2.
MiR-21 expression levels in serum samples (n = 282). A) Box plots represent serum miR-21 levels in healthy control subjects (N; n = 53) and patients with adenomatous polyps (Ad; n = 43) and different Tumor Node Metastasis (TNM) stages (I, II, III, and IV) of colorectal cancer (CRC) (n = 186). The y-axis (log10 scale) represents relative expression of miR-21 normalized to cel-miR-39. Boxes represent the interquartile range, and the horizontal line across each box indicates median values. Statistically significant differences were determined using the Mann–Whitney U test and Kruskal–Wallis tests. *P < .05; ***P < .001. Receiver operating characteristics (ROC) curve analysis using serum miR-21 for distinguishing patients with colorectal neoplasms from normal control subjects was performed. B) Serum miR-21 yielded an area under the curve (AUC) value of 0.927 (95% confidence interval [CI] = 0.89 to 0.96), with 82.8% sensitivity and 90.6% specificity in distinguishing CRC from normal control subjects. C) Serum miR-21 yielded AUC values of 0.803 (95% CI = 0.71 to 0.88) with 76.8% sensitivity and 81.1% specificity in discriminating adenomas from normal control subjects. ROC convex hull (ROCCH) curve analysis using raw data and bootstrap bias correction and accelerated (BCa) bootstrap bias-corrected data for distinguishing patients with colorectal neoplasms from normal control subjects was performed. D) AUC values derived from ROCCH curves of original and BCa bootstrap-corrected samples were 0.935 (95% CI = 0.812 to 0.982) and 0.919 (95% CI = 0.867 to 0.958), respectively, in distinguishing CRC patients from normal control subjects. E) AUC values derived from ROCCH curves of original and BCa bootstrap-corrected samples were 0.838 (95% CI = 0.619 to 0.964) and 0.813 (95% CI = 0.691 to 0.910), respectively, in distinguishing adenoma patients from normal control subjects.
Figure 3.
Figure 3.
Validation of miR-21 expression in matched tissue samples (n = 186). A) Box plots illustrating tissue miR-21 levels in different Tumor Node Metastasis (TNM) stages (I,II,III, and IV) of colorectal cancers (CRCs) (n = 166) and adjacent normal mucosa (N; n = 20). The y-axis (log10 scale) represents relative expression of miR-21 normalized to miR-16 in tissue samples. Boxes represent the interquartile range, and the horizontal line across each box indicates the median value. Statistically significant differences were determined using the Mann–Whitney U test and Kruskal–Wallis tests. *P < .05: ***P < .001. B) Scatter plots showing the correlation between relative expression of miR-21 levels in serum (y-axis: log10 scale) and matched tumor tissues (x-axis: log10 scale) obtained from 154 CRC patients. A positive correlation was found by Spearman correlation (ρ = 0.315; 95% CI = 0.17 to 0.45; P < .001).
Figure 4.
Figure 4.
Alterations in serum miR-21 expression levels in patients with colorectal cancer (CRC) before surgery (Pre) and 7 days after postsurgical removal of primary tumors (Post). A) Comparison of serum miR-21 levels from all CRC patients (n = 60). B) Comparison of serum miR-21 levels in 45 CRC patients who underwent potentially curative surgeries. C) Comparison of serum miR-21 levels in 15 CRC patients who underwent noncurative surgeries. The y-axis (log10 scale) represents relative expression of miR-21 normalized to cel-miR-39. Boxes represent the interquartile range, and the horizontal line across each box indicates the median value. Statistically significant differences were determined using the Wilcoxon test. ***P < .001; ns, not statistically significant.
Figure 5.
Figure 5.
Kaplan–Meier survival analysis in colorectal cancer (CRC) patients based upon miR-21 expression in primary tumors and matched serum samples. A) The overall survival rate in CRC patients with high miR-21 expression in tumor tissue (n = 25) was statistically significantly lower than that for those with low miR-21 expression (n = 141) (>3.7 vs ≤3.7; P = .006; log-rank test). B) The overall survival rate in CRC patients with high serum miR-21 expression (n = 126) was statistically significantly lower than that for those with low serum miR-21 expression (n = 62) (>0.003 vs ≤.003; P = .005; log-rank test). Cutoff values for miR-21 expression in serum and primary tumor tissues were determined from the receiver operating characteristic curves by using Youden’s index. The number of patients at risk are shown below each curve at various timepoints.
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