Down-Regulation of microRNA-132 is Associated with Poor Prognosis of Colorectal Cancer

Abstract

Background: Given the role of microRNA in colorectal cancer (CRC) progression, we explored the association between microRNA (miRNA) expression and CRC-related prognosis.

Methods: Three types of tissue samples (primary CRC lesions without liver metastasis, primary lesions with liver metastasis, and liver metastatic tissues) were used for miRNA profiling to identify differentially expressed miRNA. Quantitative real-time PCR was used to examine miRNA expression in CRC cells and in tumor tissues.

Results: MiR-132 was significantly down-regulated in primary CRC tissues with liver metastasis and liver metastatic lesions compared to primary lesions without liver metastasis. Multivariate analysis for overall survival indicated that low miR-132 expression was an independent prognostic factor for CRC patients (overall survival P = 0.040, disease-free survival P = 0.015). Ectopic expression of miR-132 significantly inhibited cell proliferation and cell invasion. The luciferase reporter assay revealed that anoctamin 1 (ANO1) was a direct target of miR-132. Kaplan-Meier survival curves showed that high ANO1 expression was a significant prognostic factor for overall survival of patients with CRC (P = 0.0344).

Conclusions: Down-regulation of miR-132 is associated with poor prognosis in CRC. ANO1 could be one of the crucial targets of miR-132 in CRC.

Fig. 1

Expression of miR-132 in colorectal and hepatic tissue samples. a Microarray data showed that miR-132 expression was significantly lower in liver metastatic lesions than in primary CRC tumors from patients without liver metastasis. b In extended validation cohort, qRT-PCR showed that miR-132 expression was significantly down-regulated in primary CRC lesions with liver metastasis and in liver metastatic lesions compared to primary CRC lesions without liver metastasis. c MiR-132 expression of seven pairs of primary CRC was significantly down-regulated in corresponding synchronous liver metastases. d MiR-132 expression was significantly increased in tumor tissues (n = 21) compared to their pair-matched adjacent normal colon tissues (P = 0.0268)

Fig. 2

In vitro transduction of miR-132 in CRC cells. a MiR-132-transfected cells demonstrated significantly slower growth rate than negative control-transfected cells. b Colony formation assay showed that miR-132 overexpression resulted in significant inhibition of tumor growth. c MiR-132 overexpression markedly reduced invasion ability compared to negative control. In contrast, miR-132 inhibitor increased invasion ability. Data are presented as mean ± SD. NC negative control-transfected cells, mimic miR-132 miR-132-transfected cells, inhibitor miR-132 inhibitor miR-132-transfected cells. *P < 0.05

Fig. 3

ANO1 as possible target of miR-132. a MiR-132 expression markedly suppressed ANO1 mRNA levels. b MiR-132 inhibitor resulted in up-regulation of ANO1 mRNA levels. c The 3′ UTR of ANO1 mRNA contains site for seed region of miR-132. d Activity of luciferase reporter containing predicted miR-132 binding sequence of ANO1 3′ UTR was significantly repressed by ectopic expression of miR-132. e ANO1 expression was higher in primary CRC with liver metastasis and in liver metastases than in primary tumors without metastasis (P = 0.0059 and P = 0.0001, respectively). f Significant inverse correlation was observed between miR-132 expression and ANO1 expression in validation cohort. Data are presented as mean ± SD. NC negative control-transfected cells, mimic miR-132 miR-132-transfected cells, inhibitor miR-132 inhibitor miR-132-transfected cells. *P < 0.05

Fig. 4

MiR-132 acts as prognostic marker for CRC. a Kaplan–Meier survival curves showed that patients with low miR-132 expression demonstrated poorer clinical outcome (OS, P = 0.0021, median follow-up 1653 days). b Patients with low ANO1 levels had more favorable clinical outcome (OS) than patients with high ANO1 levels. c DFS rate were significantly lower in patients with low miR-132 expression than in patients with high miR-132 expression (P = 0.0220)