Gastroenterology

Gastroenterology

Volume 145, Issue 2, August 2013, Pages 426-436.e6
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Genetic and Epigenetic Down-regulation of MicroRNA-212 Promotes Colorectal Tumor Metastasis via Dysregulation of MnSOD

https://doi.org/10.1053/j.gastro.2013.04.004Get rights and content

Background & Aims

Altered functions of microRNAs (miRNAs) have been associated with colorectal cancer (CRC). miR-212 is transcribed from a stable intron of a non-protein coding gene, and is reportedly down-regulated in different tumor types. We investigated the role of miR-212 in colorectal carcinogenesis and progression.

Methods

We analyzed the expression of miR-212 by real-time polymerase chain reaction (PCR) analysis of colorectal cell lines and 180 paired tumor samples and surrounding healthy tissue. We overexpressed and knocked down miR-212 in CRC cell lines and assessed the in vitro effects. We also studied the effects of miR-212 overexpression on metastasis of tumors grown from HCT116 cells in nude mice.

Results

Overexpression of miR-212 inhibited CRC cell migration and invasion in vitro and formation of intrahepatic and pulmonary metastasis in vivo. We identified manganese superoxide dismutase (MnSOD) messenger RNA as a direct target of miR-212, and observed an inverse correlation between the level of miR-212 and MnSOD protein in colorectal tumor samples. MnSOD was required for down-regulation of epithelial markers and up-regulation of mesenchymal markers in CRC cells, indicating that it promoted the epithelial−mesenchymal transition. Overexpression of miR-212 reduced the levels of MnSOD to block the epithelial−mesenchymal transition process. Loss of heterozygosity and promoter hypermethylation each contributed to the down-regulation of miR-212. Reduced levels of miR-212 were associated with a more aggressive tumor phenotype and short disease-free survival times of patients (P = .0045; overall survival, P = .0015).

Conclusions

miR-212 is down-regulated in human CRC tissues via genetic and epigenetic mechanisms. miR-212 might prevent tumor progression by targeting MnSOD messenger RNA; reduction of miR-212 could be a prognostic marker for patients with CRC. miR-212 and MnSOD might also be therapeutic targets for cancer.

Section snippets

Materials and Methods

The Materials and Methods can be found in the Supplementary Material.

Reduced miR-212 Is Frequently Detected in CRC Cell Lines and Tissues

We first detected the expression of miR-212 in colorectal cell lines and 30 pairs of tissue samples (tumors and normal). All 5 CRC cell lines presented lower miR-212 expression than the normal intestinal epithelial cell line FHC19 (P < .01; Figure 1A). In addition, among 30 CRC tumor tissue samples, 24 (80%) had lower miR-212 compared with the paired normal colorectal tissues (P < .05; Figure 1B).

Exogenetic Overexpression of miR-212 Suppresses CRC Cell Colony Formation, Migration, and Invasion In Vitro

To investigate the potential biological functional role of miR-212 in CRC tumorigenesis and

Discussion

In this study, we determined that miR-212 regulates the metastasis and EMT of CRC by targeting MnSOD. Several lines of evidence support our conclusion and model. First, in functional studies, introduction of miR-212 significantly repressed CRC colony formation, independent growth, migration, and invasion in vitro (Figure 1), as well as the ability to metastasize to the lung and liver in vivo (Figure 3). We did not detect significant growth inhibition in CRC cells within 72 hours (data not

Acknowledgments

The authors thank Jiemin Chen and Ling Zhou (Sun Yat-Sen University, Guangzhou, PR China) for their technical assistance. The authors thank Xinyuan Guan (The University of Hong Kong, Hong Kong, PR China) for the synthesis of fluorescence in situ hybridization probe.

Hui Wang’s Current address is: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, PR China; Yufang Zuo's Current address: Cancer Center,

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by grants from the National High Technology Research and Development Program of China (project 863, no. 2012AA02A204), the National Basic Research Program of China (973 Program, no. 2011CB504805, 2010CB912201, 2010CB529904), the National Natural Science Foundation of China (no. 81272638, 30973448) and Guangdong Innovative Research Team Program (no. 2009010058).

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