Volume 145, Issue 2, Pages 426-436.e6 (August 2013) Genetic and Epigenetic Down-regulation of MicroRNA-212 Promotes Colorectal Tumor Metastasis via Dysregulation of MnSOD  Xiangqi Meng, Jiangxue Wu, Changchuan Pan, Hui Wang, Xiaofang Ying, Yi Zhou, Hongyan Yu, Yufang Zuo, Zhizhong Pan, Ran–Yi Liu, Wenlin Huang  Gastroenterology  Volume 145, Issue 2, Pages 426-436.e6 (August 2013) DOI: 10.1053/j.gastro.2013.04.004 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 Mature miR-212 expression in colorectal cell lines and tissues, and the effects of miR-212 on colony formation, tumorigenic activity, and cell motility in CRC cell lines. mir-212 inhibited the expression of MnSOD. (A, B) Mature miR-212 expression was examined via real-time PCR in CRC cells and 30 pairs of CRC and normal tissues. ∗P < .05; ∗∗P < .01. (C) Effects of miR-212 on colony formation (i) and anchorage-independent growth (ii) in CRC cells. ∗P < .05; ∗∗P < .01. (D) The migration and invasion abilities of HCT116 (SW480) cells transfected with miR-212 or NC. Representative invasive images of HCT116 and SW480 cells are shown. ∗∗P < .01. (E) Schematic illustration of the predicted miR-212 binding sites in the MnSOD 3′UTR. nt, nucleotide; WT, wild type; MUT, mutated. (F) Ectopic expression of miR-212 decreased endogenous MnSOD in a dose-dependent manner. β-actin was used as the loading control. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 MnSOD was a direct target of miR-212 to mediate miR-212 regulated epithelial-mesenchymal cell transition in CRC metastasis progress. (A) PGL3-SOD2-WT or PGL3-SOD2-MUT was co-transfected with miR-212 mimics or NC, respectively, in the indicated cells. PGL4.73 was transfected as an internal control. ∗∗P < .01. (B) Representative images of immunohistochemistry staining for MnSOD in colorectal tumor tissues. The correlation between MnSOD protein expression and miR-212 expression in CRC tissues was analyzed. n = 30; ∗P < .05. (C) The migration and invasion abilities of HCT116 cells transfected with siMnSOD or siNC. ∗P < .05; ∗∗P < .01. (D) Migration assays using HCT116 cells co-transfected with pcDNA3.1 (+)-SOD2 or pcDNA3.1 (+) and miR-212 or NC. ∗∗P < .01. (E) HCT116 and SW480 cells were transfected with miR-control, miR-212, or miR-212 + SOD2. After 48 hours, the cell lysates were collected for the detection of epithelial markers (E-cadherin, ZO-1) and mesenchymal markers (vimentin, fibronectin). (F) Immunofluorescence was used to compare the expression levels and expression pattern of epithelial and mesenchymal markers between HCT116 (or SW480) cells transfected with miR-control, miR-212, or miR-212 + SOD2. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 Exogenous expression of miR-212 suppresses CRC cell metastasis in vivo. (A, B) Hepatic metastasis model. (A) Representative livers subjected to the indicated treatments. The metastatic nodules are indicated with arrows. (B) Representative results of H&E staining of metastatic nodules in the liver are shown. Metastatic nodules are indicated with arrows. The statistical result is on the right. n = 8; ∗P < .05. (C, D) Lung metastasis model. (C) Representative lungs subjected to the indicated treatments. The metastatic nodules are indicated with arrows. (D) Representative results of H&E staining of the metastatic nodules in the lung are shown. The metastatic nodules are indicated with arrows. The statistical result is on the right. n = 8; ∗∗P < .01. (E) Immunohistochemistry shows decreased expression of MnSOD in liver and lung tumor tissues originating from HCT116-miR212 cells compared with those originating from HCT116-NC cells. (F) Mature miR-212 expression was examined via real-time PCR in primary tumor tissues originating from HCT116-miR212 cells or HCT116-NC cells. ∗∗P < .01. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 LOH analysis in human CRC. (A) Fluorescence in situ hybridization analysis of miR-212 copy number in colorectal cells. Blue represents 4′,6-diamidino-2-phenylindole nuclear staining. None of the cell lines showed obvious copy number losses at the miR-212 locus (17p13.3), except for COLO205 cells (1000× magnification). (B) LOH of miR-212 in human CRC tissues (n = 26). Images from representative cases: no LOH (up), LOH (down) (1000× magnification). (C) miR-212/CEP17 ratios in the tumor tissues (n = 26). Ratios < 0.5 were considered to indicate LOH (38.5%). (D) Correlation between the miR-212/CEP17 ratio and miR-212 expression. (R2 = 0.2278; P < .05; n = 26). Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 miR-212 was hypermethylated in CRC cell lines and invasive CRC tissues. (A) The percentage of C+G nucleotides (CG %) and the density of CpG dinucleotides are shown for a region spanning 2-kbp upstream of miR-212. Specific primers for this CpG Island were designed (convergent arrows) and used to amplify these DNA fragments in normal and tumor samples. (B, C) A methylation-specific polymerase chain reaction (MSP) protocol was followed to specifically amplify unmethylated (U) or methylated (M) DNA. (D) Schematic illustration of the miR-212 CpG Island and the bisulfite sequencing area. (E) Bisulfite sequencing of the miR-212 up-stream region in cell lines and tissues. Open and filled circles represent unmethylated and methylated CpG sites, respectively. Circles were partially filled according to the percentage of methylation of the CpG site (the frames show the CpG pairs covered by the MSP primers). (F) Relative expression of miR-212 in colorectal cells treated with 5 μM 5-aza-2′-deoxycytidine. ∗P < .05; ∗∗P < .01. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 Reduced miR-212 expression in metastatic CRC tissues and its prognostic value in patients with CRC. (A) The expression of miR-212 between CRCs with (stage III and IV) and without (stage I and II) metastasis. The mean level of miR-212 expression in CRCs with metastasis were significantly lower than that in CRCs without metastasis. n = 180; ∗P < .05. (B, C) Correlations between miR-212 level and disease-free survival and overall survival based on Kaplan-Meier analysis in patients with high (n = 90) or low miR-212 expression (n = 90). ∗∗P < .01. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 1 Silencing of miR-212 can enhance CRC cell migration and abrogate the inhibitory effect that miR-212 caused in dual luciferase reporter assay. (A) HCT116 and SW480 cells were transfected with either anti−miR-212 or anti-NC. All cells were subjected to migration assays using fetal bovine serum as a chemoattractant. ∗P < .05; ∗∗P < .01. (B) PGL3-SOD2-WT or PGL3-SOD2-MUT was co-transfected with anti−miR-212 or anti-NC, in 293T cells. PGL4.73 was transfected as an internal control. ∗∗P < .01. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 2 miR-212 can inhibit the messenger RNA (mRNA) expression of MnSOD. And the protein expression levels of MnSOD in CRC cell lines were detected. (A) Endogenous MnSOD mRNA levels 48 hours after transfection with miR-212 mimics or NC in HCT116 and SW480 cells. GAPDH was used as a control. ∗P < .05; ∗∗P < .01. (B) The expression of MnSOD was examined by Western blot analysis in colorectal cells. β-actin was used as the loading control. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 3 Knockdown the expression of MnSOD inhibited the migration and invasion of CRC cells and overexpression of MnSOD can rescue the inhibitory effect caused by miR-212. (A) SW480 cells were transfected with either siMnSOD or siNC. All cells were subjected to migration and invasion assays using fetal bovine serum as a chemoattractant. ∗∗P < .01. (B) Anti−miR-212 was co-transfected with siSOD2 or siNC, respectively, in HCT116 cells. Then, all cells were subjected to migration assays using fetal bovine serum as a chemoattractant. ∗∗P < .01. (C) Migration assays using SW480 cells co-transfected with pcDNA3.1 (+)-SOD2 or pcDNA3.1 (+) and miR-212 or scramble RNA. ∗∗P < . 01. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 4 The expression of epithelial markers and mesenchymal markers were compared by Western blot analysis between SW480 cells transfected with siMnSOD and those transfected with siNC. β-Actin was used as a loading control. Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 5 There was no significant difference in spleen weight between the HCT116-NC and HCT116-miR212 groups (P = .3125). Gastroenterology 2013 145, 426-436.e6DOI: (10.1053/j.gastro.2013.04.004) Copyright © 2013 AGA Institute Terms and Conditions