Volume 145, Issue 4, Pages 853-864.e9 (October 2013) Decreased Levels of miR-224 and the Passenger Strand of miR-221 Increase MBD2, Suppressing Maspin and Promoting Colorectal Tumor Growth and Metastasis in Mice  Kefei Yuan, Ke Xie, John Fox, Huawei Zeng, Hongwei Gao, Canhua Huang, Min Wu  Gastroenterology  Volume 145, Issue 4, Pages 853-864.e9 (October 2013) DOI: 10.1053/j.gastro.2013.06.008 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 miR-221* and miR-224 are repressed in metastatic cancer cell lines and human cancer tissues. (A) Real-time RT-PCR of miR-221* and miR-224 in SW480 and SW620 cells. (B and C) Real-time RT-PCR of miR-221* and miR-224 with RNA extracts isolated from different CRC cells. (D) Real-time RT-PCR of miR-221* and miR-224 with RNA extracts isolated from an orthotopic CRC mouse model. RNA samples were prepared with primary tumor tissue and lung metastasis tissue, respectively (n = 6). (E and F) Real-time RT-PCR of miR-221* and miR-224 on 20 groups of normal colon, CRC, and LNM tissues (each group of normal, cancer, and metastasis tissues was collected on the same patient) (one-way analysis of variance). Average values and SDs were calculated from triplicate samples. *P < .05; **P < .01; ***P < .001. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 Prognostic value of miR-221* and miR-224. (A and B) Cumulative OS and DFS curves of patients with high or low miR-221* and miR-224 expression levels. (C) Cumulative OS and DFS curves of the combination of miR-221* and miR-224. Patients were classified into 4 groups according to their tumoral miR-221* and miR-224 expression: group I (n = 35), both high expression; group II (n = 27), high miR-221* but low miR-224 expression; group III (n = 24), high miR-224 but low miR-221* expression; group IV (n = 22), both low expression. (D) All factors adopted in ROC analysis predicted death and recurrence precisely during follow-up (P < .05 for all). The predictive value of the combination of miR-221* and miR-224 was the best one. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 Enforced expression of miR-221* and miR-224 suppresses tumor growth and metastasis in nude mouse models. (A) Whole body photos of subcutaneous CRC tumor-bearing mice (left panel). Tumor weight of subcutaneous CRC tumors (right panel). (B) Tumor mass images of orthotopic CRC tumors (left panel). Tumor weight of orthotopic CRC tumors (right panel). (C) Fluorescence images (scale bar = 1 mm) of fresh blood isolated from orthotopic CRC tumor-bearing mice for the presence of green fluorescent protein (GFP)-labeled tumor cells (left panel). Quantification of GFP-labeled tumor cells (right panel). (D) Fluorescence images of liver metastases of orthotopic CRC tumor-bearing mice (left panel). Quantification of liver metastases (right panel). (E) Bright field imaging (scale bar = 2 mm) of the lungs from SW480 and SW620 tumor-bearing mice treated with miR-221* and miR-224 mimics or miR-221* and miR-224 inhibitors at 4 weeks after tail vein injection. Arrows indicate lung metastases. (F) H&E staining of the lungs from tail vein–injected mice (left panel). Quantification of lung metastases (right panel). *P < .05; **P < .01; ***P < .001. Data are representative of 3 experiments in triplicate. Image data are representative for each group (n = 7 orthotopic group or n = 6 tail vein injection group). Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 miR-221* and miR-224 inhibit tumor metastasis by down-regulating cell migration and motility without inhibiting tumor cell growth. (A) Invasion assay showing the invasion ability of SW620 cells transfected with miR-221* mimic, miR-224 mimic, or MBD2 small interfering RNA and SW480 cells transfected with miR-221* inhibitor, miR-224 inhibitor, or pMyc-MBD2 plasmid. (B) Migration assay showing the migration ability of SW620 and SW480 cells treated as in A. (C) Growth curves of SW620 cells and SW480 cells treated as in A. (D) Flow cytometric distributions of SW620 and SW480 cells treated as in A. *P < .05, **P < .01. All data are representative of 3 experiments in triplicate. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 MBD2 is a target of miR-221* and miR-224. (A) MBD2 3′ UTRs contain one predicted miR-221* binding site and one predicted miR-224 binding site. The figure shows predicted duplex formations between MBD2 3′ UTR (bottom panel) and miR-221* or miR-224 (middle panel). The sites of target mutagenesis (top panel) are indicated in red. (B) Real-time RT-PCR of miR-221* and miR-224 in SW620 and SW480 cells after enforced expression or down-regulation of miR-221* and 224. (C) pGL3-MBD2 luciferase constructs, containing wild-type (left side of the histograms) or mutated (right side of the histograms) MBD2 3′ UTRs, were transfected into SW620 and SW480 cells. Next, SW620 cells were cotransfected with miR-221* or miR-224 mimic, whereas SW480 cells were cotransfected with miR-221* or miR-224 inhibitor. Relative repression of firefly luciferase expression was standardized to a transfection control. (D) Western blot showing MBD2 expression in SW620 and SW480 cells after miR-221* or miR-224 overexpression or down-regulation. β-actin was used for loading control. (E) Quantitative RT-PCR of MBD2 mRNA in SW620 and SW480 cells after miR-221* or miR-224 overexpression or down-regulation. All data are representative of 3 experiments in triplicate. (F) CRC and LNM tissues were analyzed for miR-221* and miR-224 (blue) expression by in situ hybridization, followed by immunohistochemistry for MBD2 (brown). MiR-159, which is not expressed in colon, was used as negative control. Scale bars = 25 μm. *P < .05, **P < .01, ***P < .001. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 MiR-221* and miR-224 increased the expression of maspin through MBD2 down-regulation. (A) Western blot showing MBD2 and maspin expression in SW620 and SW480 cells after miR-221* or miR-224 overexpression or down-regulation. β-actin was used for loading control. (B) Levels of MBD2 and maspin were assessed by Western blot in SW620 and SW480 cells after MBD2 down-regulation or overexpression. β-actin was used for loading control. (C) miR-221*, miR-224, and MBD2 were overexpressed or down-regulated in SW620 and SW480 cells. Moreover, SW620 and SW480 cells were treated with DAC (5 μmol/L) for 48 hours, TSA (0.5 μmol/L) for 24 hours, or both (DAC for 24 hours, followed by TSA for an additional 24 hours). Levels of maspin mRNA were assessed in treated SW620 and SW480 cells by quantitative RT-PCR. (D) SW620 and SW480 cells were treated as in C. Genomic DNA extracted from the cells was treated with bisulfite and then subjected to methylation-specific PCR using the methylated DNA-specific (M) and unmethylated DNA-specific (U) primer sets. (E) PCR products were sequenced for the 19 CpG sites located between nucleotides −194 and +105 of the maspin promoter. The horizontal dots represent CpG islands, and the vertical dots represent the individual 5 clones sequenced. Each black dot represents a methylated cytosine residue within the CpG islands. All data are representative of 3 experiments in triplicate. *P < .05, **P < .01. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 7 MBD2 down-regulates maspin in association with HDAC1. (A) Chromatin immunoprecipitation assay of MBD2 and HDAC1 on the promoter of maspin gene in SW620 cells. Unspecific immunoglobulin G antibody was used as negative control. Quantification of binding was represented as fold change to control immunoglobulin G. (B) miR-221*, miR-224, and MBD2 were overexpressed or down-regulated in SW620 cells. Moreover, SW620 cells were treated with DAC (5 μmol/L) for 48 hours, TSA (0.5 μmol/L) for 24 hours, or both (DAC for 24 hours, followed by TSA for an additional 24 hours). Chromatin immunoprecipitation assay of MBD2 or HDAC1 on the promoter of maspin gene was then performed. (C) MBD2 and HDAC1 were detected by Western blot in nuclear extract (Input) and in complexes that were immunoprecipitated with anti-MBD2 and anti-HDAC1 antibodies. All data are representative of 3 experiments in triplicate. **P < .01; ***P < .001. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 1 Key findings of miRNA microarray expressing profiling analysis. Heat map representation of differentially expressed miRNAs in SW620 cells compared with SW480 cells. Red indicates up-regulated miRNAs, and green indicates down-regulated miRNAs. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 2 Real-time PCR on normal colon/primary cancer/LNM samples from 20 patients with CRC. The association between miR-221* and miR-224 expression in the normal class, tumor class, and metastasis class was calculated statistically using the Pearson correlation coefficient (r) and the respective P value, all significant at P < .05. The Pearson correlation of miR-221* and miR-224 expression indicated an inverse relation between tumor class and metastasis class. However, the expression of miR-221* and miR-224 in the normal class is not correlated with their expression in the tumor class. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 3 MiR-221* and miR-224 are correlated with tumor stages. Real-time RT-PCR of miR-221* and miR-224 in human CRC samples. Clinical samples were classified into 2 subsets: stages I/II and stages III/IV. Paired-sample t test was applied to compare the average expression levels of miR-221* and miR-224 between subsets (P < .001). Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 4 Enforced expression of miR-221* and miR-224 prolonged the survival times of SW480 and SW620 tumor-bearing mice. (A) Real-time RT-PCR of miR-221* and miR-224 in subcutaneous CRC tumors of SW480 and SW620 tumor-bearing mice treated with miR-221* and miR-224 mimics or miR-221* and miR-224 inhibitors. P values were obtained using Student t test. (B) Kaplan–Meier analysis and log-rank test of subcutaneous CRC tumor-bearing mice. (C) Real-time RT-PCR of miR-221* and miR-224 in orthotopic CRC tumors of SW480 and SW620 tumor-bearing mice treated with miR-221* and miR-224 mimics or miR-221* and miR-224 inhibitors. (D) Kaplan–Meier analysis and log-rank test of orthotopic CRC tumor-bearing mice. (E) Kaplan–Meier analysis and log-rank test of tail vein–injected mice. **P < .01; ***P < .001. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 5 Down-regulation of miR-221* and miR-224 promoted the lymphogenous metastasis in nude mice models. (A) Effects of treatment on incidence of LNM. (B) Quantification of metastatic burden in lymph nodes from SW480 and SW620 tumor-bearing mice treated with miR-221* and miR-224 mimics or miR-221* and miR-224 inhibitors. Data are mean ± SEM; n = 6. *P < .05, **P < .01 by Student t test. (C) H&E staining of lymph node tissue from mice. Dotted lines indicate micrometastatic foci. Scale bar = 100 μm. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 6 MBD2 expression is inversely correlated with miR-221* and miR-224 in CRC cells. (A) Western blot of MBD2 with protein extracts isolated from different CRCs. β-actin was used for loading control. (B) Quantitative RT-PCR of miR-221* and 224 and MBD2 mRNA with RNAs isolated from different CRC cells. Average values and SDs were calculated from triplicate samples. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 7 MBD2 expression is inversely correlated with miR-221* and miR-224 in clinical CRC specimens. Quantitative RT-PCR on 8 colorectal cancer tissues and 8 LNM. Association between miR-221*/miR-224 and MBD2 mRNA was statistically analyzed using the Pearson correlation coefficient (r) and P value (all significant, P < 0.05). Correlation between miR-221* and MBD2: r = −0.758, P < .001. Correlation between miR-221* and maspin: r = 0.669, P = .034. Correlation between miR-224 and MBD2: r = −0.686, P = 0.016. Correlation between miR-224 and maspin: r = 0.711, P = .004. Correlation between MBD2 and maspin: r = −0.795, P < .001. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 8 MiR-221* and mir-224 have an additive role to control MBD2 transcription. MBD2 expression in primary tumor tissues of patients with CRC is determined by real-time PCR. Patients with CRC were divided into 4 groups according to their miR-221* and miR-224 densities: group I (n = 6), high miR-221* and miR-224 density; group II (n = 6), high miR-221* but low miR-224 density; group III (n = 6), low miR-221* but high miR-224 density; and group IV (n = 6), low miR-221* and miR-224 density. *P < .05, ***P < .001 by Student t test. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 9 A mechanism for miR-221*- and miR-224-mediated inhibition of metastasis. Maspin blocks the functional activation of HDAC1 through interaction with HDAC1. MBD2 can bind to HDAC1, leading to the detachment of maspin from HDAC1. MBD2 and HDAC1 then form a complex and repress the transcription of maspin gene in the nucleus. MiR-221* and miR-224 can increase the expression of maspin to inhibit metastasis by directly targeting MBD2. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 10 MiR-221 expression in CRC cells and clinical samples. (A) Real-time PCR of miR-221 in SW480 and SW620 cells. (B) Real-time PCR of miR-221 on normal colon/primary cancer/LNM samples of patients with CRC (n = 20). P values were obtained using Student t test. *P < .05; ***P < .001. Gastroenterology 2013 145, 853-864.e9DOI: (10.1053/j.gastro.2013.06.008) Copyright © 2013 AGA Institute Terms and Conditions