Volume 144, Issue 3, Pages 624-635.e4 (March 2013) MicroRNA-137, an HMGA1 Target, Suppresses Colorectal Cancer Cell Invasion and Metastasis in Mice by Directly Targeting FMNL2  Li Liang, Xianzheng Li, Xiaojing Zhang, Zhenbing Lv, Guoyang He, Wei Zhao, Xiaoli Ren, Yuling Li, Xiuwu Bian, Wenting Liao, Wei Liu, Guangying Yang, Yanqing Ding  Gastroenterology  Volume 144, Issue 3, Pages 624-635.e4 (March 2013) DOI: 10.1053/j.gastro.2012.11.033 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 FMNL2 is a target gene of miR-137. (A) miR-137 expressions in 8 CRC cell lines and the 293FT cell line by real-time PCR. (B) FMNL2 expressions in 8 CRC cell lines by Western blot. (C) Luciferase activities of wild-type 3′UTR-FMNL2-luc and mutant 3′UTR-FMNL2-luc constructs in HEK293A, SW620 cells after transfection of miR-137 plasmid. *P < .05. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 miR-137 inhibitor increases FMNL2 expression and promotes cell proliferation and invasion in vitro. (A) FMNL2 expression in SW480 or HCT116 cells treated with miR-137 inhibitor by Western blot. (B) Effect of miR-137 inhibitor on the invasiveness of SW480 and HCT116 cells by Boyden chamber. Morphologic comparison of cells penetrating the artificial basement membrane also was shown. (C) Effect of miR-137 inhibitor on the proliferation of SW480 and HCT116 cells by MTT assay. (D) Effect of miR-137 inhibitor on the proliferation of SW480 and HCT116 cells by colony formation assay. *P < .05. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 miR-137 decreases FMNL2 expression and suppresses cell proliferation and invasion in vitro by targeting FMNL2. (A) miR-137 expression in miR-137 overexpressing or miR-137/FMNL2 co-expressing cells by real-time PCR. (B) FMNL2 expression in miR-137 overexpressing or miR-137/FMNL2 co-expressing cells by Western blot. (C) Effects of miR-137 and miR-137/FMNL2 on cell invasion by Boyden chamber. Morphologic comparison of cells penetrating the artificial basement membrane also was shown. (D) Effects of miR-137 and miR-137/FMNL2 on cell proliferation by MTT assay. (E) Effects of miR-137 and miR-137/FMNL2 on cell proliferation by colony formation assay. *P < .05. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 miR-137 enhances tumor growth and metastasis of CRC in vivo by targeting FMNL2. (A) Fluorescence images of subcutaneous tumors of mice injected with SW620/miR-137 or SW620/miR-137/FMNL2 cells. (B) Effects of miR-137 and miR-137/FMNL2 on subcutaneous tumor growth by MTT assay. (C) Immunohistochemical (IHC) staining of FMNL2 expression in subcutaneous tumors of mice injected with SW620/miR-137 and SW620/miR-137/FMNL2 cells. (D) The whole-body fluorescence images of metastasis in mice injected with SW620/miR-137/FMNL2 cells. (E) Fluorescence images of intestinal and hepatic metastases of mice injected with SW620/miR-137 and SW620/miR-137/FMNL2 cells (n = 5). (F) Number of metastatic intestinal or hepatic nodules per mice. The number of metastatic nodules in individual mice was counted under the microscope. *P < .05. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 miR-137 is regulated directly by the transcription factor HMGA1. (A) Luciferase activity of miR-137-luc construct after transfection of HMGA1 plasmid in SW480 or HEK293A cells. (B) Chromatin immunoprecipitation assay in cells transfected with a vector expressing Myc-HMGA1. PCR was performed with primers specific for 2 binding sites within miR-137 promoter. (C) Expressions of HMGA1 and miR-137 in HMGA1 overexpressing SW480 and SW620 cells by real-time PCR. (D) Expression of FMNL2 protein in HMGA1 overexpressing SW480 and SW620 cells by Western blot. (E) Endogenous expressions of HMGA1 and miR-137 in SW480 and SW620 cells by real-time PCR. *P < .05. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 miR-137 is involved in PI3K/Akt and MAPK/ERK signaling pathways in CRC. (A) Western blot analyses of Akt, MAPK, p-MAPK, p-Akt, and VEGF in SW620/miR-137 and SW620/miR-137/FMNL2 cells. Activities of MMP-2 and MMP-9 also were examined by gelatin zymograph assay. (B) Western blot analyses of FMNL2, VEGF, p-MAPK, and p-Akt in HCT116 cells treated with miR-137 inhibitor, followed by the treatment of U0126 or LY194002. Activities of MMP-2 and MMP-9 also were examined by gelatin zymograph assay. (C) Western blot analyses of Akt, MAPK, p-MAPK, and p-Akt in SW620/mock, SW620/miR-137, SW620/IGF, SW620/miR-137+IGF, SW620/miR-137+LY194002, and SW620/miR-137+U0126 groups. (D and E) Effects of IGF, LY194002, and U0126 treatments on the invasion of SW620/miR-137 cells by Boyden chamber. Morphologic comparison of cells penetrating the artificial basement membrane also is shown. *P < .05, **P < .01, ***P < .001. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Figure 7 Expression correlations of miR-137, HMGA1, and FMNL2 in clinical CRC tissues. (A) miR-137 and HMGA1 expressions in CRC tissues and the corresponding normal mucosa by real-time PCR. (B) Real-time PCR analysis of miR-137 expression in 8 paired CRC tissues. (C) Real-time PCR and (D) Western blot analyses of HMGA1 expression in the same 8 paired CRC tissues. (E) Western blot analysis of FMNL2 expression in the same 8 paired CRC tissues. *P < .05. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 1 Bioinformatic prediction and initial screening of potential miRNAs targeting FMNL2. (A) Bioinformatic prediction of potential miRNAs targeting FMNL2 by 4 common databases. (B) Incomplete complementation of the base of miR-137 or miR-142-3p to the 3′UTR region of FMNL2 mRNA. (C) miR-137 expression was detected by real-time PCR in 4 CRC cell lines. (D) miR-142-3p expression was measured by real-time PCR in 4 CRC cell lines. The relative mRNA levels of miR-137 or miR-142-3p in SW480 cells were normalized to 1. (E) FMNL2 expression was determined by Western blotting in 4 CRC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown as a control. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 2 Construction of mutant 3′UTR-FMNL2-luc vector. (A) The FMNL2 3′UTR fragment was amplified by PCR. (B) Two miR-137 binding sites in the 3'UTR region of FMNL2 were mutated by PCR. (C) The sequence of 2 mutated binding site miR-137 in the 3′UTR region of FMNL2. Gastroenterology 2013 144, 624-635.e4DOI: (10.1053/j.gastro.2012.11.033) Copyright © 2013 AGA Institute Terms and Conditions