MicroRNA-137, an HMGA1 Target, Suppresses Colorectal Cancer Cell Invasion and Metastasis in Mice by Directly Targeting FMNL2

GASTROENTEROLOGY 2013;144:624 – 635

MicroRNA-137, an HMGA1 Target, Suppresses Colorectal Cancer Cell Invasion and Metastasis in Mice by Directly Targeting FMNL2 LI LIANG,1,2,* XIANZHENG LI,1,3,* XIAOJING ZHANG,1,4,* ZHENBING LV,1,2 GUOYANG HE,1,2 WEI ZHAO,1,2 XIAOLI REN,1,2 YULING LI,1,2 XIUWU BIAN,5 WENTING LIAO,1,2 WEI LIU,6 GUANGYING YANG,7 and YANQING DING1,2 1 Department of Pathology, Nanfang Hospital, 2Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong Province, People’s Republic of China; 3Prenatal Diagnosis Center & Genetic Disease Diagnosis and Treatment Center, Guangdong Women and Children Hospital, Guangzhou, Guangdong Province, People’s Republic of China; 4Department of Pathology, Shenzhen University, Shenzhen, People’s Republic of China; 5Institute of Pathology and Southern Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, People’s Republic of China; 6Department of Oncology, Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China; 7Department of Pathology, People’s Hospital of Zhengzhou University, Zhengzhou, Henan Province, People’s Republic of China

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BACKGROUND & AIMS: Formin-like (FMNL)2 is upregulated in colorectal tumors and has been associated with tumor progression, but little is known about regulatory mechanisms. We investigated whether microRNAs regulate levels of FMNL2 in colorectal cancer (CRC) cells. METHODS: We used real-time polymerase chain reaction and immunoblot analyses to measure levels of miR137, high-mobility group AT-hook (HMGA)1, and FMNL2 in CRC cells and tissue samples from patients (n ⫽ 50). We used luciferase reporter assays to determine the association between miR-137 and the FMNL2 3= untranslated region, and HMGA1 and the miR-137 promoter. Chromatin immunoprecipitation assays were used to assess direct binding of HMGA1 to the miR-137 promoter. RESULTS: miR-137 and miR-142-3p were predicted to bind FMNL2 based on bioinformatic data. Only the level of miR-137 had a significant inverse correlation with the level of FMNL2 protein in CRC cell lines and tissues. FMNL2 messenger RNA was targeted by miR-137; expression of miR-137 inhibited proliferation and invasion by CRC cells in vitro, and metastasis to liver and intestine by CRC xenografts in nude mice. HMGA1 bound to the promoter of miR-137 and activated its transcription, which reduced levels of FMNL2 in CRC cells. Ectopic expression of miR-137 in CRC cells inhibited phosphorylation of mitogen-activated protein kinase (MAPK) and Akt, which reduced levels of matrix metalloproteinase 2, matrix metalloproteinase 9, and vascular endothelial growth factor; it also reduced invasiveness of CRC cells, inhibiting signaling via phosphatidylinositol-4,5-bisphosphate 3-kinase, Akt, and MAPK. CONCLUSIONS: Levels of miR-137 and HMGA1 are reduced, and levels of FMNL2 are increased, in CRC samples compared with adjacent normal mucosa. In CRC cells, miR-137 targets FMNL2 messenger RNA and is regulated by the transcription factor HMGA1. Expression of miR-137 reduces CRC cell invasion in vitro and metastasis of tumor xenografts in mice. FMNL2 appears to activate phosphatidylinositol-4,5-bisphosphate 3-kinase, protein kinase B (Akt), and MAPK signaling pathways. Keywords: Mouse Model; Gene Expression; Tumor Suppressor Activity; Signaling Pathway.

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ormins are widely expressed proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling actin exerted through the formin homology domains.1 They frequently are deregulated during pathologic situations such as tumor cell transformation and metastasis.2 Formin-like 2 (FMNL2), a novel member of Diaphanousrelated formins (DRFs), recently was identified3 and, until now, only 6 articles have reported the role of FMNL2 in cancer.4 –9 Our previous study established a specifically enhanced hepatic metastatic subline of human colorectal carcinoma (CRC) SW480 cell line named SW480/M5 by repeated in vivo selection.10,11 The gene expression profiles of highly metastatic M5, SW620 cells, and low metastatic SW480 cells were analyzed by complementary DNA microarray.11 Among substantially up-regulated genes in M5 and SW620 cells, FMNL2 was screened as a potential metastasisassociated gene by using gene clusters with literature profiles (GenCLiP) software (http://www.geneclip.com).12 Overexpression of FMNL2 in CRC tissues and cell lines was found to be associated with invasion and lymphatic metastasis.4,5 We also documented that FMNL2 promoted CRC cell proliferation, motility, in vitro invasion, and in vivo metastasis,6 and enhanced the invasive potential of CRC by inducing epithelial-mesenchymal transition.7 Kitzing et al8 identified FMNL2 as a RhoC effector. In melanoma cells, FMNL2 colocalizes with F-actin dots at the tips of cellular protrusions.9 However, little is known about the underlying mechanisms responsible for the increase of FMNL2 expression in the progression of CRC. MicroRNAs (miRNAs) are small (19 –25 nt), noncoding, regulatory RNAs that regulate gene expression by comple*Authors

share co-first authorship.

Abbreviations used in this paper: Akt, protein kinase B; CRC, colorectal cancer; ERK, extracellular signal–regulated kinase; FMNL2, formin-like 2; HMGA, high-mobility group AT-hook; MAPK, mitogenactivated protein kinase; miRNA, microRNA; MMP, matrix metalloproteinase; mRNA, messenger RNA; NC, negative control; PCR, polymerase chain reaction; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; UTR, untranslated region; VEGF, vascular endothelial growth factor. © 2013 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2012.11.033

mentary base pairing with the 3=-untranslated region (UTR) of target messenger RNAs (mRNAs), causing their degradation or suppressing mRNA translation.13,14 miRNAs also were reported to be transcribed by RNA polymerase II to produce a primary microRNA (primiRNA). This process also has been reported to be regulated by known transcription factors.15 miRNAs have been implicated in cancer and metastasis by targeting oncogenes or tumor-suppressor genes, thus establishing them as a relatively new and important class of oncogenes or tumor suppressors.16,17 Although miRNAs have been the subject of extensive research in recent years, the molecular regulatory mechanisms of miRNAs and their effects on cancer are not well understood. Herein, we report miRNA-mediated repression as a potential mechanism for increased FMNL2 expression in CRC. We provide evidence that microRNA-137 (miR-137), induced by its upstream transcription factor high mobility group protein (HMGA1), can suppress CRC invasion and metastasis by targeting FMNL2, at least in part through inhibiting phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase (ERK) pathways.

Materials and Methods Construction of Plasmids and Transfection MiR-137 lentivirus-expressing vector pEZX-MR01/miR137 containing the enhanced green fluorescent protein (EGFP) gene (GeneCopier, Rockville city, MD) was transfected into lentiviral packaging cell lines 293T. Then 1 mL of viral supernatant containing 4 Attogram (Ag) of polybrene was added into CRC cell lines for stable transduction. After 14 days, puromycinresistant cell pools were established. pGC FU-GFP-LVFMNL2-CT lentiviral-expressing vector constructed in our previous study contains functional FMNL2-CT fragment without the 3=UTR region, which was used to show FMNL2 as a positive regulator of CRC invasion and metastasis.6 To obtain the mir137/FMNL2 co-expressing cells, 3 ␮L of FMNL2-CT lentivirusexpressing vector concentrated solution was added into mir-137 overexpressing SW620 and Lovo cells. Five to approximately 8 ␮g/mL of polybrene then was mixed in those cells. After 72 hours, Western blot was performed to detect the expression of FMNL2. Cells were transfected with miR-137 inhibitor or inhibitor negative control (NC) by using Lipofectamine 2000 (Invitrogen, Foster city, CA).

Methyl Thiazolyl Tetrazoliym Assay The cells were seeded in 96-well plates (1 ⫻ 104 cells/mL) with 100-␮L cell suspension in each well and incubated for 7 days. Methyl thiazolyl tetrazoliym (MTT) assay was performed by adding 20 ␮L of MTT (5 mg/mL; Promega, Madison, WI) for 4 hours until a purple precipitate was visible. Precipitates were dissolved in 150 ␮L of dimethyl sulfoxide. The absorbance value of each well was measured with a microplate reader set at 570 nm. Each experiment was repeated 3 times.

Plate Colony Formation Test About 1 ⫻ 102 cells were added to each well of a 6-well culture plate. The cells were incubated at 37°C for 14 days, then

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were washed twice with phosphate-buffered saline and stained with Giemsa solution. The number of colonies containing 50 cells or more was counted under a microscope (plate clone formation efficiency ⫽ [number of colonies/number of cells inoculated] ⫻100%).

In Vitro Invasion Assay The Boyden invasion chambers were rehydrated with RPMI 1640 (serum-free) for 2 hours at 37°C. RPMI 1640 with 100 mL/L fetal bovine serum was added to the lower compartment as the chemotactic factor. Then 1.5 ⫻ 105 tumor cells in serum-free Dulbecco’s modified Eagle medium were added to the upper compartment of the chamber. After incubation for 48 hours, the noninvasive cells were removed with a cotton swab. Cells that had migrated through the membrane and stuck to the lower surface of the membrane were stained with hematoxylin and counted under a light microscope in 5 random visual fields (200⫻). Each experiment was repeated 3 times.

Animal Models To evaluate in vivo tumor growth, 1 ⫻ 107 cells were injected subcutaneously into the left flank or right flank of nude mice (n ⫽ 6 per group). Then the fluorescence emitted by cells was collected and imaged through a whole-body GFP imaging system (Lighttools, Encinitas, CA). Tumors were measured with calipers to estimate volume from day 5 to day 28 after injection. For orthotropic metastasis assay, nude mice were anesthetized and their cecum was exteriorized by laparotomy (n ⫽ 5 per group). The subcutaneous tumors were cut into small pieces and embedded into the mesentery at the tail end of cecum. After injection, the gut was returned to the abdominal cavity and closed with surgical drapes. Six weeks later, the mice were killed and all organs were removed for examination. Hepatic and intestinal metastases were detected by H&E staining and quantified by counting metastatic lesions in each section.

Luciferase Activity Assay For the binding of miR-137 to FMNL2 3=UTR, the 3=UTR segment of the FMNL2 gene was amplified by polymerase chain reaction (PCR) and inserted into the vector. A mutant construct in 2 mir-137 binding sites of FMNL2 3=UTR region also was generated using Quick Change Site-Directed Mutagenesis Kit (Agilent, Roseville City, CA). Co-transfections of FMNL2 3=UTR or mutFMNL2-2 3=UTR plasmid with miR-137 lentivirus vector into the cells were accomplished by using Lipofectamine 2000 (Invitrogen). For the binding of HMGA1 to miR-137 promoter, the coding region of HMGA1 and the 1-kb region directly upstream of the miR-137 transcription binding site were amplified by polymerase chain reaction and then inserted into the vectors, respectively. Luciferase activity was measured 48 hours after transfection by the Dual-Luciferase Reporter Assay System (Promega). Each assay was repeated in 6 independent experiments.

Chromatin Immunoprecipitation Assay According to the chromatin immunoprecipitation assay kit (Millipore, Bedford, MA) protocol, HMGA1-expressing SW480 cells were lysed using sodium dodecyl sulfate lysis buffer and DNA was sheared by sonication to lengths between 200 and 1000 base pairs. Protein-DNA complexes were precipitated by anti-HMGA1 antibody (Abcam, Boston City, MA) and control IgG, respectively, followed by the elution of the complex from the antibody. Cross-links in protein-DNA complexes then were

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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 realtime PCR. (B) FMNL2 expressions in 8 CRC cell lines by Western blot. (C) Luciferase activities of wild-type 3=UTRFMNL2-luc and mutant 3=UTRFMNL2-luc constructs in HEK 293A, SW620 cells after transfection of miR-137 plasmid. *P ⬍ .05.

reversed by NaCl. PCR was performed with primers specific for human miR-137. All authors were involved in writing the paper and had final approval of the submitted and published versions.

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Results Bioinformatic Prediction and Initial Screening of Candidate miRNAs Targeting FMNL2 To associate miRNAs with the regulation of FMNL2 expression, a bioinformatics search was performed for potential miRNAs targeting mRNA of FMNL2 by using 4 common databases such as microRNA.org, TargetScan, Pictar, and miRanda. At least 3 of the databases predicted miR-137 and miR-142-3p as the potential miRNAs to target FMNL2 (Supplementary Figure 1A). The base of these 2 miRNAs incompletely complemented to the target 3=UTR region of FMNL2 mRNA (Supplementary Figure 1B). The expression levels of miR-137 and miR-142-3p were assessed in highly metastatic Lovo, SW620 cell lines, and lowly metastatic HT29, SW480 cell lines. Real-time PCR results showed SW620 and Lovo cells expressing lower miR-137 compared with HT29 and SW480 cells (P ⬍ .05; Supplementary Figure 1C). The expression of miR-142-3p was highest in SW620 cells, gradually decreasing in SW480, HT29, and Lovo (Supplementary Figure 1D). Western blot analyses showed upregulation of FMNL2 in SW620 and Lovo cells compared with HT29 and SW480 cells (Supplementary Figure 1E). Because miRNAs are believed to function by inhibiting translation of their mRNA targets, these observations imply that miR-137 might be a negative regulator of FMNL2.

Inverse Correlation Between miR-137 Expression and FMNL2 Protein Amounts in 8 CRC Cell Lines The expression levels of miR-137 and FMNL2 protein in a series of CRC cell lines were determined to

ascertain whether miR-137 could functionally affect FMNL2 expression. MiR-137 expression was highest in HCT116 cells, followed by SW480 (P ⬍ .05; Figure 1A). In the cell lines SW620, Lovo, and Caco-2, with no or very low endogenous miR-137 expression, a high amount of FMNL2 protein was observed, whereas HCT116, SW480, and Cola205 cells with high miR-137 expression showed no or weakly expressed FMNL2 protein (Figure 1B). Across the 8 cell lines tested we detected a significant inverse correlation between miR137 and FMNL2 protein levels (P ⬍ .01; Spearman correlation, ␥ ⫽ -0.753).

The 3=UTR Region of FMNL2 mRNA Is a Direct Target of miR-137 To confirm whether miR-137 directly targets the 3=UTR region of FMNL2, we subcloned a 1949-bp fragment of the 3=UTR region of FMNL2 mRNA that included the predicted miR-137 recognition site and then inserted it into a luciferase reporter plasmid (Supplementary Figure 2A). Two miR-137 binding sites in the 3= UTR region of FMNL2 were mutated to obtain 3=UTR-MutFMNL2-2-luc plasmid (Supplementary Figure 2B and C). 293FT and SW620 cells were chosen for transient transfection. Transient transfection of wildtype FMNL2-luc reporter with miR-137– expressing vector into 293FT and SW620 cells led to a significant decrease in luciferase activity compared with NC or blank control (P ⬍ .01). However, miR-137 could not decrease the luciferase activity of a mutant construct3=UTR-MutFMNL2-2-LUC in the miR-137 binding site compared with NC or blank (P ⬎ .05; Figure 1C). The results make it evident that miR-137 affects FMNL2 expression by directly binding to the 3=UTR region of FMNL2 and validate that FMNL2 is a direct target of miR-137.

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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.

Inhibition of miR-137 Led to Increased FMNL2 Expression and Increased Invasive or Proliferative Capacity of CRC Cells In Vitro A loss-of-function assay was performed to ascertain whether miR-137 could functionally target FMNL2, thereby regulating invasion and metastasis in CRC. Transfection of SW480 and HCT116 cells with miR-137 inhibitor caused increased FMNL2 protein expression (Figure 2A) and consequently enhanced cell invasiveness compared with parental or NC cells (P ⬍ .001; Figure 2B). MiR-137 inhibitor–treated SW480 and HCT116 cells showed higher proliferative capacities than parental or NC cells by MTT assay (P ⬍ .001; Figure 2C). MiR-137 inhibitor also strongly impaired the ability to form colonies in 2 cell lines (P ⬍ .01; Figure 2D). These data clearly substantiate that down-regulation of miR-137 contributes to enhanced FMNL2 expression, cell proliferation, and invasiveness in vitro.

Ectopic Expression of miR-137 Decreases the Invasive or Proliferative Capacity of CRC Cells In Vitro by Down-Regulating FMNL2 The functional effect of miR-137 overexpression on cell behaviors in vitro in CRC cells lines was assessed by transfecting MiR-137 lentivirus-expressing vector into SW620 and Lovo cell lines and generating stable transfectants. To ascertain whether a reduced FMNL2 level ac-

counts for the changes of cell behaviors observed after miR-137 expression, a construct encoding the entire FMNL2-CT coding region, but lacking the 3=UTR of FMNL2, specifically was established, yielding an mRNA resistant to miR-137–mediated inhibition of translation. Real-time PCR showed that miR-137 expression in SW620/miR-137 or Lovo/miR-137 cells was higher than that in mock or blank cells (P ⬍ .05), but there was no significant difference when compared with miR-137/ FMNL2 cells (P ⬎ .05; Figure 3A). Western blot results revealed a sharp decrease of FMNL2 protein in SW620/ miR-137 or Lovo/miR-137 cells, whereas co-transfection of miR-137/FMNL2 rescued FMNL2 expression (Figure 3B). In the Matrigel (BD Biosciences, Franklin lakes, NJ) invasion assay, SW620/miR-137 and Lovo/miR-137 cells showed significantly less invasiveness compared with mock or blank cells (P ⬍ .001). Strikingly, exogenous FMNL2 expression almost completely rescued the invasive phenotype in SW620/miR-137 and Lovo/miR-137 cells (P ⬍ .001; Figure 3C). In both cell lines, ectopic expression of miR-137 had an inhibitory effect on proliferation in vitro as evidenced by MTT (P ⬍ .001; Figure 3D) and colony formation assays (P ⬍ .001; Figure 3E). The resulting constitutive expression of FMNL2 also rescued miR-137–induced inhibition on cell proliferation (P ⬍ .001). These data make it obvious that down-regulation of FMNL2 is necessary for

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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.

miR-137–mediated repression of invasion and proliferation in vitro.

Ectopic Expression of miR-137 Inhibits Tumor Growth and Metastasis by Orthotropic Implantation In Vivo To assess the effect of miR-137 on tumor growth in vivo, SW620/miR-137 cells, SW620/mir-137/FMNL2 cells, and control cells were implanted subcutaneously into nude mice, and the growth of resultant primary tumors then was monitored. Mice injected with SW620/miR-137 cells developed smaller tumors than those injected with control or miR-137/FMNL2 cells (P ⬍ .01). Tumors in mice injected with miR-137/FMNL2 cells showed no significant difference from blank cells (P ⬎ .05; Figure 4A and B). Immunohistochemistry staining verified positive expression of FMNL2 in the miR-137/FMNL2–xenografted tumors compared with negative expression in miR-137–xenografted tumors (Figure 4C). To determine the effect of miR-137 on metastasis of CRC in vivo, SW620/miR-137 cells, SW620/miR-137/ FMNL2 cells, SW480/FMNL2 cells, and control cells were

implanted into the cecum terminus, and the organs subsequently were scanned for metastasis by a whole-body visualization system. The results showed metastatic lesions only in the intestine and liver (Figure 4D and E). In the group of mice injected with SW620/miR-137 cells, only 20% (1 of 5) and 40% (2 of 5) of mice had hepatic and intestinal metastases, respectively. However, 3 of 5 mice injected with miR-137/FMNL2 or SW620/mock cells had hepatic metastatic lesions, and all mice had extensive intestinal metastases (Figure 4D). Sixty percent (3 of 5) and 80% (4 of 5) of mice injected with SW480/FMNL2 had hepatic and intestinal metastases, respectively, whereas only 20% (1 of 5) of mice injected with SW480/mock cells had intestinal metastases. Many large hepatic metastatic nodules were discovered in miR-137/FMNL2, SW620/mock, and SW480/ FMNL2 groups, yet a few small nodules were detected in the SW620/miR-137 group (Figure 4E). The number of hepatic or intestinal metastatic lesions in mice injected with SW620/miR-137 cells obviously was reduced compared with control cells (P ⬍ .05), whereas

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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.

enhanced expression of FMNL2 increased the number of hepatic or intestinal metastases of mice and blocked the inhibitory effects of miR-137 (P ⬍ .05; Figure 4F). Based on these results it would be reasonable to conclude that miR-137 inhibits tumor growth and metastasis in vivo by down-regulating FMNL2.

MiR-137 Is Regulated Directly by the Transcription Factor HMGA1 To understand how miR-137 expression was regulated by transcription factor, we analyzed the 1-kb region directly upstream of miR-137 and found the presence of the 4 most possible binding motifs for HMGA1 within the

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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.

⫺892 to ⫺877, ⫺890 to ⫺875, ⫺679 to ⫺664, and ⫺667 to ⫺662 regions in the promoter of miR-137. The miR137 promoter was subcloned into a pGL3-basic vector, and a dual-luciferase reporter assay was performed to study the functionality of interaction between HMGA1 and miR-137. It was observed that transient expression of HMGA1 effectively stimulated transcription of miR-137 in SW480 and HEK293A cells (P ⬍ .01; Figure 5A). Then the chromatin immunoprecipitation assay was used to determine whether HMGA1 was recruited at these binding sites. Because ⫺892 to ⫺877, ⫺890 to ⫺875, ⫺679 to ⫺664, and ⫺667 to ⫺662 regions in the promoter of miR-137 were overlapping, the interactions of HMGA1 within the region of ⫺892 to ⫺875 (binding site 1) and ⫺679 to ⫺662 (binding site 2) were examined, with the results that HMGA1 could bind the region of ⫺892 to ⫺875 in the promoter of miR-137 (Figure 5B). Transient expression of HMGA1 led to increased expression of miR-137, with decreased expression of FMNL2 in SW480 and SW620 cells (Figure 5C and D). Finally, we analyzed the levels of HMGA1, miR-137, and FMNL2 in SW480 cells (weakly metastatic cells), and SW620 (highly invasive and metastatic cells). The FMNL2 protein level progressively was up-regulated from SW480 (weakly metastatic cells) to SW620 cells (highly invasive and metastatic cells) (Figure

1B), whereas miR-137 and HMGA1 levels were down-regulated from SW480 to SW620 cells (Figure 5E). These results suggest that HMGA1 up-regulates the level of miR137 and consequently affects the functions of the miR137–FMNL2 pathway in CRC cells.

MiR-137 Is Involved in PI3K/Akt and MAPK/ERK Signaling Pathways in CRC HMGA1 participates in cellular invasion through PI3K/Akt-dependent modulation of matrix metalloproteinase (MMP)-9 activity.18 MAPK and PI3K signaling pathways evidently regulate thrombin-induced migration and MMP-9 expression of glioma cells.19 Thus, we wondered whether miR-137 participates in the invasion and metastasis of CRC by activating or inhibiting PI3K/Akt and MAPK/ ERK pathways. Results of Western blot showed low phosphorylation levels of p-Akt and p-MAPK, with down-regulation of MMP-9, MMP-2, and vascular endothelial growth factor (VEGF) in SW620/miR-137 cells compared with blank or mock cells, with no change in the total protein amount of Akt and MAPK. However, ectopic expression of FMNL2 rescued those inhibitory effects (Figure 6A). miR-137 inhibitor transfected HCT116 cells and then they were treated with MAPK/MAP kinase kinase (MEK) inhibitor U0126 or PI3K/ Akt inhibitor LY294002 for 2 days, which showed high

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phosphorylation levels of p-Akt and p-MAPK, with up-regulation of MMP-9, MMP-2, and VEGF. U0126 treatment inhibited activation of p-MAPK, but not p-Akt, whereas treatment with LY294002 resulted in opposite effects. Inhibitors of both MAPK and Akt significantly blocked the activities of MMPs and VEGF (Figure 6B). We further examined the involvement of 2 pathways in CRC cell invasion induced by miR-137. Because both PI3K/Akt and MAPK signaling cascade are known to be involved in the transmission of IGF-1/IGF-1R signaling,20,21 recombinant IGF-1 was used to activate PI3K/Akt and MAPK/ERK signaling pathways simultaneously in CRC cells. IGF-1 stimulation increased the phosphorylation levels of p-Akt and p-MAPK in SW620/ mock and SW620/miR-137 cells, respectively, with no change in the total protein amount of Akt and MAPK. Moreover, treatment with IGF-1 in SW620/miR-137 cells promoted activations of p-Akt and p-MAPK compared with LY294002 or U0126 treatment (Figure 6C). Results of Boy-

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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 pAkt in SW620/mock, SW620/ miR-137, SW620/IGF, SW620/ miR-137⫹IGF, SW620/miR-137 ⫹LY194002, and SW620/miR137⫹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.

den chamber experiments showed that IGF-1 stimulation significantly enhanced cell invasiveness in SW620/mock and SW620/miR-137 cells (P ⬍ .05; Figure 6D and E). LY294002 or U0126 treatment could not inhibit the invasiveness of SW620/miR-137 cells (P ⬎ .05), but showed reduced cell invasiveness compared with IGF-1 treatment (P ⬍ .01; Figure 6D and E). All these results certainly indicate an inhibitory role of miR-137 in CRC invasion, at least in part by inhibiting PI3K/Akt and MAPK/ERK pathways, followed by the suppression of MMP-9, MMP-2, and VEGF.

Correlations of miR-137 With HMGA1, FMNL2 Expressions, Tumor Grade, and Metastatic Status in Clinical CRC Tissues The expressions of miR-137, HGMA1, and FMNL2 were detected in a matched collection of 26 lymph node– positive and 24 lymph node–negative human CRC tissues. miR-137 expression obviously was lower in CRC tissues

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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.

than adjacent normal mucosa by real-time reverse-transcription PCR (P ⬍ .01; Figure 7A and B). However, there was no significant difference in miR-137 expression between CRC tissues with lymphatic metastasis and those without lymphatic metastasis (P ⬎ .05; Figure 7A). The relationship between the relative miR-137 expression and patients’ clinical characteristics is shown in Supplementary Table 1. As evident from the analyzed data, miR-137 expression did not appear to be related to age, sex, tumor size, serosal invasion, differentiation, lymphatic metastasis, lymph node ratio, or Duke stage (P ⬎ .05). Results of real-time reverse-transcription PCR and Western blot showed that HMGA1 was down-regulated significantly in CRC tissues compared with adjacent normal mucosa (P ⬍ .01; Figure 7A–D). There was a positive relationship between the miR-137 and HMGA1 expression levels by Spearman correlation analysis (r ⫽ 0.306; P ⬍ .01). FMNL2 protein expression was investigated by Western blot and found to be up-regulated in 42 cases of CRC

tissue samples compared with adjacent normal mucosa (Figure 7E). Only 8 CRC tissues showed almost identical or down-regulated FMNL2 compared with adjacent normal mucosa. Spearman correlation analysis showed a negative relationship between the miR-137 expression level and the FMNL2 protein level (r ⫽ ⫺0.723; P ⬍ .05). These data verify that HMGA1 induces miR-137 expression and consequently suppresses its direct target HMGA1. The decreased expression of miR-137 can be one of the causes of high expression of FMNL2 in CRC tissues.

Discussion Our study investigated the potential involvement of a miRNA-mediated mechanism in increased expression of FMNL2 in CRC. We performed a bioinformatics search for potential miRNAs targeting FMNL2 mRNA by using 4 common databases, and identified miR-137 and miR-142-3p with highest predictive scores. Although bioinformatics

databases have played a central role in predicting miRNA targets, virtually all available programs generate some levels of false predictions.13 Therefore, we further investigated the expressive tendency between these 2 miRNAs and FMNL2 protein in 4 CRC cell lines. Only miR-137 expression seemed to have an inverse correlation with FMNL2 protein level and metastatic potential of CRC cells. Because most miRNAs are believed to function by inhibiting translation of their mRNA targets,16 we proposed that mir-137 might be a novel negative regulator for FMNL2. Luciferase activity assay confirmed that miR-137 directly could target the 3=UTR of FMNL2. Because a single miRNA potentially can target many genes, our study added FMNL2 as one more bonafide target of miR-137, which also has been implicated in targeting Cdc42, CDK6, MITF, and CtBP1.22–25 To date, down-regulation of miR-137 has been found in several different tumor types, including gastric cancer, glioblastoma multiforme, melanoma, and CRC.22,25–27 Aberrant epigenetic regulation of miR-137 promoter, such as by DNA hypermethylation and/or histone modification, may represent a key mechanism of down-regulation of miR-137 in several human cancers.26 –29 Moreover, methylation silencing of miR-137 in colorectal adenomas suggests it to be an early event, which has prognostic and therapeutic implications.27 However, the association of miR-137 with tumor metastasis has not yet been reported. Gain-of-function and loss-of-function assays were performed to assess the effect of miR-137 on CRC invasion and metastasis. Results showed that silencing of miR-137 up-regulated FMNL2 and strengthened cell proliferation and invasion in vitro whereas overexpression of miR-137 inhibited FMNL2 expression as well as cell proliferation, invasion in vitro. MiR-137 was reported to induce cellcycle G1 arrest and inhibit invasion in cancer cells by targeting Cdc42,22 which could explain the inhibition of miR-137 in proliferating cancer cells. Xenograft tumor experiments provided additional support for involvement of miR-137 in inhibiting CRC cell growth and hepatic or intestinal metastasis in vivo. Our previous study had identified FMNL2 as a positive regulator of cell invasion and metastasis of CRC.4,6 The in vitro and in vivo FMNL2 rescue experiments proved that miR-137 regulated invasion and metastasis of CRC cells mainly by targeting FMNL2. Hence, miR-137 is an important suppressor oncomir in CRC invasion and metastasis, and FMNL2 seems to be a major downstream effector of miR-137 in its target network. miRNAs have been shown to be regulated by the upstream transcription factors.13,30 Our observation that miR-137 was down-regulated in highly metastatic CRC cell lines raised the possibility of transcription factors activating miR-137 expression. We analyzed the promoter region of miR-137 and found the 4 most possible binding motifs of transcription factor HMGA1 within the promoter region of miR-137. Because HMGA1 has been shown to participate in cancer metastasis31,32 and FMNL2 expression is up-regulated during the progression of CRC

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cells to more invasive phenotypes,6 we reasoned that HMGA1 might have a role in miR-137 expression and in turn FMNL2 regulation. Results verified that HMGA1 stimulated the transcription activity of miR-137 by binding directly to the promoter of miR-137, leading to upregulation of miR-137 expression and down-regulation of FMNL2. Thus, miR-137 is a target regulated by the transcription factor HMGA1. Having established miR-137 as a metastasis suppressor miRNA in CRC, we investigated possible pathways by which miR-137 could be involved in CRC. FMNL2 is known to drive tumor cell motility as a downstream effector of RhoC.8 RhoC promotes tumor metastasis in prostate cancer by sequential activation of Pyk2, FAK, MAPK, and Akt, followed by up-regulation of MMP-2 and MMP-9.33 RhoC expression has been correlated positively with VEGF and MMP-9 in ovarian cancer.34 PI3K/Akt and MAPK/ERK signaling pathways have important implications in modulating tumor cell proliferation, invasion, epithelial-mesenchymal transition, and metastasis.35,36 It also is well established that MMP-2 and MMP-9 have important functions in acceleration of cancer invasion and metastasis.37 We hypothesized that miR-137 might participate in invasion and metastasis of CRC by inhibiting the PI3K/Akt or MAPK/ERK signaling pathways. Results validated that miR-137 resulted in low phosphorylation levels of p-Akt and p-MAPK, followed by the suppression of MMP-9, MMP-2, and VEGF. Ectopic expression of FMNL2 was sufficient to rescue the suppression induced by miR-137. IGF-1 has been shown to be implicated in tumor progression in a variety of human neoplasms.19 Its mitogenic and anti-apoptotic signal is mediated preferentially through the IGF-1 receptor, a tyrosine kinase linked to the PI3K/Akt and MAPK/ERK pathways.20 Our results showed that IGF-1 stimulation increased the phosphorylation levels of p-Akt and p-MAPK, and subsequently enhanced cell invasiveness in SW620/mock and SW620/miR-137 cells. Therefore, miR137 inhibits PI3K/Akt and MAPK/ERK signaling pathways in CRC invasion, followed by the suppression of MMP-9, MMP-2, and VEGF. Finally, we detected the expression correlations of miR137, HMGA1, and FMNL2 in the same 50 paired cases of human CRC tissues. Expressions of miR-137 and HMGA1 were up-regulated, whereas FMNL2 was up-regulated in CRC tissues. There was a positive relationship between the miR-137 and HMGA1 expression levels, and a negative relationship between miR-137 and FMNL2. This evidence clearly validates that HMGA1 induces miR-137 expression and consequently suppresses its direct target FMNL2 in CRC tissues. To conclude, we show a novel regulatory mechanism of FMNL2 expression in CRC wherein transcription factor HMGA1 induces expression of miR-137, which suppresses its direct target FMNL2, in turn regulating CRC invasion and metastasis. MiR-137 negatively regulates invasion and metastasis of CRC cells, at least in part by inhibiting PI3K/Akt and MAPK/ERK pathways. Identification of

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cancer-specific miRNAs and their targets is critical for understanding their role in the progression of cancer and important for defining novel therapeutic targets. MiR-137 may constitute a promising therapeutic target for inhibition of CRC metastasis.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http:// dx.doi.org/10.1053/j.gastro.2012.11.033.

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22.

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1. Faix J, Grosse R. Staying in shape with formins. Dev Cell 2006; 10:693–706. 2. Sahai E. Mechanisms of cancer cell invasion. Curr Opin Genet Dev 2005;15:87–96. 3. Katoh M, Katoh M. Identification and characterization of human FMNL1, FMNL2 and FMNL3 genes in silico. Int J Oncol 2003;22: 1161–1168. 4. Zhu XL, Liang L, Ding YQ. Overexpression of FMNL2 is closely related to metastasis of colorectal cancer. Int J Colorectal Dis 2008;23:1041–1047. 5. Zhu XL, Liang L, Ding YQ. Expression of FMNL2 and its relation to the metastatic potential of human colorectal cancer cells. Nan Fang Yi Ke Da Xue Xue Bao 2008;28:1775–1778. 6. Zhu XL, Zeng YF, Guan J, et al. FMNL2 is a positive regulator of cell motility and metastasis in colorectal carcinoma. J Pathol 2011; 224:377–388. 7. Li Y, Zhu X, Zeng Y, et al. FMNL2 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition. Mol Cancer Res 2010;8:1579 –1590. 8. Kitzing TM, Wang Y, Pertz O, et al. Formin-like 2 drives amoeboid invasive cell motility downstream of RhoC. Oncogene 2010;29: 2441–2448. 9. Gardberg M, Talvinen K, Kaipio K, et al. Characterization of diaphanous-related formin FMNL2 in human tissues. BMC Cell Biol 2010;11:55. 10. Zhang YF, Liu L, Ding YQ. Isolation and characterization of human colorectal cancer cell subline with unique metastatic potential in the liver. Nan Fang Yi Ke Da Xue Xue Bao 2007;27:126 –130. 11. Wang S, Zhou J, Wang XY, et al. Down-regulated expression of SATB2 is associated with metastasis and poor prognosis in colorectal cancer. J Pathol 2009;219:114 –122. 12. Huang XZ, Sun Q, Ding YQ, et al. Mining microarray gene expression data of metastatic colorectal cancer by literature profiling. Di Yi Jun Yi Da Xue Xue Bao 2003;23:1195–1197. 13. Ambros V, Chen X. The regulation of genes and genomes by small RNAs. Development 2007;134:1635–1641. 14. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15–20. 15. Fujita S, Ito T, Mizutani T, et al. miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism. J Mol Biol 2008;378:492–504. 16. Shenouda SK, Alahari SK. MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer Metastasis Rev 2009;28:369 – 378. 17. Baranwal S, Alahari SK. miRNA control of tumor cell invasion and metastasis. Int J Cancer 2010;126:1283–1290. 18. Liau SS, Jazag A, Whang EE. HMGA1 is a determinant of cellular invasiveness and in vivo metastatic potential in pancreatic adenocarcinoma. Cancer Res 2006;66:11613–11622. 19. Kim J, Lee JW, Kim SI, et al. Thrombin-induced migration and matrix metalloproteinase-9 expression are regulated by MAPK and

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Received December 17, 2011. Accepted November 13, 2012. Reprint requests Address requests for reprints to: Li Liang, PHD, or Yanqing Ding, MD, Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Repulic of China. e-mail: [email protected] or dyq@fimmu.com; fax: (86) 2061642148. Acknowledgments The authors thank Professors Geevan and Reddy for editing the English writing.

Conflicts of interest The authors disclose no conflicts. Funding Supported by National Natural Science Foundation of China (81272759, 81172382, 81071735); Major projects of National Natural Science Foundation of China (81090422); National Basic Research Program of China (973 Program, 2010CB529402, 2010CB529403); the key NSFC-Guangdong Joint Project of China

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(U1201226); the Science and Technology Planning Project of Guangdong Province (2010B031500012); the Key Scientific and Technical Innovation Project of Higher Education of Guangdong Province (GXZD1016); Natural Science Foundation of Guangdong Province (S2012010009669); Research Fund for Subject and Profession development of Higher Education of Guangdong Province (2012KJCX0026); Research Fund for the Science and technology Star of Zhujiang of Guangzhou City (2011J2200074).

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Supplementary Materials and Methods Cell Lines, Human Tissue Samples, and Animals Human CRC cell lines LoVo, SW620, HT29, SW480, CaCo2, CoLo205, HCT116, LS174T, and human embryonal kidney cells 293FT cells were obtained from Cell Bank of Type Culture Collection (Shanghai City, China). All cell lines were grown in Dulbecco’s modified Eagle medium (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah, USA) at 37°C under 5% CO2. For inhibitor treatment, 10 mmol/L MEK inhibitor U0126 or 10 mmol/L PI3K inhibitor LY294002 (Cell Signal Technology, Danvers, MA) was added in the cultured cells every 2 days. For recombinant human IGF-1 (Pepro Tech, Inc, UK) treatment, 50 ng/mL IGF-1 was added in the cultured cells every 2 days. Fresh CRC tissues and the corresponding normal tissues were collected from 50 patients who underwent CRC resection without prior radiotherapy and chemotherapy at the Department of General Surgery in Nanfang Hospital (Guangzhou, Guangdong Province, People’s Republic of China) in 2009. These samples were collected immediately after resection, snap-frozen in liquid nitrogen, and stored at -80°C until needed. Four- to 6-week-old male athymic BALB/c nu/nu mice were purchased from the Central Laboratory of Animal Science at Southern University (Guangzhou, China). The mice were maintained at our laboratory in a specific pathogen-free environment. All protocols for animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at our University.

Real-time Reverse Transcription PCR Total RNA was extracted using TRIzol reagent (Invitrogen) and complementary DNA was synthesized by oligo dT primed reverse-transcription using an access reverse-transcription system (Promega). Real-time PCR was performed using the Mx3000P real-time PCR System (Stratagene, La Jolla, CA) and SYBR PremixEx Taq (TaKaRa, Shiga, Japan). The primers were selected as follows: has-mir-137, 5=-CAA ATT CGT GAA GCG TTC CAT AT-3=; has-mir-142-3p, 5=-GCT GTA GTG TTT CCT ACT TTA TGG AAA-3=; U6, 5=-TAT TGC TTA AGA ATA CGC GGT AGA AA-3=. U6 was amplified as an internal control. The PCR condition was 95°C for 10 minutes, followed by 40 cycles of amplification (95°C for 10 s, 60°C for 20 s, and 72°C for 34 s). The comparative quantification was determined using the 2-⌬⌬Ct method. Each sample was tested 3 times.

Western Blotting Analysis Proteins were extracted with RIPA buffer (1⫻ phosphate-buffered saline, 1% NP40, 0.1% sodium dodecyl sulfate, 5 mmol/L EDTA, 0.5% sodium deoxycholate, and 1 mmol/L sodium orthovanadate) with protease inhibitors and quantified by bicinchoninic acid method.

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Protein lysates (50 ␮g) were resolved on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes (Immobilon P; Millipore). Membranes were immunoblotted overnight at 4°C with anti-FMNL2 monoclonal antibody (Abnova, Taibei City, Taiwan), anti-MAPK, anti– p-MAPK, anti-Akt, anti–p-Akt antibody (Cell Signaling Technology, Inc), anti-VEGF monoclonal antibody (Santa Cruz Biotechnology), followed by their respective horseradish-peroxidase– conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence reaction performed according to the manufacturer’s instructions (Alpha Innotech, San Leandro, CA). Image density of the immunoblotting was determined by gel densitometry (Bio-Rad, Philadelphia, PA).

Promoter Analysis The 1-kb region directly upstream of miR-137 was predicted on the National Center for Biotechnology Information website. Analysis of putative transcription factor binding sites on the miR-137 promoter was performed by using the TF prediction program Consite http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite, Transcriptional regulatory element database http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process⫽home, and MAPPER 2.0 http://genome.ufl.edu/mapperdb.

Gelatin Zymograph Assay For the zymography assay, cells (2.5 ⫻ 105) were seeded in 12-well plates and incubated for 48 hours. Supernatants were collected and mixed with sample buffer followed by electrophoresis on an 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis containing 5 mg/mL of gelatin. The gel was washed for 2 hours and further incubated in the reaction buffer (50 mmol/L Tris-HCl, 5 mmol/L CaCl2, 1 mol/L ZnCl2, and 1% Triton X-100 (Sigma, Saint Louis, MO)) for an additional 18 hours at room temperature. The gel then was stained with 0.5% Coomassie blue for 9 hours and subsequently immersed with destaining buffer (30% methanol, 10% acetic acid) for 12 hours. The image was photographed and the intensity of each band was quantified digitally.

Immunohistochemical Staining Four-micrometer–thick histology sections from xenograft tumors were cut, deparaffinized using xylene, and hydrated through graded alcohol to water. Antigen retrieval was performed by boiling at 100°C for 10 minutes in 10 mmol/L citrate buffer (pH 6.0). In brief, the sections were incubated in polyclonal antibody against human FMNL2 (Abnova) overnight at 4°C. Subsequently, the horseradish-peroxidase– conjugated antigoat secondary antibody (Dako Cytomation, Glostrup, Denmark) was applied and incubated for 1 hour at room temperature. The visualization signal was developed with 3,3=-diaminobenzidine tetra hydrochloride staining, and

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the slides were counterstained in hematoxylin. The extent of immunostaining was evaluated by the intensity of staining (score, 1–3) and staining density (score, 1– 4). The intensity score was multiplied by the density score to calculate an overall score (1–12). Negative samples were scored as “0.”

Statistical Analysis The MTT method, plate colony formation assay, and in vitro invasion assay were tested using 1-way analysis of variance for factorial design. A paired t test was

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used to investigate the difference of the miR-137 expression level between normal and cancerous tissues. A 2-sample t test was used to analyze the clinicopathologic characteristics of mir-137 expression in CRC patients. The Pearson correlation coefficient was used to measure the degree of the linear relationship between the expression levels of miR-137 and FMNL2 or HMGA1 in CRC cells and tissues. Data were presented as the mean with 95% confidence intervals of at least 3 independent experiments. A P value less than .05 was considered statistically significant.

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.

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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.

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Supplementary Table 1. Clinicopathologic Characteristics of miR-137 Expression in CRC Patients 2-Ct, mean ⫾ SD

P/t

20 30

0290 ⫾ 0.227 0.295 ⫾ 0.271

t ⫽ -0.072 P ⫽ .943

31 19

0.319 ⫾ 0.257 0.251 ⫾ 0.247

t ⫽ 0.921 P ⫽ .362

23 27

0.276 ⫾ 0.211 0.264 ⫾ 0.220

t ⫽ 0.159 P ⫽ .874

39 11

0.263 ⫾ 0.241 0.295 ⫾ 0.233

t ⫽ 0.363 P ⫽ .718

10 30 10

0.345 ⫾ 0.232 0.287 ⫾ 0.280 0.258 ⫾ 0.192

t ⫽ 0.309 P ⫽ .736

24 26

0.260 ⫾ 0.223 0.323 ⫾ 0.278

t ⫽ -0.894 P ⫽ .376

45 5

0.254 ⫾ 0.241 0.408 ⫾ 0.301

t ⫽ -1.277 P ⫽ .208

24 26

0.260 ⫾ 0.223 0.323 ⫾ 0.278

t ⫽ -0.894 P ⫽ .376

Features

Number

All cases Age, y ⱕ50 ⬎50 Sex Male Female Tumor size, cm ⬍5 ⱖ5 Serosal invasion N Y Differentiation Well Moderate Poor Lymphatic metastasis N Y Lymph node ratio N Y Duke stage A⫹B C⫹D

50

NOTE. The lymph node ratio is defined as the number of positive lymph nodes divided by the total number of lymph nodes examined.

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