IJC International Journal of Cancer
MicroRNA-409-3p suppresses colorectal cancer invasion and metastasis partly by targeting GAB1 expression Rongpan Bai1,3,4, Chunhua Weng1, Haojie Dong1,3,4, Siqi Li1,3,4, Guangdi Chen1,3,4* and Zhengping Xu1,2,3,4* 1
Institute of Environmental Health, Zhejiang University School of Public Health, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China 3 Research Center of Molecular Medicine, Zhejiang University School of Medicine, Hangzhou, China 4 Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
Cancer Cell Biology
2
Colorectal cancer (CRC) is one of the most common cancers worldwide and its metastasis accounts for the majority of deaths. However, the molecular mechanisms underlying CRC progression are not well characterized. In this study, we identified miR409-3p as a tumor suppressor of CRC. MiR-409-3p expression was significantly downregulated in CRC tissue compared to adjacent non-tumor tissue, and reduced miR-409-3p expression was correlated with CRC metastasis. In vitro and in vivo studies revealed that miR-409-3p negatively regulated CRC metastatic capacities, including suppressing cancer cell migration, invasion and metastasis. To explore the mechanism of action of miR-409-3p, we adopted a pathway and pathophysiological event-based target screening and validation approach, and found nine known metastasis-related genes as potential targets. The 3’-UTR binding assays between the candidates and miR-409-3p suggested that only GAB1, NR4A2 and LMO4 were directly regulated by the miRNA. However, endogenous expression analysis revealed that only GAB1 was modulated by miR-409-3p in CRC cells at both the mRNA and protein levels. Furthermore, we provided evidence to conclude that GAB1 was partially responsible for miR-409-3p-mediated metastasis. Taken together, our data demonstrate that miR-409-3p is a metastatic suppressor, and post-transcriptional inhibition of the oncoprotein GAB1 is one of the mechanisms of action of this miRNA. Our finding suggests miR-409-3p might be a novel target for CRC metastasis treatment.
Key words: colorectal cancer, tumor metastasis, microRNA-409-3p, GAB1 Abbreviations: CRC: colorectal cancer; FBS: fetal bovine serum; GAB1: Grb2-associated binding protein; LMO4: LIM Domain Only 4; miRNA: microRNA; miR-409-3pi: microRNA-409-3p inhibitor; Mut: mutant; NC: negative control mimic; NCi: negative control inhibitor; NR4A2: nuclear receptor subfamily 4, group A, member 2; RT-qPCR: real-time quantitative PCR; RT-CIM: realtime cell invasion and migration; WT: wild-type Additional Supporting Information may be found in the online version of this article. Conflict of interest: There are no conflicts of interest to disclose. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 81372303 and 31170721; Grant sponsor: National High-tech R&D Program of China (863 Program); Grant number: 2008AA02Z101 DOI: 10.1002/ijc.29607 History: Received 31 Dec 2014; Accepted 11 May 2015; Online 19 May 2015 Correspondence to: Zhengping Xu, Ph.D., Zhejiang University School of Medicine, 866 Yu-Hang-Tang Road, Hangzhou 310058, China, Tel.: 186 571 88208008; Fax: 186 571 88208163, E-mail: [email protected] (or) Guangdi Chen, M.D., Ph.D., Zhejiang University School of Medicine, 866 Yu-Hang-Tang Road, Hangzhou 310058, China, Tel.: 186 571 88208169; Fax: 186 571 88208163, E-mail: [email protected]
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
Colorectal cancer (CRC) is the third most common cancer in men (746,000 cases, 10.0% of the total) and the second in women (614,000 cases, 9.2% of the total) worldwide.1,2 Despite a good outcome of treatment for early CRC, the clinical output for advanced CRC is unsatisfactory.1,2 Therefore, elucidating the molecular mechanisms underlying CRC progression is critical for developing new therapeutic strategies to improve the prognosis of patients with advanced CRC. Metastasis is the end-product of a multistep process termed the invasion-metastasis cascade wherein cancer cells spread from primary sites, disseminate to lymph nodes and/ or distant organs, and finally adapt to the microenvironment of inhabited foreign tissue.3,4 It accounts for as much as 90% of cancer-associated mortality, yet remains the most poorly understood process of cancer pathogenesis.3 Recent studies have indicated that microRNAs (miRNAs), a class of small (19–25 nucleotides) non-coding RNAs, can regulate various genes and play pivotal roles in the process of metastasis.5–8 Dysregulation of miRNA expression is reported to be associated with CRC metastasis,5 including escape from apoptosis (e.g., miR-34a and miR-18a),9,10 the epithelial-mesenchymal transition (e.g., miR-200c and miR-132),11,12 angiogenesis (e.g., miR-107 and miR-1246),13,14 and migration, invasion and proliferation (e.g., miR-124, miR-139-5p and miR181a).6,8 However, considering the complexity of the process, these CRC-related miRNAs are insufficient to elucidate the mechanism of CRC metastasis.5,15–17 Meanwhile, most of the
2311
Bai et al.
What’s new? What happens on the molecular level when cancer metastasizes? In some cases, small, noncoding microRNAs influence gene regulation and can prompt metastasis. In this paper, the authors show that one microRNA, miR-409-3p, acts as a tumor suppressor in colorectal cancer. They found less of the miRNA in CRC cells than the surrounding tissue; they went on to demonstrate that miR-409-3p stifles metastasis by suppressing expression of a protein, GAB1, which plays a role in migration and invasion. Thus, miR-409-3p could prove useful in devising a therapy to thwart metastasis.
Material and Methods Human samples
Paraffin embedded and fresh CRC specimens were provided by the tissue bank of the Second Affiliated Hospital of Zhejiang University School of Medicine. Total RNAs of paraffin embedded specimens were isolated from four 20-lm sections of each sample (Recover All Total Nucleic Acid Isolation Kit, Ambion, Austin, TX) and that of fresh tissues were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). The study was approved by the Ethics Committee of Zhejiang University School of Medicine. Cells and cell culture
The CRC cell lines HCT116, RKO, DLD1, SW480 were purchased from the cell bank at the Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI 1640 (Gibco, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Hyclone, Beijing, China). The 293T/17 cell was purchased from the cell bank at the Chinese Academy of Sciences (Shanghai) and maintained in Dulbecco’s modified Eagle’s medium with 10% FBS (Hyclone). All cells were incubated at 378C in a humidified 5% CO2 atmosphere.
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
Plasmid construction
The GAB1 coding region was amplified with a cDNA library from SW480 cells and inserted into the pCDH vector (System Bioscience, Mountain View, CA) between the EcoR I and Xba I sites. For the luciferase assay, the sequence of a 200-bp segment containing the predicted miR-409-3p binding site on the 3’UTR of GAB1, NR4A2, CTNND1, PDK1, ROD1, SPRED1, GRHL3, GATA2 or LMO4 was synthesized by Biosea (Hangzhou, China) and cloned into the Xho I and Not I sites of the psiCHECK-2 dual luciferase reporter vector (Promega, Madison, WI). Site-direct mutagenesis of the predicted miR-409-3p binding site on the 3’UTR of each gene was performed using a FAST mutagenesis system kit (Transgene, Beijing, China). All constructs were verified by sequencing. Oligonucleotide and plasmid transfection
Transfection of oligonucleotide and plasmid was performed with Lipofectamine 2000 (Invitrogen). Negative control mimic, negative control inhibitor, miR-409-3p mimic, miR409-3p inhibitor and siRNA for GAB1 were synthesized by Genepharma (Shanghai, China) and 100 lM was used for each transfection. For plasmid transfection, the final concentration was 1 lg/ml. The oligonucleotide sequences were listed in Supporting Information Table 1. RNA isolation and RT-qPCR
Total RNAs were extracted using TRIzol reagent (Invitrogen). The expression of mature miRNA was evaluated using TaqMan MiRNA Assay Kit (Applied Biosystems, Foster City, CA) and the U6 small nuclear RNA was used as an internal control. The mRNA level of each miR-409-3p-targeting gene was evaluated by reverse transcription (RT) reaction with Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen), followed by conventional quantitative PCR (qPCR) with SYBR Premix Ex Taq (TaKaRa, Dalian, China). The qPCR was performed on a Roche 480 real-time PCR system (Roche, Basel, Switzerland). Luciferase activity analysis
Luciferase reporter assay was performed in 293T/17 cells. Briefly, the cells were seeded in 96-well plates at 2.5 3 104 per well. After overnight incubation, the cells at 70% confluence were co-transfected with 25 ng psiCHECK-2-3’UTR and
Cancer Cell Biology
miRNAs have been identified from a small number of CRC patient samples, and usually only determine one target gene to explain its mechanism of action in CRC progression.5,15,17 Therefore, it is warranted to explore more CRC metastasisrelated miRNAs and fully elucidate its targeting genes and mediating pathways and biological events.16 MiR-409-3p has attracted much attention due to its involvement in various processes including tumor cell proliferation, apoptosis, vascularization, migration and invasion.18–22 Recent studies have indicated that miR-409-3p represses metastasis in various types of cancers, such as gastric cancer,18,20 fibrosarcoma,19 lung adenocarcinoma23 and bladder cancer.24 However, it remains unclear whether miR409-3p is involved in CRC progression. In this study, we evaluated the miR-409-3p expression levels in CRC tissue and adjacent non-tumor tissue, assessed the correlation between the miR-409-3p expression level and CRC metastasis, examined the effects of miR-409-3p on CRC cell metastatic ability in vitro and in vivo, and identified the potential targets of miR-409-3p using a metastatic-pathway-based target screening and validation approach.
miR-409-3p suppresses CRC invasion and metastasis
Cancer Cell Biology
2312
Figure 1. MiR-409-3p is downregulated in CRC and is associated with metastasis. (a) Expression of miR-409-3p in 82 pairs of samples from CRC and corresponding non-tumorous tissue. (b) Paired comparison of miR-409-3p expression levels between CRC and corresponding nontumorous tissue (Tumor/Normal). (c) Expression of miR-409-3p in CRC samples with or without metastasis. (d) Percentages of CRC samples with downregulated miR-409-3p in metastasis and non-metastasis tissues.
6 pmol RNA oligonucleotide (negative control mimic or miR-409-3p mimic). To evaluate the dose-dependent luciferase reporter activity, different concentrations of RNA oligonucleotide (negative control mimic or miR-409-3p mimic, from 0 to 10 pmol) were applied in the transfections. Wholecell lysates were prepared 24 hrs after transfection, and the luciferase activity was measured using a Dual-Luciferase Reporter Assay System kit (Promega). The Renilla luciferase activity was normalized to that of firefly luciferase. Cell proliferation and colony-formation
Cell proliferation was evaluated using a cell-counting kit (Dojindo Molecular Technologies, Kumamoto, Japan). Briefly, 24 hrs after transfection with miRNAs, cells were collected and counted by the hemocytometer method, then reseeded into the 96-well plates at 2,000 per well and incubated overnight. Cell proliferation was assessed as OD450 on Days 1–4 after seeding. For colony-formation assay, the cells were plated in six-well plates at 2,000 per well and maintained in RPMI 1640 containing 10% FBS. After 2 weeks, the cells
were washed twice with PBS, fixed in 4% paraformaldehyde, and stained with 0.5% crystal violet (Beyotime, Shanghai, China). The colony number was counted under a microscope (Olympus, Tokyo, Japan) at 1003. Migration and invasion
Migration and invasion assays were completed in transwell chambers (8-lm pore size, Millipore, Billerica, MA). For migration assay, cells were washed twice with serum-free growth medium and re-suspended at a final density of 50 3 104 per ml, then 200 ll was seeded in chambers and cultured for 24 hrs in 24-well plates (BD Biosciences, San Jose, CA) containing 1,300 ll 10% serum medium as a migratory stimulant. Cells that migrated through the chamber were fixed in 4% paraformaldehyde and stained with crystal violet (Beyotime). Images were captured and cell numbers were counted with a microscope (Olympus) at 2003. For invasion assay, transwell chambers were coated with 100 ll Matrigel plug (BD Biosciences) at a final concentration of 250 ng/ml. The subsequent steps were performed as for the migration assay.
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
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Cancer Cell Biology
Bai et al.
Figure 2.
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
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Real-time migration monitoring
Cancer Cell Biology
RT-CIM assay was performed on CIM-16 plate with an 8lm pore membrane (Roche). The xCELLigene DP system (Roche) was used to measure cell migration. This system uses a special culture plate that contains a gold electrode array on the bottom of each individual well. The impedance increases when there have more cellular contacts on the electrode surface. This impedance value is measured by the DP system and is reported in arbitrary unit of cell index. The cellular invasion and migration (CIM) plate is a two-chambered system similar to a standard Boyden chamber. The upper chamber is sealed at the bottom with a microporous polyethylene terephthalate membrane containing a microfabricated gold electrode array on the lower side. The median pore size of this membrane is 8 lm and chamber diameter is 5.0 mm. Cells were washed twice with serum-free growth medium, resuspended at a final density of 1 3 106 per ml, then 100 ll was added into the top chamber. The lower chamber was filled with 165 ll 10% serum medium as a migratory stimulant. The cell index was recorded every 15 min. Western blotting
Cells and fresh CRC tissues were lysed with RIPA lysis buffer consisting of 150 mM NaCl, 10 mM Tris, pH 7.5, 1% NP40, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 1 3 protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Cell and CRC tissue lysates were separated by electrophoresis on 10–15% SDS-polyacrylamide gels, transferred to nitrocellulose membranes (Whatman, Freiburg, Germany), blocked with 3% bovine serum albumin (BSA) in Trisbuffered saline and Tween 20, and blotted with the primary antibody followed by the IRDye 800CW-labeled goat antirabbit secondary antibody (Li-COR Biosciences, Lincoln, NE). The blot was visualized by an Odyssey infrared imaging system (Li-COR) and then quantified using densitometry. The primary rabbit anti-GAB1 (1:2,000) antibody was from Cell Signaling Technology (CST, MA); the mouse anti-CMet, RDX, PHF10, LMO4, GAPDH and b-actin (1:2,000) were from Santa Cruz Biotechnology (Santa Cruz, CA).
miR-409-3p suppresses CRC invasion and metastasis
transfected with negative control mimic or miR-409-3p mimic. After 8 weeks, all the mice were sacrificed and the lungs were collected and fixed in 10% formalin. After the process of paraffin embedding, the lung samples were sectioned and stained with hematoxylin and eosin. For in vivo tumorigenesis assay, 4 to 5-week-old female BALB/c-nu/nu mice (five mice per group) were injected subcutaneously with 1 3 106 cells transfected with negative control mimic or miR-409-3p mimic. The tumor growth rate was monitored by measuring tumor diameter every 4 days, and tumor volume was calculated as 1/2 3 length 3 width2 and recorded in mm3. When mice were killed, the tumors were collected and weighed. Immunohistochemistry
The hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) assays were performed as described previously.19 For immunohistochemistry, the section was deparaffinized, rehydrated, and boiled in 10 mM citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase was blocked by 3% H2O2. The slide was blocked in serum, incubated with the anti-Ki67 primary antibody (Abcam, Cambridge, UK) at a dilution of 1:100 at 48C overnight, then incubated with anti-rabbit secondary antibody, and visualized with diaminobenzadine (DAB kit, Invitrogen). Then, hematoxylin was used to counterstain the slide to label the nucleus. Negative control was obtained by omission of the primary antibody. The imagines of H&E and IHC staining were photographed at 1003 or 4003 with microscope (Olympus). Statistical analysis
All analyses were performed with SPSS version 11.5 (SPSS, Chicago, IL). The difference between two groups was assessed using Student’s t test. Data are presented as the mean with the standard error of the mean (SEM). The difference between variables was assessed by the v2 test or Fisher’s exact test. Two-tailed test was used and p values < 0.05 was considered to be statistically significant.
Animal study
Results
The animal research used a protocol approved by the Medical Experimental Animal Care Commission of Zhejiang University. For in vivo pulmonary metastasis assay, 4 to 5week-old female BALB/c-nu/nu mice (seven mice per group) were injected through the lateral tail vein with 1 3 106 cells
Downregulation of miR-409-3p expression is associated with CRC metastasis
To establish the association between miR-409-3p and CRC, we first assessed the miR-409-3p expression levels in 82 paired samples of CRCs and their adjacent non-tumorous
Figure 2. MiR-409-3p suppresses HCT116 cell migration and invasion without affecting its proliferation. (a) Representative images and quantification of transwell migration and invasion assays in HCT116 cells overexpressing miR-409-3p (NC, negative control mimic). (b) Representative images and quantification of transwell migration and invasion assays when the endogenous miR-409-3p was blocked in HCT116 cells (NCi, negative control inhibitor). (c) RT-CIM assays showing that over-expression of miR-409-3p decreased the migration of HCT116 cells while inhibition of endogenous miR-409-3p slightly enhanced it. (d) Neither overexpression nor inhibition of miR-409-3p influenced colony-formation in HCT116 cells. (e) Quantification of colony-formation number. (f) CCK8 assays showing that miR-409-3p did not affect the viability of HCT116 cells. *p
MicroRNA-409-3p suppresses colorectal cancer invasion and metastasis partly by targeting GAB1 expression Rongpan Bai1,3,4, Chunhua Weng1, Haojie Dong1,3,4, Siqi Li1,3,4, Guangdi Chen1,3,4* and Zhengping Xu1,2,3,4* 1
Institute of Environmental Health, Zhejiang University School of Public Health, Hangzhou, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China 3 Research Center of Molecular Medicine, Zhejiang University School of Medicine, Hangzhou, China 4 Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
Cancer Cell Biology
2
Colorectal cancer (CRC) is one of the most common cancers worldwide and its metastasis accounts for the majority of deaths. However, the molecular mechanisms underlying CRC progression are not well characterized. In this study, we identified miR409-3p as a tumor suppressor of CRC. MiR-409-3p expression was significantly downregulated in CRC tissue compared to adjacent non-tumor tissue, and reduced miR-409-3p expression was correlated with CRC metastasis. In vitro and in vivo studies revealed that miR-409-3p negatively regulated CRC metastatic capacities, including suppressing cancer cell migration, invasion and metastasis. To explore the mechanism of action of miR-409-3p, we adopted a pathway and pathophysiological event-based target screening and validation approach, and found nine known metastasis-related genes as potential targets. The 3’-UTR binding assays between the candidates and miR-409-3p suggested that only GAB1, NR4A2 and LMO4 were directly regulated by the miRNA. However, endogenous expression analysis revealed that only GAB1 was modulated by miR-409-3p in CRC cells at both the mRNA and protein levels. Furthermore, we provided evidence to conclude that GAB1 was partially responsible for miR-409-3p-mediated metastasis. Taken together, our data demonstrate that miR-409-3p is a metastatic suppressor, and post-transcriptional inhibition of the oncoprotein GAB1 is one of the mechanisms of action of this miRNA. Our finding suggests miR-409-3p might be a novel target for CRC metastasis treatment.
Key words: colorectal cancer, tumor metastasis, microRNA-409-3p, GAB1 Abbreviations: CRC: colorectal cancer; FBS: fetal bovine serum; GAB1: Grb2-associated binding protein; LMO4: LIM Domain Only 4; miRNA: microRNA; miR-409-3pi: microRNA-409-3p inhibitor; Mut: mutant; NC: negative control mimic; NCi: negative control inhibitor; NR4A2: nuclear receptor subfamily 4, group A, member 2; RT-qPCR: real-time quantitative PCR; RT-CIM: realtime cell invasion and migration; WT: wild-type Additional Supporting Information may be found in the online version of this article. Conflict of interest: There are no conflicts of interest to disclose. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 81372303 and 31170721; Grant sponsor: National High-tech R&D Program of China (863 Program); Grant number: 2008AA02Z101 DOI: 10.1002/ijc.29607 History: Received 31 Dec 2014; Accepted 11 May 2015; Online 19 May 2015 Correspondence to: Zhengping Xu, Ph.D., Zhejiang University School of Medicine, 866 Yu-Hang-Tang Road, Hangzhou 310058, China, Tel.: 186 571 88208008; Fax: 186 571 88208163, E-mail: [email protected] (or) Guangdi Chen, M.D., Ph.D., Zhejiang University School of Medicine, 866 Yu-Hang-Tang Road, Hangzhou 310058, China, Tel.: 186 571 88208169; Fax: 186 571 88208163, E-mail: [email protected]
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
Colorectal cancer (CRC) is the third most common cancer in men (746,000 cases, 10.0% of the total) and the second in women (614,000 cases, 9.2% of the total) worldwide.1,2 Despite a good outcome of treatment for early CRC, the clinical output for advanced CRC is unsatisfactory.1,2 Therefore, elucidating the molecular mechanisms underlying CRC progression is critical for developing new therapeutic strategies to improve the prognosis of patients with advanced CRC. Metastasis is the end-product of a multistep process termed the invasion-metastasis cascade wherein cancer cells spread from primary sites, disseminate to lymph nodes and/ or distant organs, and finally adapt to the microenvironment of inhabited foreign tissue.3,4 It accounts for as much as 90% of cancer-associated mortality, yet remains the most poorly understood process of cancer pathogenesis.3 Recent studies have indicated that microRNAs (miRNAs), a class of small (19–25 nucleotides) non-coding RNAs, can regulate various genes and play pivotal roles in the process of metastasis.5–8 Dysregulation of miRNA expression is reported to be associated with CRC metastasis,5 including escape from apoptosis (e.g., miR-34a and miR-18a),9,10 the epithelial-mesenchymal transition (e.g., miR-200c and miR-132),11,12 angiogenesis (e.g., miR-107 and miR-1246),13,14 and migration, invasion and proliferation (e.g., miR-124, miR-139-5p and miR181a).6,8 However, considering the complexity of the process, these CRC-related miRNAs are insufficient to elucidate the mechanism of CRC metastasis.5,15–17 Meanwhile, most of the
2311
Bai et al.
What’s new? What happens on the molecular level when cancer metastasizes? In some cases, small, noncoding microRNAs influence gene regulation and can prompt metastasis. In this paper, the authors show that one microRNA, miR-409-3p, acts as a tumor suppressor in colorectal cancer. They found less of the miRNA in CRC cells than the surrounding tissue; they went on to demonstrate that miR-409-3p stifles metastasis by suppressing expression of a protein, GAB1, which plays a role in migration and invasion. Thus, miR-409-3p could prove useful in devising a therapy to thwart metastasis.
Material and Methods Human samples
Paraffin embedded and fresh CRC specimens were provided by the tissue bank of the Second Affiliated Hospital of Zhejiang University School of Medicine. Total RNAs of paraffin embedded specimens were isolated from four 20-lm sections of each sample (Recover All Total Nucleic Acid Isolation Kit, Ambion, Austin, TX) and that of fresh tissues were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). The study was approved by the Ethics Committee of Zhejiang University School of Medicine. Cells and cell culture
The CRC cell lines HCT116, RKO, DLD1, SW480 were purchased from the cell bank at the Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI 1640 (Gibco, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Hyclone, Beijing, China). The 293T/17 cell was purchased from the cell bank at the Chinese Academy of Sciences (Shanghai) and maintained in Dulbecco’s modified Eagle’s medium with 10% FBS (Hyclone). All cells were incubated at 378C in a humidified 5% CO2 atmosphere.
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
Plasmid construction
The GAB1 coding region was amplified with a cDNA library from SW480 cells and inserted into the pCDH vector (System Bioscience, Mountain View, CA) between the EcoR I and Xba I sites. For the luciferase assay, the sequence of a 200-bp segment containing the predicted miR-409-3p binding site on the 3’UTR of GAB1, NR4A2, CTNND1, PDK1, ROD1, SPRED1, GRHL3, GATA2 or LMO4 was synthesized by Biosea (Hangzhou, China) and cloned into the Xho I and Not I sites of the psiCHECK-2 dual luciferase reporter vector (Promega, Madison, WI). Site-direct mutagenesis of the predicted miR-409-3p binding site on the 3’UTR of each gene was performed using a FAST mutagenesis system kit (Transgene, Beijing, China). All constructs were verified by sequencing. Oligonucleotide and plasmid transfection
Transfection of oligonucleotide and plasmid was performed with Lipofectamine 2000 (Invitrogen). Negative control mimic, negative control inhibitor, miR-409-3p mimic, miR409-3p inhibitor and siRNA for GAB1 were synthesized by Genepharma (Shanghai, China) and 100 lM was used for each transfection. For plasmid transfection, the final concentration was 1 lg/ml. The oligonucleotide sequences were listed in Supporting Information Table 1. RNA isolation and RT-qPCR
Total RNAs were extracted using TRIzol reagent (Invitrogen). The expression of mature miRNA was evaluated using TaqMan MiRNA Assay Kit (Applied Biosystems, Foster City, CA) and the U6 small nuclear RNA was used as an internal control. The mRNA level of each miR-409-3p-targeting gene was evaluated by reverse transcription (RT) reaction with Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen), followed by conventional quantitative PCR (qPCR) with SYBR Premix Ex Taq (TaKaRa, Dalian, China). The qPCR was performed on a Roche 480 real-time PCR system (Roche, Basel, Switzerland). Luciferase activity analysis
Luciferase reporter assay was performed in 293T/17 cells. Briefly, the cells were seeded in 96-well plates at 2.5 3 104 per well. After overnight incubation, the cells at 70% confluence were co-transfected with 25 ng psiCHECK-2-3’UTR and
Cancer Cell Biology
miRNAs have been identified from a small number of CRC patient samples, and usually only determine one target gene to explain its mechanism of action in CRC progression.5,15,17 Therefore, it is warranted to explore more CRC metastasisrelated miRNAs and fully elucidate its targeting genes and mediating pathways and biological events.16 MiR-409-3p has attracted much attention due to its involvement in various processes including tumor cell proliferation, apoptosis, vascularization, migration and invasion.18–22 Recent studies have indicated that miR-409-3p represses metastasis in various types of cancers, such as gastric cancer,18,20 fibrosarcoma,19 lung adenocarcinoma23 and bladder cancer.24 However, it remains unclear whether miR409-3p is involved in CRC progression. In this study, we evaluated the miR-409-3p expression levels in CRC tissue and adjacent non-tumor tissue, assessed the correlation between the miR-409-3p expression level and CRC metastasis, examined the effects of miR-409-3p on CRC cell metastatic ability in vitro and in vivo, and identified the potential targets of miR-409-3p using a metastatic-pathway-based target screening and validation approach.
miR-409-3p suppresses CRC invasion and metastasis
Cancer Cell Biology
2312
Figure 1. MiR-409-3p is downregulated in CRC and is associated with metastasis. (a) Expression of miR-409-3p in 82 pairs of samples from CRC and corresponding non-tumorous tissue. (b) Paired comparison of miR-409-3p expression levels between CRC and corresponding nontumorous tissue (Tumor/Normal). (c) Expression of miR-409-3p in CRC samples with or without metastasis. (d) Percentages of CRC samples with downregulated miR-409-3p in metastasis and non-metastasis tissues.
6 pmol RNA oligonucleotide (negative control mimic or miR-409-3p mimic). To evaluate the dose-dependent luciferase reporter activity, different concentrations of RNA oligonucleotide (negative control mimic or miR-409-3p mimic, from 0 to 10 pmol) were applied in the transfections. Wholecell lysates were prepared 24 hrs after transfection, and the luciferase activity was measured using a Dual-Luciferase Reporter Assay System kit (Promega). The Renilla luciferase activity was normalized to that of firefly luciferase. Cell proliferation and colony-formation
Cell proliferation was evaluated using a cell-counting kit (Dojindo Molecular Technologies, Kumamoto, Japan). Briefly, 24 hrs after transfection with miRNAs, cells were collected and counted by the hemocytometer method, then reseeded into the 96-well plates at 2,000 per well and incubated overnight. Cell proliferation was assessed as OD450 on Days 1–4 after seeding. For colony-formation assay, the cells were plated in six-well plates at 2,000 per well and maintained in RPMI 1640 containing 10% FBS. After 2 weeks, the cells
were washed twice with PBS, fixed in 4% paraformaldehyde, and stained with 0.5% crystal violet (Beyotime, Shanghai, China). The colony number was counted under a microscope (Olympus, Tokyo, Japan) at 1003. Migration and invasion
Migration and invasion assays were completed in transwell chambers (8-lm pore size, Millipore, Billerica, MA). For migration assay, cells were washed twice with serum-free growth medium and re-suspended at a final density of 50 3 104 per ml, then 200 ll was seeded in chambers and cultured for 24 hrs in 24-well plates (BD Biosciences, San Jose, CA) containing 1,300 ll 10% serum medium as a migratory stimulant. Cells that migrated through the chamber were fixed in 4% paraformaldehyde and stained with crystal violet (Beyotime). Images were captured and cell numbers were counted with a microscope (Olympus) at 2003. For invasion assay, transwell chambers were coated with 100 ll Matrigel plug (BD Biosciences) at a final concentration of 250 ng/ml. The subsequent steps were performed as for the migration assay.
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
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Cancer Cell Biology
Bai et al.
Figure 2.
C 2015 UICC Int. J. Cancer: 137, 2310–2322 (2015) V
2314
Real-time migration monitoring
Cancer Cell Biology
RT-CIM assay was performed on CIM-16 plate with an 8lm pore membrane (Roche). The xCELLigene DP system (Roche) was used to measure cell migration. This system uses a special culture plate that contains a gold electrode array on the bottom of each individual well. The impedance increases when there have more cellular contacts on the electrode surface. This impedance value is measured by the DP system and is reported in arbitrary unit of cell index. The cellular invasion and migration (CIM) plate is a two-chambered system similar to a standard Boyden chamber. The upper chamber is sealed at the bottom with a microporous polyethylene terephthalate membrane containing a microfabricated gold electrode array on the lower side. The median pore size of this membrane is 8 lm and chamber diameter is 5.0 mm. Cells were washed twice with serum-free growth medium, resuspended at a final density of 1 3 106 per ml, then 100 ll was added into the top chamber. The lower chamber was filled with 165 ll 10% serum medium as a migratory stimulant. The cell index was recorded every 15 min. Western blotting
Cells and fresh CRC tissues were lysed with RIPA lysis buffer consisting of 150 mM NaCl, 10 mM Tris, pH 7.5, 1% NP40, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 1 3 protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Cell and CRC tissue lysates were separated by electrophoresis on 10–15% SDS-polyacrylamide gels, transferred to nitrocellulose membranes (Whatman, Freiburg, Germany), blocked with 3% bovine serum albumin (BSA) in Trisbuffered saline and Tween 20, and blotted with the primary antibody followed by the IRDye 800CW-labeled goat antirabbit secondary antibody (Li-COR Biosciences, Lincoln, NE). The blot was visualized by an Odyssey infrared imaging system (Li-COR) and then quantified using densitometry. The primary rabbit anti-GAB1 (1:2,000) antibody was from Cell Signaling Technology (CST, MA); the mouse anti-CMet, RDX, PHF10, LMO4, GAPDH and b-actin (1:2,000) were from Santa Cruz Biotechnology (Santa Cruz, CA).
miR-409-3p suppresses CRC invasion and metastasis
transfected with negative control mimic or miR-409-3p mimic. After 8 weeks, all the mice were sacrificed and the lungs were collected and fixed in 10% formalin. After the process of paraffin embedding, the lung samples were sectioned and stained with hematoxylin and eosin. For in vivo tumorigenesis assay, 4 to 5-week-old female BALB/c-nu/nu mice (five mice per group) were injected subcutaneously with 1 3 106 cells transfected with negative control mimic or miR-409-3p mimic. The tumor growth rate was monitored by measuring tumor diameter every 4 days, and tumor volume was calculated as 1/2 3 length 3 width2 and recorded in mm3. When mice were killed, the tumors were collected and weighed. Immunohistochemistry
The hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) assays were performed as described previously.19 For immunohistochemistry, the section was deparaffinized, rehydrated, and boiled in 10 mM citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase was blocked by 3% H2O2. The slide was blocked in serum, incubated with the anti-Ki67 primary antibody (Abcam, Cambridge, UK) at a dilution of 1:100 at 48C overnight, then incubated with anti-rabbit secondary antibody, and visualized with diaminobenzadine (DAB kit, Invitrogen). Then, hematoxylin was used to counterstain the slide to label the nucleus. Negative control was obtained by omission of the primary antibody. The imagines of H&E and IHC staining were photographed at 1003 or 4003 with microscope (Olympus). Statistical analysis
All analyses were performed with SPSS version 11.5 (SPSS, Chicago, IL). The difference between two groups was assessed using Student’s t test. Data are presented as the mean with the standard error of the mean (SEM). The difference between variables was assessed by the v2 test or Fisher’s exact test. Two-tailed test was used and p values < 0.05 was considered to be statistically significant.
Animal study
Results
The animal research used a protocol approved by the Medical Experimental Animal Care Commission of Zhejiang University. For in vivo pulmonary metastasis assay, 4 to 5week-old female BALB/c-nu/nu mice (seven mice per group) were injected through the lateral tail vein with 1 3 106 cells
Downregulation of miR-409-3p expression is associated with CRC metastasis
To establish the association between miR-409-3p and CRC, we first assessed the miR-409-3p expression levels in 82 paired samples of CRCs and their adjacent non-tumorous
Figure 2. MiR-409-3p suppresses HCT116 cell migration and invasion without affecting its proliferation. (a) Representative images and quantification of transwell migration and invasion assays in HCT116 cells overexpressing miR-409-3p (NC, negative control mimic). (b) Representative images and quantification of transwell migration and invasion assays when the endogenous miR-409-3p was blocked in HCT116 cells (NCi, negative control inhibitor). (c) RT-CIM assays showing that over-expression of miR-409-3p decreased the migration of HCT116 cells while inhibition of endogenous miR-409-3p slightly enhanced it. (d) Neither overexpression nor inhibition of miR-409-3p influenced colony-formation in HCT116 cells. (e) Quantification of colony-formation number. (f) CCK8 assays showing that miR-409-3p did not affect the viability of HCT116 cells. *p
