Loss of miR-638 in vitro promotes cell invasion and a mesenchymal-like transition by influencing SOX2 expression in colorectal carcinoma cells
- Kelong Ma†1, 2, 3,
- Xiaorong Pan†1,
- Pingsheng Fan†4,
- Yinghua He1,
- Jun Gu1,
- Wei Wang1,
- Tengyue Zhang4,
- Zonghai Li1Email author and
- Xiaoying Luo1Email author
© Ma et al.; licensee BioMed Central Ltd. 2014
Received: 21 December 2013
Accepted: 19 May 2014
Published: 23 May 2014
Colorectal carcinoma (CRC) is a major cause of cancer mortality. The aberrant expression of several microRNAs is associated with CRC progression; however, the molecular mechanisms underlying this phenomenon are unclear.
miR-638 and SRY-box 2 (SOX2) expression levels were detected in 36 tumor samples and their adjacent, non-tumor tissues from patients with CRC, as well as in 4 CRC cell lines, using real-time quantitative RT-PCR (qRT-PCR). SOX2 expression levels were detected in 90 tumor samples and their adjacent tissue using immunohistochemistry. Luciferase reporter and Western blot assays were used to validate SOX2 as a target gene of miR-638. The regulation of SOX2 expression by miR-638 was assessed using qRT-PCR and Western blot assays, and the effects of exogenous miR-638 and SOX2 on cell invasion and migration were evaluated in vitro using the HCT-116 and SW1116 CRC cell lines.
We found that miR-638 expression was differentially impaired in CRC specimens and dependent on tumor grade. The inhibition of miR-638 by an antagomiR promoted cell invasion and a mesenchymal-like transition (lamellipodium stretching increased and cell-cell contacts decreased, which was accompanied by the suppression of the epithelial cell marker ZO-1/E-cadherin and the upregulation of the mesenchymal cell marker vimentin). A reporter assay revealed that miR-638 repressed the luciferase activity of a reporter gene coupled to the 3′-untranslated region of SOX2. miR-638 overexpression downregulated SOX2 expression, and miR-638 inhibition upregulated SOX2 expression. Moreover, miR-638 expression levels were correlated inversely with SOX2 mRNA levels in human CRC tissues. The RNAi-mediated knockdown of SOX2 phenocopied the invasion-inhibiting effect of miR-638; furthermore, SOX2 overexpression blocked the miR-638-induced CRC cell transition to epithelial-like cells.
These results demonstrate that the loss of miR-638 promotes invasion and a mesenchymal-like transition by directly targeting SOX2 in vitro. These findings define miR-638 as a new, invasion-associated tumor suppressor of CRC.
MicroRNAs (miRNAs) play pivotal roles in physiological and pathological processes via their regulation of a wide variety of genes, predominantly through their interaction with the 3′-untranslated regions (3′ UTR) of their corresponding mRNA targets[1, 2]. More than 4,665 mature miRNA products have been annotated in the human genome, according to the most recent version of the miRBase program (Release 20: June 2013; http://www.mirbase.org/), and increasing evidence has shown that the deregulation of miRNAs is involved in the pathogenesis of a wide range of diseases, such as human cancers[3, 4]. However, the roles of most miRNAs in tumor initiation and progression are still unknown.
Colorectal carcinoma (CRC) is the fourth-most common cause of cancer-related mortality worldwide. Approximately 715,000 deaths from CRC are estimated to occur annually, accounting for 8% of all cancer deaths. Because of advancements in CRC treatment regimens, there has been substantial progress in the treatment for colorectal cancer, and survival rates have improved over the past 40 years. Metastasis is the major concern in cancer therapy; cell invasion and the epithelial-to-mesenchymal transition (EMT) is the primary step in this process.
The EMT is a biological process in which a polarized epithelial cell, which normally interacts with the basement membrane via its basal surface, undergoes multiple biochemical changes that cause the epithelial cell to assume a mesenchymal cell phenotype. These phenotypic changes include enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and a greatly increased production of ECM components. Mounting evidence suggests that the EMT occurs in CRC[9, 10]. Recent studies have revealed that miRNAs are involved in the EMT process in CRC cells; for example, miR-101, miR-212, miR-155, miR-130b, and miR-34 have been found to be involved in the EMT process in CRC cells. One study has provided evidence that miR-638 is downregulated at the invasive front of CRC; however, its expression and function were not addressed.
In the present study, we sought to determine the role of miR-638 in CRC progression. We defined miR-638 as a new, invasion-associated tumor suppressor miRNA in vitro. Moreover, we identified SOX2, a factor that can induce pluripotent stem cells, as a direct, functional target of miR-638.
Materials and methods
Patients and tissue microarray
Participants who provided samples also provided written, informed consent to participate in this study. The Ethics Committee of the Shanghai Cancer Institute approved the study, the consent procedure, and the tissue array study. All of the research was performed in China. Paired colorectal tumor tissues and their corresponding adjacent non-tumor colorectal tissues (5 cm away from the lesions) were collected from patients who underwent curative surgery for CRC at Anhui Medical University, Anhui Province, China. Normal colon tissue was collected from patients with non-cancerous colon disease. A CRC diagnosis was confirmed by histological examination, and the relevant clinical and pathological information was retrieved from the hospital database (Additional file1: Table S1a). Glass-slide tissue arrays for CRC were purchased from the Shanghai Outdo Biotech Co., Ltd. (Shanghai, China) (Additional file1: Table S2a), and immunostaining (SOX2, ab75485, 1:100, Abcam, Cambridge, MA; vimentin, #5741, 1:50, Cell Signaling Technology, Beverly, MA) was performed on the tissue microarray slides. Staining was analyzed based on the percentage of positively stained cells and staining intensity by a pathologist or using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Bethesda, MD) (Additional file2).
Four human CRC cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). HCT-116 cells (ATCC No. CCL-247) were maintained in McCoy’s 5A medium, LoVo cells (ATCC No. CCL-229) were maintained in F-12 K medium (Kaighn’s Modification of Ham’s F-12 Medium), and SW480 cells (ATCC No. CCL-228) and SW1116 cells (ATCC No. CCL-233) were maintained in Leibovitz’s l-15 medium. The media were supplemented with 10% fetal bovine serum, and the cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
miRNA mimics and miRNA antagomiRs were designed and synthesized by RiboBio (Guangzhou, China). The miRNA antagomiRs were composed of nucleotides with a 2′-O-methylmodification. The SOX2 siRNAs (sense, 5′-GGAAUGGACCUUGUAUAGAUC-3′; anti-sense, 5′-UCUAUACAAGGUCCAUUCCCC-3′) were synthesized by RiboBio (Guangzhou, China), and the SOX2 overexpression construct was obtained from Origene Inc. (Beijing, China). The miRNA mimics, miRNA antagomiRs, SOX2-targeted siRNA, and SOX2 overexpression construct were transiently co-transfected with GFP (transfection efficiency control) using an Amaxa Nucleofector (Amaxa, Koeln, Germany) according to the manufacturer’s protocol.
RNA extraction and quantitative real-time RT-PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. qRT-PCR with miRNA was performed using the TaqMan Reverse Transcription Kit (Applied Biosystems), TaqMan MicroRNA Assays (Applied Biosystems), and a LightCycler TaqMan Master (Roche Diagnostics, Mannheim, Germany) according to the manufacturers’ instructions. The miRNA expression levels were calculated using the delta-delta Ct method with RNU6B as an internal control. A Ct value of 35 was set as the cut-off value for defining as non-detected.
cDNA was reverse-transcribed from 1 μg of RNA using the SYBR®Prime Script™ RT-PCR kit (Takara Biochemicals, Tokyo, Japan), and the reactions were performed on an ABI PRISM®7900HT Real-Time PCR System. The thermal cycling conditions were as follows: an initial step at 95°C for 15 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Each experiment was performed in a 20-μl reaction volume containing 10 μl of SYBR® Prime Ex Taq™ II (2×), 0.8 μl of forward primer and reverse primer (10 μM each), 0.4 μl of ROX Reference Dye or Dye II (50×), 2 μl of cDNA, and 6 μl of H2O. β-Actin was used as an internal control. The quantification of the mRNA was calculated using the comparative Ct (the threshold cycle) method according to the following formula: Ratio = 2-ΔΔ ct = 2- [Δ Ct(sample) - Δ Ct(calibrator)], where ΔCt is equal to the Ct of the target gene minus the Ct of the endogenous control gene (β-actin). The primers for SOX2 (SOX2RTF: 5′-CGAGATAAACATGGCAATCAAAAT-3′; SOX2RTR: 5′-AATTCAGCAAGAAGCCTCTCCTT-3′) have been described previously. The internal control actin primers were designed as previously described[19, 20].
Western blot analysis
Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% non-fat milk and incubated with the following antibodies: the epithelial cell marker ZO-1 (1:200) and E-cadherin (1:1000), the mesenchymal cell marker vimentin (1:50,000), SOX2 (1:2,000), Myc (1:2,000), β-actin (1:2,000, Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:10,000; Kang-Chen Bio-tech Shanghai, China). And the quantification of Western Blot exerted by ImagineJ software (NIH, USA).
Cell migration and invasion assays
We transfected the miR-638 mimics and SOX2 siRNA into HCT-116 and SW1116 cells using an Amaxa Nucleofector (Amaxa, Koeln, Germany). Cell migration and invasion were evaluated using Matrigel-uncoated and -coated transwell chambers (cat. 3422, Corning, NY). Briefly, 5 × 104 cells were suspended in 200 μl of DMEM without serum and placed into cell culture inserts (8-μm pore size; BD Falcon, San Jose, CA) of a companion plate (BD Falcon San Jose, CA) in pre-warmed culture medium containing 10% fetal bovine serum in the well. The cells were incubated overnight at 37°C in 5% CO2 and then fixed in 4% paraformaldehyde in PBS. Cell migration and invasion were determined by staining the cells with 0.1% crystal violet (Sigma, St Louis, MO) and counting the cells under a light microscope (100× magnification) in eight randomly selected areas.
Luciferase reporter assay
Luciferase activity was detected using the Dual Luciferase Assay (Promega, USA) according to the manufacturer’s protocols. The transfected cells were lysed in tissue culture dishes with lysis buffer, and the lysates were centrifuged at maximum speed for 1 min in an Eppendorf microcentrifuge. The relative luciferase activity was determined using a Modulus TD20/20 Luminometer (Turner Biosystems, Sunnyvale, CA), and the transfection efficiency was normalized to Renilla activity.
Transfected SW1116 cells were seeded at a density of 2 × 104 onto poly-L-lysine-coated glass coverslips in a 6-well plate. After further culture overnight, the cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO). For filamentous actin (F-actin) staining, the coverslips were incubated with TRITC-labeled phalloidin (Sigma-Aldrich, St. Louis, MO) at room temperature, and the cell nuclei were counterstained with DAPI. The cells were co-transfected with 40 ng of pEGFP plasmid as a control.
All experiments were performed in triplicate. The data are presented as the mean values ± standard error of the mean (SEM) and were analyzed using Student’s t-test. p values less than 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA).
The accession numbers for miR-638 is MIMAT0003308, and that for SOX2 is NM_003106.2.
miR-638 shows reduced expression in colorectal carcinoma
miR-638 suppresses cell invasion and migration
Loss of miR-638 promotes a mesenchymal-like transition in CRC cells
miR-638 directly inhibits SOX2 expression by interacting with its 3′-UTR
SOX2 is highly expressed in CRC tissues
Knockdown of SOX2 expression suppresses cellular invasion in CRC cells
SOX2 overexpression rescues the miR-638-induced inhibition of invasion and mesenchymal-like transition
Taken together, these data indicate that SOX2 can rescue the miR-638-induced inhibition of cell invasion and mesenchymal-like transition.
Tumor metastasis is the major cause of death in CRC patients and occurs in >30% of patients at diagnosis and, subsequently, in >50% of patients after surgery with curative intent. Unfortunately, because of the lack of knowledge regarding the mechanism underlying colorectal tumorigenesis, no effective therapies that block the development and progression of metastasis have been identified. Therefore, the 5-year survival rate of patients with colon cancer is less than 10% if the cancer has metastasized[26, 27]. Invasion and a mesenchymal-like transition are the primary processes involved in metastasis. In this study, we aimed to find a new microRNA associated with invasion and the transition to mesenchymal-like cells.
miRNAs are a class of gene expression-regulating molecules that are associated with cancer development and progression. The deregulation of miRNAs is a common event in human cancers, and many miRNAs, such as miRNA-143, miR-9, and miR-137, have been found to be deregulated in CRC and involved in cancer invasion or migration. In a previous study, we found 23 apparently downregulated miRNAs in CRC tissues using a microarray assay. After validating the expression of these miRNAs through qRT-PCR in CRC tissues, we found that miR-638, which has been reported to be downregulated in human gastric cancers, basal cell carcinomas, and chronic lymphocytic leukemias, was obviously downregulated in CRC cancer tissues compared with adjacent tissues. Subsequently, we examined the effect of miR-638 on CRC cell invasion and migration in vitro and found that miR-638 particularly inhibited cell invasion and migration, which suggested that miR-638 is an anti-oncomiR and invasion-related miRNA in CRC. Furthermore, miR-638 was correlated with tumor differentiation grade. These data suggest that miR-638 may be involved in the interaction between or conversion of epithelial and mesenchymal cells. Further data showed that miR-638 loss induced a mesenchymal-like transition (such as reduced cell-to-cell contacts, stretched lamellipodia, and upregulation of the mesenchymal cell marker vimentin).
miR-638 plays certain roles in pathology and physiology. miR-638, which was downregulated in non-small-cell lung cancer (NSCLC) tissues, aggravated DNA damage (induced by benzo (a) pyrene) by suppressing breast cancer 1 (BRCA1). Moreover, one study indicated that miR-638 is downregulated at the invasive fronts of colorectal liver metastases based on microarray analysis. In this study, we found that miR-638 exhibited reduced expression in CRC tissues, and this loss of expression promoted cell invasion and a mesenchymal-like transition.
The regulatory functions of miRNAs are mediated by their target genes; therefore, it is essential to identify the specific genes that are targeted by those miRNAs. The mirSVR score that is provided at mircrorna.org ranks microRNA target sites using a downregulation score. This score is used to identify target genes or predict the extent of their downregulation at the mRNA or protein levels[35, 36]. Using this method, SOX2 was chosen as a candidate target gene. Using a series of assays (for example, a reporter assay to validate that SOX2 is the only and direct target, miR-638 alterations drove SOX2 expression, miR-638 expression was inversely correlated with SOX2 expression in CRC tissues, SOX2 knockdown phenocopied the overexpression of miR-638, and SOX2 overexpression in miR-638-transfected cells rescued miR-638-induced function), SOX2 was finally identified as the functional target gene of miR-638 in CRC.
SOX2 is a Yamanaka factor that can induce pluripotent stem cells from mouse embryonic and adult fibroblast cultures. The EMT process is accompanied by increased SOX2 expression and can be induced via the SOX2 pathway. The overexpression of SOX2 promotes dedifferentiation and induces the EMT process, whereas the knockdown of SOX2 induces the MET process[40, 41]. Furthermore, SOX2 expression (by conventional IHC) is correlated with lymph node metastasis; therefore, it can serve as a metastasis marker for CRC. In the present study, we used a more precise assay (ompare to Conventional immunohistochemical expression analysis), IHC in tissue arrays, and found that SOX2 was overexpressed in CRC tissues and was correlated with tumor grade.
In summary, our results suggest that miR-638 is a potential invasion-associated tumor suppressor in CRC. In this study, we confirmed that miR-638 exhibited reduced expression in CRC, and its expression was correlated with tumor differentiation grade. Furthermore, the inhibition of miR-638 induced CRC cell lines to develop mesenchymal-like cell features (e.g., reduced cell-cell contact, increased lamellipodium stretching, decreased expression of the epithelial cell marker ZO-1/E-cadherin, increased expression of the mesenchymal cell marker vimentin, and increased cell migration and invasion), and we confirmed that SOX2 is a direct target of miR-638. These findings may facilitate the development of new CRC therapeutics.
This work was supported by the Natural Science Foundation of the Shanghai Science and Technology Committee (No. 12ZR1430200), the Natural Science Foundation of Anhui Provincial Education Bureau (No. KJ2013Z184), the research fund of the State Key Laboratory of Oncogenes and Related Genes (No. 91-11-05), the Natural Science Foundation of Anhui University of Chinese Medicine (No. 2012zr008), the Young Scientists Foundation of the Shanghai Cancer Institute (No. SB11-10), and the National Natural Science Foundation of China (Nos. 81201576, 81350005, and 31000565).
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