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MicroRNA-143 down-regulates Hexokinase 2 in colon cancer cells
© Gregersen et al.; licensee BioMed Central Ltd. 2012
Received: 25 October 2011
Accepted: 15 May 2012
Published: 12 June 2012
MicroRNAs (miRNAs) are well recognized as gene regulators and have been implicated in the regulation of development as well as human diseases. miR-143 is located at a fragile site on chromosome 5 frequently deleted in cancer, and has been reported to be down-regulated in several cancers including colon cancer.
To gain insight into the role of miR-143 in colon cancer, we used a microarray-based approach in combination with seed site enrichment analysis to identify miR-143 targets.
As expected, transcripts down-regulated upon miR-143 overexpression had a significant enrichment of miR-143 seed sites in their 3'UTRs. Here we report the identification of Hexokinase 2 (HK2) as a direct target of miR-143. We show that re-introduction of miR-143 in the colon cancer cell line DLD-1 results in a decreased lactate secretion.
We have identified and validated HK2 as a miR-143 target. Furthermore, our results indicate that miR-143 mediated down-regulation of HK2 affects glucose metabolism in colon cancer cells. We hypothesize that loss of miR-143-mediated repression of HK2 can promote glucose metabolism in cancer cells, contributing to the shift towards aerobic glycolysis observed in many tumors.
MicroRNAs (miRNAs) represent an abundant group of small non-coding RNAs that repress translation and promote degradation of their mRNA targets through binding to partially complementary regions in the 3'UTR [1–3]. The target recognition is mediated by the RNA-induced silencing complex (RISC) with AGO2 as a key component. AGO2 presents the miRNA to its targets in such a way that the nucleotides at position 2–8 of the mature miRNA, also known as the seed region, are able to base pair with complementary regions in the 3'UTR .
Past years research on miRNAs has revealed a role of miRNAs in the regulation of numerous cellular functions including development and differentiation, cell cycle regulation, metabolism and apoptosis [4, 5]. A large number of miRNAs are encoded by genes located in regions frequently exposed to changes in cancer cells  and alterations of miRNA expression levels have been associated with various types of cancer . In addition, miRNA signatures of cancer have also in some cases been shown to correlate with the prognosis and progression of cancer . By down-regulation of protein-encoding genes either promoting or inhibiting cell proliferation, several miRNAs have been shown to function as tumorsuppressors and oncogenes [5, 8–11].
miR-143 is located at a fragile site often deleted in cancers  and has accordingly been found down-regulated in a number of cancers [13–22]. Furthermore, miR-143 overexpression has been demonstrated to have a growth inhibitory effect in several cell lines, indicating that loss of miR-143 expression could contribute to the development of cancer [13–15, 18, 22, 23].
During development miR-143 expression has been reported to be induced during differentiation of adipocytes and vascular smooth muscle cells [24–26]. In vascular smooth muscle cells miR-143 inhibition was found to increase the proliferative potential 2-fold, but by itself miR-143 overexpression was not able to induce vascular smooth muscle differentiation . This suggests that miR-143 may normally function to restrict the proliferative potential of differentiated cells, explaining why down-regulation or loss of miR-143 can contribute to the formation and/or growth of cancer.
To investigate the function of miR-143 as a putative tumorsuppressor, we sought to understand the mechanistic basis for the involvement of miR-143 in cancer by the identification of miR-143 targets. We chose to focus our study on colon cancer, since miR-143 has been frequently reported as down-regulated in colon cancers [14, 15, 17, 18, 20]. In order to identify miR-143 targets we used a microarray-based approach. Potential miR-143 targets were identified as genes containing miR-143 seed sites in the 3'UTRs that were down-regulated upon miR-143 overexpression. Here, we report that miR-143 targets and down-regulates the glycolytic enzyme hexokinase 2 (HK2) in colon cancer cell lines. Furthermore we show, that re-introduction of miR-143 leads to a decrease in lactate secretion, indicating that miR-143-mediated downregulation of HK2 impairs the rate of glycolysis.
Cell cultures and cell proliferation assays
Cells were cultured as previously described . Overexpression of miR-143 was achieved by transient transfection with a miR-143 duplex that mimics the mature miR-143 duplex (PM10883; Ambion, Austin, TX, USA). Transfection with a scrambled negative control siRNA (1027281, Qiagen, GermantownMD, USA) was used as control. All transfections were carried out using Lipofectamine™ 2000 Transfection Reagent (11668-019, Invitrogen, Burlington, ON, Canada) according to the manufactures protocol using a final concentration of 50nM of oligonucleotides. Crystal violet assays were performed as previously described .
Total RNA was isolated with TRIZOL (15596-026, Invitrogen) and treated with DNaseI (DNase-free kit™, AM1906, Ambion). Primer sequences used for quantitative PCR (Q-PCR) are listed in Additional file 1: Table S1. Hypoxanthine phosphoribosyltransfease (HPRT) or beta-actin (ATCB) served as housekeeping normalization controls. Mature miR-143 levels were quantified using TaqMan® MicroRNA Assay (4373134, Applied Biosystems, Austin, TX, USA) and normalized to the U6 small nuclear B non-coding RNA (4373381, Applied Biosystems).
DLD-1 cells were transfected with miR-143 duplex or mock transfected in four biological replicates. Total RNA was isolated with TRIZOL 24 h after transfection. Affymetrix microarray analysis (HG-U133 Plus 2.0 human) was performed at the Microarray Center, Rigshospitalet, Copenhagen University Hospital as previously described . Data processing and word analysis are described in a separate section below.
Vector construction and reporter assays
The miR-143 luciferase reporter vector was cloned by inserting a site with perfect complementarity to miR-143 into HindIII/SpeI sites of pMIR-REPORT (AM5795, Applied Biosystems). Antisense and sense oligonucleotide sequences (with restriction overhangs indicated in lower case) are as follows:
miR-143 AS: 5'-ctagtGAGCTACAGTGCTTCATCTCAGCTCAGCA-3',
miR-143 S: 5'-agcttGCTGAGCTGAGATGAAGCACTGTAGCTCA-3',
3' UTR fragment of HK2 was PCR amplified from DLD-1 genomic DNA and cloned into the pGL3+ vector described previously . The primer sequences used for PCR amplification were as follows (restriction sites indicated in lower case):
HK2 3'UTR BglII FW: 5'-GGGagatctGGAGGGATGAGAGTGGCTTA-3'
HK2 3'UTR XhoI RV: 5'-GGGctcgagAATGACAACATCTTCACTAGACTGAG-3'
The miR-143 8mer seed site, TCATCTCA, in the 3'UTR of HK2 was converted into TCATGACA using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Mutagenesis primers used were as follows:
HK2 mut FW: 5'-GTGTGATGAATAGCGAATCATGACAAATCCTTGAGCACTCAGTC-3'
HK2 mut RV: 3'-GACTGAGTGCTCAAGGATTTGTCATGATTCGCTATTCATCACAC-5'
Luciferase assays were performed as previously described . Briefly, cells were co-transfected with indicated luciferase reporters and a Renilla normalization control, pRL-TK (E2241, Promega, Madison, WI, USA) vector alone or with miR-143 duplex or a scrambled negative control. Firefly and Renilla luminescence was measured 24 h after transfection using the Dual-Glo luciferase kit (E2940, Promega).
HK2 siRNA knockdown experiments
Knockdown experiments were performed by transient transfection of HK2 siRNA using lipofectamine as described above. Cells were double transfected with 50nM siRNA for 6 h on two subsequent days and the cell lysates were harvested 48 h after the first transfection for protein and RNA extraction. The sequence of the HK2 siRNA is as previously published : HK2 sense 5'-GGAUAAGCUACAAAUCAAA[dT][dT]-3',
Antibodies and western blot analysis
For western blotting DLD-1 or HCT116 cells were double transfected for 6 h on two subsequent days. Cells were harvested 48 h after the first transfection, washed twice in PBS, and lysed in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl at pH 8, 2 mM EDTA) containing protease inhibitor cocktail (04693124001, Roche, Basel, Switzerland)) and phosphatase inhibitors (1 mM NaVO3, 10 mM NaF and 1 mM β-glycerolphosphat). 30 μg protein/lane was separated on polyacrylamide gels, transferred to a nitrocellulose membrane and incubated with antibodies against HK2 (1:1000, 2106, Cell Signaling Technology, Danvers, MA, USA) or antibodies against Tubulin (ab11304, Abcam, Cambridge, MA, USA) serving as a loading control. Band intensities were quantified using TotalLab image analysis software.
DLD-1 cells were double transfected for 6 h on two subsequent days. To measure the secretion of lactate, media samples were removed in 6 h intervals following the addition of fresh media after the second transfection and stored at −80°C until measurement. Lactate was measured using the Lactate Acid Assay Kit (K607-100, BioVison, Mountain View, CA, USA).
Analysis of microarray profiles, seed site enrichment and word analysis
The microarray data was processed as previously described . Non-specific filtering was used to remove genes with low variance between arrays using a cutoff of 0.25. This left 1241 genes that were used for the following analysis. Differentially expressed genes were found using limma . Genes with a FC above 1.1 or below −1.1 were used to define the up and down set, respectively. The no-change set was selected from genes with a logFC centered on 0. The microarray data has been deposited in the GEO database accession number GSE33420.
Seed site enrichment was calculated by scanning the 3'UTR sequences in the up, down and no-change sets for the presence of 6mer, 7mer, 7mer-A1 and 8mer seed sites.
We used Gene Set Enrichment Analysis (GSEA) to detect significantly enriched biological functions/pathways from the KEGG pathway , Biocarta pathway (http://www.biocarta.com) and MSigDB  databases for the down-regulated gene sets after over-expressing miR-143. GSEA can detect an overall change in a gene set for up- and down-regulated genes even though individual genes in the set may not be significantly differentially expressed. The analysis was based on expression fold changes between miR-143 and mock transfection of all genes on the array without any cutoffs, and p-value less than 0.01 was used for statistical significance. The package “gage”  in Bioconductor was used for the analysis.
For the word analysis we used a non-parametric statistical framework for scoring and ranking oligonucleotide words based on their overrepresentation in a ranked list of sequences as previously described .
TCGA colorectal adenocarcinoma expression correlation
Data was obtained from the public open-access HTTP directory at the TCGA website (http://tcga-data.nci.nih.gov/) for the colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) projects. Level 3 normalized Agilent microarray mRNA expression data and miRNA expression sequencing data summarized for each mature miRNA was obtained for 184 colon and rectum adenocarcinoma samples.
We next sought to identify functionally relevant targets that could explain the underlying role of miR-143 in cancer. To achieve this, DLD-1 cells were transfected with miR-143 duplex or mock transfected. Total RNA was harvested 24 h post-transfection and analyzed on Affymetrix HG-U133 Plus 2.0 human arrays.
Enriched KEGG pathways among miR-143 down-regulated gene sets
Ubiquitin mediated proteolysis
Pathogenic Escherichia coli infection - EHEC
Base excision repair
Vibrio cholerae infection
p53 signaling pathway
Regulation of actin cytoskeleton
Wnt signaling pathway
Biosynthesis of unsaturated fatty acids
Glycolysis / Gluconeogenesis
Enriched BioCarta pathways among miR-143 down-regulated gene sets
HIV-I Nef: negative effector of Fas and TNF
mTOR Signaling Pathway
Ras-Independent pathway in NK cell-mediated cytotoxicity
The IGF-1 Receptor and Longevity
Inhibition of Cellular Proliferation by Gleevec
Role of ERBB2 in Signal Transduction and Oncology
Cell Cycle: G1/S Check Point
In addition we also performed a search for enriched transcription factor and miR-143 binding motifs among miR-143 down-regulated genes. The second most significantly enriched motif in the down-regulated gene set was the miR-143 seed site (p-value = 7·10−10), while the most significantly enriched motif was binding site of the transcription factor E2F (Additional file 3: Table S2).
In addition to seed site enrichment analysis, we also performed an unbiased word analysis of words present in 3’UTRs of transcripts ranked according to their FC. The 7mer and the 7mer-1A seed sites of miR-143 were identified as the most significantly enriched 7mer words in the 3'UTRs of transcripts down-regulated after miR-143 overexpression (Figure 2B). Sequence variations of the miR-143 seed site were also among the highest scoring words. Similarly, a 6mer word analysis indentified the miR-143 6mer seed site as the most enriched 6mer word in 3'UTRs of down-regulated transcripts (Additional file 2: Figure S4). The overrepresentation of miR-143 seed sites in 3'UTRs of down-regulated transcripts can be visualized by plotting the running sum of the overrepresentation scores of the seed sites in transcripts ranked according to their logFC. As seen in Figure 2C the overrepresentation scores of the miR-143 7mer seed site are highest among 3'UTRs of down-regulated transcripts (black line). This was not the case, when the same analysis was performed for 100 permutations of the ranked transcript list (grey lines).
As miR-143 possesses a tumor-suppressor function, we would expect a down-regulation of oncogenes and genes promoting cell proliferation upon miR-143 overexpression. Putative miR-143 targets, defined as genes down-regulated upon miR-143 overexpression with a FC < −1.1 and containing either a least one 7mer, 7mer-1A or 8mer seed site in their 3'UTR are listed in Additional file 4: Table S3. Among the down-regulated genes containing miR-143 seed sites in their 3'UTRs we found a number of genes that have previously been implicated in tumorigenesis. This include the Steroid 5-alpha-reductase SRD5A1, the CCR4-NOT component RQCD1 and the Rab11 effector protein RAB11FIP1 which have all been reported as up-regulated in breast cancers [35–37]. Other miR-143 responsive genes with a miR-143 seed site in their 3'UTR were SEMA5A, SLC35B2 and KLF5 which have all been shown to be up-regulated in cancers and to promote cell proliferation [38–41]. Among the putative miR-143 targets we also found the deubiquitinating enzyme USP22, which have been reported to be associated with a poor prognosis of colorectal cancer  and invasive breast cancer . In addition we also observed a reduced expression of the glycolytic enzyme hexokinase 2 (HK2) upon miR-143 overexpression. HK2 catalyzes the first step of glycolysis by phosphorylation of glucose into glucose-6-phosphate. HK2 is often found upregulated in cancer and facilitates a high rate of glucose metabolism necessary for tumor growth .
Among genes motioned above, three genes have also been predicted by a target prediction model built on 12 transfection datasets with good prediction posterior probabilities and low FDR (<25%) . This includes HK2 (posterior probability = 0.93; adjusted p-value = 0.17), RAB11FIP1 (posterior probability = 0.92; adjusted p-value = 0.17) and SEMA5A (posterior probability = 0.9; adjusted p-value = 0.22). This adds supportive evidence that these genes are direct targets of miR-143 beyond a simple seed match search.
As a validation of the microarray data we selected 7 transcripts identified as down-regulated by miR-143 in the microarray analysis for Q-PCR validation. All 7 transcripts including HK2 were found to be down-regulated, confirming the microarray data (Figure 2D). In accordance with previous reports we also find KRAS downregulated upon miR-143 overexpression (Figure 2D) . KRAS was also found down-regulated in the microarray analysis but because it had a borderline logFC of −0.14, it is not included in our list of potential miR-143 targets as listed in Additional file 4: Table S3.
To further strengthen the connection between miR-143 and HK2 we surveyed the expression levels of both miR-143 and HK2 in data from The Cancer Genome Atlas (TCGA) consortium. TCGA is currently profiling the genomes of a large cohort of colon and rectum adenocarcinomas. We found a significant negative correlation between miR-143 and HK2 (P = 0.002, r = −0.22, Pearson correlation) in 184 public TCGA colorectal adenocarcinoma tumor samples with miRNA and mRNA expression data available (Figure 3D). This observation supports that miR-143 could target and repress HK2 expression in-vivo and that HK2 expression could be upregulated in a subset of tumors due to lower levels of miR-143.
Next, to determine if downregulation of HK2 mediated by miR-143 resulted in an impairment of glycolysis, lactate production was measured in mock transfected cells and cells transfected with miR-143 duplex or HK2 siRNA. Cells transfected with a HK2 siRNA showed a marked decrease in the rate of lactate secretion over a period of 48 h (Figure 4D). Importantly, a decrease in the lactate secretion was also observed upon miR-143 overexpression (Figure 4D), confirming that miR-143 downregulation of HK2 has a functional effect on the glucose metabolism. The observed decrease in lactate secretion caused by miR-143 overexpression is less pronounced than for HK2 siRNA mediated inhibition. However, this might be explained by the more efficient down-regulation of HK2 mediated by the HK2 siRNA than by overexpression of miR-143.
In accordance with numerous reports of miR-143 down-regulation in cancer, we observed low or undetectable expression levels of miR-143 in human cancer cell lines. This was in contrast to non-tumorigenic fibroblast cell lines, which had a relatively high expression level of miR-143. In addition, we confirmed the growth inhibitory effect of miR-143 reported by others in DLD-1 colon cancer cells [14, 15, 18].
Using a microarray based experimental approach we have identified a number of putative miR-143 targets that are down-regulated at the transcript level by miR-143 overexpression and contain miR-143 seed sites in their 3'UTRs. This target identification approach provides a way to indentify functionally relevant miRNA targets in colon cancer cells without any assumption concerning the conservation of miR-143 binding sites, but by means of detecting expression changes of potential miR-143 targets at the transcript level. Even though miRNAs repress the protein output of their target genes as a combined effect of mRNA destabilisation and translational repression, a recent study has reported transcript destabilization to be the main contribution to miRNA target deregulation . Therefore, target identification based on detection of changes at the transcript level should in principal be able to detect the majority of miRNA targets, thus justifying our approach to identify miRNA targets. As a further confirmation of this strategy to identify miRNA targets, we observed a highly significant enrichment of miR-143 seed sites in the 3'UTRs of genes down-regulated upon miR-143 overexpression.
Among the putative miR-143 targets we found a number of genes known to promote cell proliferation, including SRD5A1, RQCD1, RAB11FIP1, SEMA5A, KLF5, USP22, SLC35B2 and HK2. Three of these genes have also been predicted as miR-143 targets by an independent miRNA target prediction algorithm . Previous studies have identified ERK5 and KRAS as miR-143 targets in colon cancer [14, 15]. In our study we also observed down-regulation of KRAS upon miR-143 transfection. However the degree of down-regulation was above the logFC of −1.1 used to define our set of putative miR-143 target. In the case of ERK5, we did not observe any change in expression in our microarray experiment. This is in agreement with a study of miR-143 in liposarcoma that also did not identify ERK5 as a miR-143 target, but did observe a down-regulation of HK2 in response to miR-143 overexpression .
miR-143 mediated down-regulation of one or more of the above mentioned genes in colon cancer cells could account for the growth inhibitory effect of miR-143. However, the tumor suppressive function of miR-143 is likely a result of the combined effect of miR-143-mediated down-regulation of several genes rather than a single gene. Considering miR-143 down-regulated genes in our study, including both direct and potential secondary effects, we found an enrichment of genes involved in cell cycle regulation as well as cellular metabolism. This suggests that miR-143 targets genes involved in a number of cellular pathways, including pathways controlling cell growth and metabolism which mediates downstream gene expression changes of genes in these pathways.
We chose to focus on HK2 as a potential target of miR-143 for further functional analysis, because we hypothesized that miR-143 mediated regulation of HK2 may account for the changes in glucose metabolism observed in many cancer cells. Alterations in glucose metabolism in cancer cells have been known for a long time. This was first reported by Warburg, who noted that cancer cells take up high amounts of glucose which is converted primarily into lactate and has hence been coined the Warburg effect . Whereas non-proliferating cells mainly produce energy by oxidative phosphorylation, proliferative cells and cancer cells also get a significant part of their energy from aerobic glycolysis . During aerobic glycolysis cancer cells convert pyruvate into lactate—a process normally inhibited by the presence of oxygen. HK2 is overexpressed in many human cancers and has been reported to be involved in maintenance of the malignant state of tumors . The overexpression of HK2 in cancer is thought to provide cancer cells with a growth advantage due to increase glycolytic flux by promoting the first step of glycolysis and thus promoting/inducing the shift towards aerobic glycolysis. This type of catabolism of glucose with lactate as the end product produces significantly less ATP than oxidative phosphorylation, but even though the ATP production is reduced, this shift is thought to provide rapidly dividing cancer cells with certain advantages, such as the biosynthesis of nucleic acids as well as providing the cofactor NADPH for synthesis of phospholipids and fatty acids though the pentose phosphate pathway . In addition to creating an acid environment protecting against the immune system and favouring invasion of surrounding tissue . Finally, the Warburg effect also makes the cells less dependent on oxygen, which ensures survival during hypoxic and anoxic conditions.
Here, we reported the identification of HK2 as a target of miR-143, confirming the down-regulation of HK2 upon miR-143 overexpression of transcript cells as well as protein level in both DLD-1 and HCT116 colon cancer cell lines. Interestingly the expression level of HK2 is markedly different between DLD-1 and HCT116 cells. This might be due to different mutations in the two cell lines that are giving rise to the tumorigenic phenotype or different metabolic adaptations to the need for a fast proliferation. By mutation of the miR-143 binding site in the 3'UTR of HK2 we showed that the target interaction between miR-143 and HK2 is direct. We further showed that inhibition of HK2 results in a reduction in cellular proliferation of DLD-1 colon cancer cells, an effect that resembles the effect of miR-143 overexpression. Interestingly, the decreased cell proliferation observed upon HK2 siRNA-mediated knockdown was not as strong as for miR-143 overexpression. This suggests that additional miR-143 targets besides HK2 may also be responsible for the growth inhibitory effect of miR-143. In support of miR-143’s role in glucose metabolism we showed that overexpression of miR-143 in DLD-1 cells leads to a reduced lactate secretion. However, the decrease in lactate secretion as a result of miR-143 overexpression is not as marked as the decrease observed upon HK2 siRNA mediated knockdown. This might be explained by the fact that miR-143 only mediates a relatively moderate reduction of HK2 protein level compared with the siRNA mediated knockdown of HK2.
Here, we have identified a number of putative miR-143 targets in colon cancer cells. We verified HK2 as a direct target of miR-143 and show that miR-143 mediated down-regulation of HK2 results in a decreased lactate secretion. We speculate that loss of miR-143 in cancer cells might promote the metabolic shift towards aerobic glycolysis due to up-regulation of HK2.
Work in the authors’ laboratories are supported by EC FP7 funding (ONCOMIRS, grant agreement number 201102; this publication reflects only authors’ views; the commission is not liable for any use that may be made of the information herein), the Novo Nordisk Foundation, The Lundbeck Foundation, The Danish National Research Foundation, The Danish Medical Research Council, The Danish Cancer Society and the Danish National Advanced Technology Foundation.
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