FOXO3a-mediated microRNA-622 overexpression suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in ERK-responsiveness of lung cancer
Chun-Wen Cheng1,2,*, Po-Ming Chen1, Yi-Hsien Hsieh1, Chung-Chih Weng1, Chia-Wei Chang1, Chung-Chin Yao3, Ling-Yueh Hu4, Pei-Ei Wu4, Chen-Yang Shen4,5,*
1 Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung,
2 Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, 3 Department of Surgery, Chung Shan Medical University Hospital, Taichung, 4 Institute of Biomedical Sciences, Academia Sinica, Taipei,
5 Graduate Institute of Environmental Science, China Medical University, Taichung, Taiwan.
*Correspondence should be addressed:
Dr. Chun-Wen Cheng, Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung (40201), Taiwan, Tel: 2473-0022 ext 11677, Fax: +886-4-2324-8195, E-mail: [email protected] or Dr. Chen-Yang Shen, Institute of Biomedical Sciences, Academia Sinica, Taipei (11529), Taiwan, Tel: 2789-9036, Fax: +886-2-2782-3047, E-mail: [email protected]
Key words: Lung cancer, HIF-1α, miR-622, EMT, FOXO3a, ERK 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
ABSTRACT
Metastatic spread of cancer cells portends the clinical relevance of poor prognosis and mortality of lung cancer patients. Hypoxia-inducible factor-1α (HIF-1α) enhances tumor cell motility through activating epithelia-mesenchymal transition (EMT) which is considered a prerequisite to retain metastatic potential. Recent studies of microRNA involvement in cancer invasion and metastasis have highlighted the prognostic role in tumor development. However, validation of potent tumor suppressor miRNAs by
targeting HIF-1α to down-modulate EMT program and thereby counteract aggressiveness and metastasis of lung cancer cells has been scantily described. Hereby, we identified the 3’-untranslated region (3’-UTR) of HIF-1α mRNA as a target of the miR-622 and
established that miR-622-mediated down-modulation of HIF-1α correlates with decreased levels of mesenchymal proteins, including Snail (SNAI1), -catenin, and vimentin. Functional analyses showed increased miR-622 expression inhibited invasion and migration of lung cancer cells in vitro. We also demonstrated that miR-622 inhibited the genesis of metastatic lung nodules in lung cancer xenograft model in nude mice
transplanted A549 cells carried miR-622. Mechanistic analyses showed that EGF significantly decreased the miR-622 level in A549 cells, and the reduced miR-622 level was rescued by administrating U0126, an inhibitor of ERK. Moreover, miR-622
overexpression mediated by the transcription factor FOXO3a decreased lung tumor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
invasion by inhibiting HIF-1α through inactivation of ERK signaling in U0126-treated A549 cells. These findings highlight the pivotal role of the FOXO3a/miR-622 axis in inhibiting HIF-1α to intercept metastatic cancer cell programming, and this information may contribute to development of novel therapeutic strategies for treating aggressive lung cancer. 1 2 3 4 5 6
INTRODUCTION
Tumor growth and transformation to a malignant state, which is comprised of invasion and metastasis, are key steps in cancer progression. Developing new strategies that decrease malignancy of cancer cells is therefore considered the most challenging issue facing therapeutic interventions against cancer mortality. Factors associated with increased tumor-specific angiogenesis, which is required to resolve hypoxia in tumor
microenvironments, promote expansion of tumor cells . Increasing evidence has demonstrated that hypoxia itself enhances pro-survival mechanisms underlying tumor outgrowth orchestrated by the activation of the hypoxia-inducible factor-1 alpha (HIF-1α) encoded by HIF-1 . HIF-1α is essential for enabling angiogenesis and metastasis in a variety of solid cancers including lung cancer . The epithelial-to-mesenchymal transition (EMT), which can be induced by hypoxia, is considered to be prerequisite that leads to the typical tumor phenotypes of angiogenesis, cell motility, and extracellular matrix invasion. Regulation of the mesenchyme-specific transcription factor gene Snail (SNAI1), which is activated via an HIF-1α signaling cascade, enhances expression of the mesenchymal markers β-catenin and vimentin in hepatocellular carcinoma in response to hypoxia . Importantly, HIF-1α is a key factor responsible for the transcriptional regulation of genes that facilitate the stemness properties of cancer cells and enhance their metastatic potential in leukemia, prostate, and breast carcinomas . Accumulating lines of in vitro evidence 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
indicate that HIF-1α is overexpressed in tumors to induce VEGF expression through activation of the signaling pathway mediated by the MAPK/ERK . Thus, HIF-1α is an established target for the development of cancer therapeutics.
MicroRNAs (miRNAs) are small noncoding regulatory molecules averaging 22 nucleotides in length that principally recognize target sequences of cognate mRNAs via a less-than-perfect complementarity with the 3’-untranslated region (3’-UTR), leading to cleavage of the target mRNA or repression of its translation . More than 30% of protein-coding genes are predicted to be regulated by miRNAs based on bioinformatic algorithms . Intensive studies of lung cancer using gene expression profiling to investigate
tumorigenesis and tumor progression have revealed that miRNAs function as tumor suppressors by negatively regulating oncogenes . However, validation of the potent tumor suppressor miRNAs through targeting HIF-1α to down-modulate EMT program and thereby counteract the aggressiveness and metastasis of lung cancer cells has been scantily described. More importantly, the regulation to retrieve critical molecular information of the miRNA expression in metastatic lung tumor cells that imprint on tumor progression were lesser clarified. To address this deficiency, we predicted that the 3'-UTR of HIF-1α mRNA contains a sequence that directs miR-622-mediated translational repression, and indeed we validated HIF-1α mRNA as a target of miR-622. We thus used lentivirus-mediated
transduction to establish two stable clones of the lung cancer cell lines A549 and H1299 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
that expressed miR-622 to validate the ability of this miRNA to suppress cancer cell motility both in vitro and in vivo. We also detailed the novel mechanism underlying the miR-622-mediated regulation of HIF-1α, which event is activated by the transcription factor FOXO3a. Our findings hereby suggest invaluable clues in which FOXO3a-mediated miR-622 overexpression regulates lung cancer progression may drive towards a candidate miRNA that leads optimization process for the design and evaluation of potential
therapeutic treatment in metastatic lung cancer.
RESULTS
miR-622 represses HIF-1α expression by directly targeting the 3’-UTR
We used a combined computational prediction algorithm approach, including use of the programs: miRBase (http://www.mirbase.org/), miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/), and TargetScan (http://www.targetscan.org/), to examine that miR-622 contains a sequence complementary to the 3’-UTR of HIF-1α (Figure 1A), which is located at human chromosome 14q23.2 (Figure 1B). Toward this end, we used the pGL4.13-luciferase reporter to generate a construct encoding the full-length 3’-UTR of HIF-1α (wild-type HIF-1α 3’-UTR-luc) as well as a 3’-UTR/Mutant-luc with a mismatched version of the miR-622 complementary sequence (Figure 1C). We found that miR-622 significantly reduced the luciferase activity of the HIF-1α 3’-UTR-luc 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
pGL4.13 reporter construct containing the mutation in the 3’-UTR of HIF-1α (Figure 1D). Furthermore, HIF-1α protein repression was more prominent in miR-622-transfected A549 lung cancer cells compared with control (Figure 1E). These results clearly demonstrated that miR-622 decreases HIF-1α expression by directly binding its 3’-UTR.
miR-622 represses HIF-1α to inhibit invasiveness of lung cancer cells
As to tumor hypoxia induces EMT, which leads to invasion and metastasis by
repressing the expression of the epithelial marker, E-cadherin , we therefore examined the suppressive function of miR-622 in lung cancer progression of two lung cancer cell lines in
vitro. As expected, our results demonstrated that the migration and invasion abilities of
miR-622-transfected A549 and H1299 lung cancer cells were diminished more than 50% compared with mock-transfected controls (Figure 2E and Supplementary Figure S1). In addition, miR-622 overexpression in these cell lines reduced HIF-1α level, which resulted in decreased levels of Snail, β-catenin, and vimentin and increased level of E-cadherin (Figure 2D).
Further, to underscore the contribution of miR-622 to the molecular mechanism that enables mesenchymal tumor cells may regain the cobblestone-like epithelial phenotype to inhibit cancer cell aggressiveness through during mesenchymal-to-epithelial transition (MET) transversion of lung cancer cells, by using a lentivirus expression system and the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
stably expressing miR-622 transcripts; the A549-pLKO/miR-622-transfected cells had a ~4.5-fold higher level of miR-622 compared with control (Figure 3A). After the third day of incubation, the proliferation rate of A549-miR-622 cells was significantly lower compared with control (Figure 2C). Interestingly, A549 cells with the increased miR-622 level underwent a morphological change from elongated and spindle-like fibroblasts to a cobblestone-like epithelial phenotype (Figure 3B). These data indicated that miR-622 overexpression in A549 cells resulted in altered cell morphology and diminished tumor cell proliferation as a consequence of the repression of HIF-1α expression. On the other hand, the decreased cancer cell mobility upon miR-622 overexpression was restored upon
transfection of A549 cells with a miR-622 inhibitor, and vimentin level was restored to that of the control (Figure 2D-2F). In parallel, results from HIF-1α silencing experiments with two short hairpin RNAs targeting HIF-1α mRNA (shHIF-1α) revealed dramatic decreases in the migration and invasion of lung cancer cells (Figure 2G-2I), lending support to our theory that miR-622 inhibits tumor motility via repression of HIF-1α to downmodulate the EMT axis.
miR-622 suppresses metastasis in a xenograft-transplantation model of lung cancer Because we found that miR-622 plays a critical upstream mediator role in
regulating lung cancer invasion and migration and repressing HIF-1α expression in vitro (Figure 2), we explored whether miR-622-associated metastatic suppression occurs in vivo. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
To examine whether miR-622 downregulates the HIF-1α axis with consequent effects on tumor motility, we established a xenograft model of human lung cancer cells in nude mice. In our mouse model, A549 cells containing pLKO (control) or pLKO/miR-622 were injected into the tail vein (5 × 106 cells/mouse). After 6 weeks, the average body weight in the control group was significantly reduced compared with the miR-622 overexpression group (p = 0.008, Figure 3A and 3B). Mice were sacrificed and their lungs dissected to evaluate tissue morphology by hematoxylin and eosin staining. We found that mice injected with A549-pLKO/miR-622 cells had only a few pulmonary metastatic nodules on average and significantly fewer than the number of nodules formed in the control group (p = 0.001, Figure 3C). The control mice had significantly larger tumors and greater neovasculature development and the metastatic lung cancer tissues that lack miR-622 overexpression had intensively positive staining for the HIF-1α protein in the immunohistochemical analysis (Figure 3D), which supports our hypothesis of a suppressive function for miR-622 in lung cancer metastasis in vivo.
miR-622 is downregulated by ERK in lung cancer
miRNAs are frequently downregulated by promoter hypermethylation in different types of solid cancers . We therefore examined whether miR-622 reduction was ascribable to DNA hypermethylation in lung cancer cells. However, we found no significant change in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
miR-622 level in A549 cells treated with 5-aza-2'-deoxycytidine (Figure 5A). Alternatively, evidence has been shown that induction of angiogenesis by HIF-1α in cancer cells is activated by EGF/AKT/ERK signaling during hypoxia and our analysis of miR-622-overexpressing cells revealed decreased HIF-1α-dependent cell invasion, we therefore hypothesized that lung cancer cell aggressiveness could be rescued by EGF/AKT/ERK signaling to compensate for the downregulation of HIF-1α which is regulated by miR-622. Support for our hypothesis came from our observation that A549 cells treated with 10% serum or EGF (20 nM) had a reduced level of miR-622 (Figure 5C); this lower miR-622 level led to increased HIF-1α as a consequence of ERK phosphorylation in the EGF-treated A549 cells (Figure 5B). This inhibitory effect on miR-622 transcription was comparable to that of the ERK inhibitor, U0126, and inactivation of phosphorylated ERK (p-ERK) in the lung cancer cells resulted in a higher miR-622 level in a dose-dependent manner (Figure 5D). Beside, monitoring phosphorylation of Akt at T308 improves the assessment of Akt activation, and show that Akt activation is a poor prognostic factor for lung cancer , we thus examined the activation of AKT in association with miR-622 expression level during hypoxia; however, the T308 residue of AKT kinase is not phosphorylated in response to EGF treatment in A549 cells (Figure 5E). In addition, although phosphorylation of ERK reduced the miR-622 level, miR-622 level was not affected by HIF-1α knockdown in A549 cells (Figure 5F). We conclude that a block of EGF/ERK signaling may inhibit lung cancer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
metastasis via activation of the miR-622-repressed HIF-1α axis, in which axis dose not proceed a feedback inhibition of miR-622 level by HIF-1α.
FOXO3a upregulates miR-622 level
Inhibition of FOXO proteins by ERK depends on ERK phosphorylation that leads to ubiquitination of FOXOs and their subsequent degradation in proteasomes . Based on bioinformatics, we found that primary microRNA transcript of miR-622 (pri-miR-622) is located at chromosome 13q31.3, and an in silico analysis with ALLGGEN_PROMO predicted three putative FOXO3a-binding sites in the pri-miR-622 promoter region; thus, we speculated that FOXO3a might regulate miR-622 expression. To test this possibility, we designed a FOXO3a knockdown experiment in combination with altered pri-miR-622 promoter constructs. In the gene encoding miR-622, three fragments upstream of the 5’-UTR of were amplified and cloned into the vector pGL4.21-Basic-Luc; these were named (–845/+1)-Luc, (–845/–376)-Luc, and (–376/+1)-Luc (Figure 6A). In the presence of U0126 (40 μM), A549 cells having either promoter construct (–845/+1)-Luc or miR-622 (– 376/+1)-Luc showed an approximately 2-fold increase in luciferase activity compared with the negative control (Figure 6A). Additionally, chromatin immunoprecipitation assays performed with an antibody against FOXO3a showed that FOXO3a binds the upstream promoter region of the miR-622 gene in response to ERK (Figure 6B). Further, because 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
U0126-mediated ERK inactivation in A549 cells stabilized endogenous FOXO3a mRNA (Supplementary Figure S2A), as demonstrated with western blotting (Supplementary Figure S2B), we designed a FOXO3a knockdown experiment in U0126-treated A549 cells and found that the effect of FOXO3a on miR-622 elevation was abrogated by shFOXO3a (Figure 6D). Concomitantly, in the presence of ERK inactivation, the decreased level of HIF-1α in the miR-622-overexpressing A549 cells was restored upon transfection with FOXO3-specific short hairpin RNA (shRNA-FOXO3)(Figure 6C), which also resulted in enhanced tumor cell invasiveness compared with control oligonucleotide-transfected cells (Figure 6E). Taken together, the inhibitory effect of FOXO3a-mediated miR-622
expression on inhibition of cancer cell invasion is controlled by suppressing ERK-HIF-1α signaling.
ERK activation downregulates FOXO3a-miR-622 axis to increase HIF-1α expression in lung cancer cell invasion
As shown in Figure 6, FOXO3a upregulates miR-622 to repress HIF-1α expression through inactivation of ERK phosphorylation in inhibiting invasion of A549 cancer cells. By introducing knockdown of ERK gene, we found that increase in FOXO3 protein in combination with decreased level of HIF-1α in A549-pLKO/miR-622 cells with treatment of shERK1/2. Further, we used a luciferase gene construct encoding the promoter region of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
HIF-1α (pGL4.21 (–376/+1), shown in Figure 6A) and promoter activity values showed that increased luciferase activity in cells with ERK1/2 gene knockdown normalized
luciferase activities of the reporter in control group of short hairpin RNA. On the contrary, this elevation was reduced in shERK1/2-cells treated with shFOXO3a by >60% (Figure 7B). In addition, for an immunoprecipitation with chromatin using an antibody against FOXO3a, capture of the DNA fragment was shown in A549-pLKO/miR-622 cells transfected with shERK1/2 as compared with cells containing the shFOXO3a or shRNA negative control (Figure 7C). Concomitantly, we detected the expression levels of miR-622 in A549 cells with shERK1/2 treatment (Figure 7D), and showed the expression of miR-622 was significantly higher in cell when compared to shRNA (NC), and the expression level of miR-622 in the shFOXO3-treated cells was much lower than those in the shERK1/2 cells, indicating that miR-622 downregulation correlates with ERK-FOXO3a axis of HCC. Likewise, in miR-622 stably expressing A549 cells, we confirmed that the number of invading cells was significantly reduced upon shERK1/2 treatment. Moreover, the decreased level of cell invasion in the shERK1/2-transfected A549-pLKO/miR-622 cells was restored upon transfection with shFOXO3a, again, confirmed FOXO3a-miR-622 in repressing HIF-1α expression to account for ERK activation in human lung cancer cell invasion (Figure 7E and 7F).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
DISCUSSION
Down-regulation of miR-622 promotes cellular invasion and tumor metastasis in different types of cancer, including, brain, esophageal, and gastric cancer , however, the molecular mechanism involved in lung cancer metastasis and the target gene that miR-622 modulated remain unknown yet. In the hypoxic microenvironment of solid tumors, HIF-1α expression in certain clones affords a selective advantage through multiple mechanisms that rely on angiogenesis and EMT, which ultimately increases tumor aggressiveness. HIF-1α overexpression in lung tumor cells is associated with increased invasion capacity and metastasis, and HIF-1α serves as a biomarker of poor prognosis in human lung cancer . The most prescient findings of the present study are that miR-622 directly targets HIF-1α as assessed with three matching algorithms and that the resultant repression of HIF-1α expression can inhibit cancer cell migration and invasion as assessed in vitro in two lung tumor cell lines under hypoxia environments. Our xenotransplantation study of the tumorigenicity of human lung cancer cells in mice confirmed that the number of lung nodules was significantly decreased in animals that received cells overexpressing miR-622, which facilitated downregulation of HIF-1α level. These findings not only describe a relationship between the miR-622-mediated suppression of lung cancer metastasis (based on targeting HIF-1α) but also delineate a mechanism underlying the inhibitory effect of miR-622 on lung cancer progression via repression of the transcription factor of Snail 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
which is responsible for the mesenchymal phenotype progression during EMT. Recent studies have shown that miRNA expression in tissues is regulated by methylation of DNA sequences upstream of the corresponding miRNA gene and that cancer cells are commonly hypermethylated compared with normal cells . We conjectured that this modification could be involved in miR-622-mediated silencing in lung cancer, and thus we investigated global DNA methylation patterns of the miR-622 promoter in A549 cells. Our results, however, showed that miR-622 reduction in lung cancer did not
encounter by DNA hypermethylation in promoter sequence of itself. In contrast, expression levels of certain miRNAs are tightly regulated during the cellular response to hypoxia in poorly differentiated solid tumors, and indeed EGF acts as an inhibitor to decrease the levels of tumor-suppressor miRNAs . Intriguingly, we found a correlation between EGF treatment and miR-622 reduction under hypoxia, which led to an elevated HIF-1α level and promoted tumor cell invasion (Figure 5). Hypoxia upregulates the EGF receptor and prolongs the activation of ERK and AKT signaling, which contributes to tumor-related angiogenesis and tumorigenesis . Our present data showed that, although treatment with EGF in A549 cells did not activate the AKT kinase, it enhanced the phosphorylation of ERK which led to decreased FOXO3a level. Notably, blocking ERK activation with U0126 enhanced FOXO3a-mediated miR-622 transcription that resulted in inhibition of HIF-1α expression in cancer cells, leading to decreased tumor invasion and metastasis (Figure 8). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Going beyond the current study of the inhibitory effects of miR-622 on lung cancer progression owing to HIF-1α reduction, our results suggest a mechanism underlying down-modulation of HIF-1α by miR-622 that then inactivates EMT pathway genes, leading to reduced levels of Snail, β-catenin, and vimentin. In response to hypoxia, HIF-1α and a broad array of its downstream targets are synthesized de novo as a consequence of defects displayed by a variety of tumor suppressors in concert with organ-specific cancer cell invasion and migration. For example, deficiency of von Hippel-Lindau function counters the degradation of HIF-1α under normoxia , HIF-1α represses the transcription of the E-cadherin gene, contributing to EMT in von Hippel-Lindau-null renal cell carcinomas . In brain and breast cancers, HIF-1α overexpression increases angiogenesis by upregulating the levels of vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor . In more recent, association studies between miRNA markers and lung cancer development have demonstrated that the miRNAs miR-18, miR-199, and miR-519c can suppress HIF-1α expression for assessing cancer prognosis , Therefore, it would be informative to be able to validate additional genetic factors, including miRNAs that are involved in modulating HIF-1α level in collaboration with mesenchymal markers to potentially help predict risk of aggressive lung cancer.
MiRNAs post-transcriptionally regulate the expression of hundreds of tumor-suppressor genes that control a wide range of biological and physiological events, leading 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
to inhibition of tumorigenesis and progression as well as the promotion of tumor cell death in different cancer types . To our knowledge, the present study is the first to suggest a potential prognostic significance of FOXO3a-mediated miR-622 transcription that then downregulates HIF-1α and decreases tumor aggressiveness in response to EGF-activated ERK signaling in lung cancer. Our data demonstrate a role for miR-622 in repressing metastasis through inhibition of HIF-1α-related EMT signaling and thus suggest that miR-622 could be utilized as a promising target for the development of a lung cancer
therapeutic. 1 2 3 4 5 6 7 8
MATERIALS AND METHODS
Cell lines, virus generation, and infection
The lung cancer cell lines A549 and H1299 were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Life Technologies) containing 0.1 mM sodium pyruvate, 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The lung cancer cell line A549 was obtained from the American Type Culture Collection and tested positive for the presence of EGF receptor expression in the current genetic analysis. For the incubation of cells under hypoxic mimics, cells were treated with deferoxamine mesylate (DFX, Sigma-Aldrich, St. Louis, MO, USA) or by culturing cells in a hypoxia chamber (1% O2, 5% CO2 and 94% N2 atmosphere) at 37°C. For transfection, cells were carried out in the Lipofectamine™ 2000 transfection reagent according to manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. Briefly, 2 × 106 HEK293T (human embryonic kidney) cells were cotransfected with 10 μg of the lentiviral vector pLKO (control) or pLKO/miR-622, 9 μg of pCMV△R8.91 (packaging plasmid), and 2.5 μg of pMDG (envelope plasmid) were purchased from National RNAi Core Facility at Academic Sinica, Taiwan. At 24 h post-transfection, virus-containing supernatants were collected Lentiviral infection was performed by adding virus-containing solution to A549 cells (1 × 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
106) at the desired multiplicity of infection in the presence of 8 ng/mL polybrene. After 48 h, stable transfectants were selected under 5 µg/mL puromycin. miR-622 expression was confirmed by real-time PCR.
Reverse transcription and real-time PCR
Primers specific for miR-622 and random hexamers [supplied with the TaqMan miRNA assay were obtained from Applied Biosystems. Real-time PCR was performed using an Applied Biosystems 7000 Fast Real-time PCR system with miR-622 primers and TaqMan Universal PCR Master Mix and AmpErase UNG (uracil-N-glycosylase; Applied
Biosystems). Values represent the average of two independent experiments, normalized to the endogenous control gene (RNU-6B).
Cell proliferation assay
Lung cancer cells (2 × 103 cells) were cultured in 96-well flat-bottomed microtiter plates supplemented with DMEM containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cell viability was determined by the MTT (methyl thiazolyl tetrazolium) assay (absorbance read at 570 nm), and cell viability is expressed as a percentage of viability measured for the relevant control cells.
Western blotting
Detailed procedure for western blot was described elsewhere . Western blotting used 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
8312), p-AKT (sc-16646-R), ERK (sc-94), p-ERK (sc-7383), vimentin (sc-6260), and vascular endothelial growth factor (sc-53462); each antibody was purchased from Santa Cruz Biotechnology, CA). Antibodies against HIF-1α (Cell Signaling) and E-cadherin (cat. 610182, BD Biosciences, Franklin Lakes, NJ) were also used. For western blotting, proteins separated by SDS-PAGE were transferred to a Hybond-C Extra membrane (GE Healthcare, Little Chalfont, UK) that was then subjected to western blotting with an appropriate primary antibody as indicated. Anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody for detection using an ECL western blot detection system and the intensities of the bands quantified by densitometric analysis (Digital Protein DNA Imagineware, Huntington Station, NY).
Site-directed mutagenesis
Site-directed mutagenesis was performed to generate the mutant HIF-1α 3’-UTR sites of the luciferase construct using complementary oligos (Figure 1). Plasmids containing multiple point mutations of the sites were generated using the QuikChange site-directed mutagenesis system (Stratagene, Santa Clara, CA). Different concentrations of expression plasmids were transiently transfected into lung cancer cells (1 × 106) using Transfast reagent (Thermo, Waltham, MA). After 48 h, the cells were harvested, and whole-cell extracts were assayed in subsequent experiments.
Flow cytometry analysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
pellets were washed twice with PBS and fixed with 70% ethanol at 4°C for 30 min. After centrifugation at 200 g for 5 min, the cell pellets were washed with PBS to remove any residual ethanol. Finally, the cells were resuspended in 1 mL of solution containing 0.5 mg/mL RNase A, 1% (w/v) Triton X-100, and 40 μg/mL propidium iodide and incubated at 37°C for 30 min. The cells were filtered through a 40-μm nylon mesh before flow cytometry analysis of cell cycle distribution using a FACSCalibur flow cytometer (BD Biosciences).
Migration/invasion assay
The cells were trypsinized and collected from the dishes. Samples consisting of 5 104 cells were seeded into 24-well modified Boyden chambers with polycarbonate
membranes (8 μm pore size) to evaluate their migration (without Matrigel) and invasion (with Matrigel) capability for 12 h.
Xenograft tumor formation
All mice were housed in the animal facility at the Chung Shan Medical University Experimental Animal Center, Taichung, Taiwan. Ethical approval was obtained for the use of animals and all experiments were performed in accordance with the guidelines for animal care of the Institutional Animal Care and Use Committee of Chung Shan Medical University. Five-week-old female immunodeficient nude mice (BALB/c nu/nu) were injected with A549-pLKO or A549-pLKO/miR-622 cells via the tail vein (5 × 105 cells in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
metastatic lung tumors was confirmed with hematoxylin and eosin staining under a dissecting microscope.
Statistical analysis
Statistical significance of the experimental data grouped by one variable was assessed by the unpaired two-tailed Student’s t-test, one-way analysis of variance, or Dunnett's test where appropriate. All statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). A value of p < 0.05 was considered to indicate statistical
significance.
FUNDING AND ACKNOWLEDGEMENTS
This work was supported by research grant NSC 102-2628-B-040-002-MY3 from the Ministry of Science and Technology, Taipei, Taiwan, ROC.
LIST OF ABBREVIATIONS
HIF-1α: HIF-1α; miRNA: microRNA; EMT: Epithelial-to-mesenchymal transition; EGF: epidermal growth factor; ERK: extracellular signal-regulated kinase; UTR: 3’-untranslated region, ChIP: Chromatin immunoprecipitation;
CONFLICT OF INTERESTS STATEMENT The authors declare they have no conflict of interest. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
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FIGURE LEGENDS
Figure 1: HIF-1α is a direct target of miR-622. (A) miR-622 contains a sequence complementary to the 3’-UTR of HIF-1α by using three matching algorithms. (B) The predicted miR-622-target sequence is located at position 869-875 of the HIF-1α 3’-UTR in human chromosome 14q23.2. (C) Schematic representation of the luciferase reporter constructs. The three bolded nucleotides represent mutant sites in the HIF-1α mRNA created by site-directed mutagenesis to yield mismatches with the complementary sequence in miR-622. (D) Cells were cultured in 24-well plates and transfected with 100 ng of wild-type or mutated HIF-1α 3’-UTR construct to A549-pLKO control cells (the negative control) and A549-pLKO/miR-622 cells. The firefly luciferase/Renilla luciferase activity ratio of each sample was measured in the dual-luciferase reporter assay system (Promega Company, WI, USA). Each bar represents the mean ± standard deviation of three independent experiments; * p < 0.05. (E) HIF-1α level in A549-pLKO control cells and its reduced level in A549-pLKO/miR-622 cells overexpressing miR-622, as revealed by western blotting.
Figure 2: Inhibitory effect of miR-622 on the migration and invasion of lung cancer cell lines. (A)-(D) Boyden chamber assay for cells that were transiently transfected with miR-622 mimic or with mock transfection (miR-mimic, control) and then plated on 8 µm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
cell culture coated without (A) or with (B) matrigel. Migration (C) and invasion (D) assays for miR-622-transfected HCC cells were treated with miR-622 inhibitor or negative control of anti-miRNA (anti-miR (NC)). (A)-(D), cells in five random fields of view at 100× magnification were counted and expressed as the average number of cells per field of view. Results represent the average of three independent experiments (mean ± S.D.). *P < 0.05, **P < 0.01, and ***P < 0.001. (E) and (F) Western blot of the protein expression of HIF-1α and epithelial-mesenchymal transition markers (Snail, E-Cadherin, β-catenin, and vimentin) in HCC cells of A549 (left panel) and H1299 (right panel) transiently expressing 622 mimic or miRNA-mimic (control group) in (E) and miR-622-transfected HCC cells treated with miR-622 inhibitor (anti-miR-622) or negative control of anti-miRNA (anti-miR (NC)) in (F), respectively. β-actin was used as an internal control.
Figure 3: Overexpression of miR-622 inhibits the migration and invasion through repression of HIF-1α in lung cancer cells. (A) Real-time PCR detection of the expression levels of miR-622 in A549 cells stably transfected with pLKO (Control) or A549-pLKO/miR-622 as quantified by. The RNU6B small nuclear RNA was used as an internal control. (B) Proliferation of A549-pLKO (control) and A549-pLKO/miR-622 cells as assessed with the MTT assay. (C) Representative images of phenotypic change 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
from mesenchymal (A549/pLKO) to cobblestone-like epithelial state (arrow indicated) as seen in A549 cells overexpressing miR-622 (A549-pLKO/miR-622). (D) Western blot of the protein expression of HIF-1α and epithelial (E-Cadherin) and mesenchymal markers (vimentin) in pLKO/miR-622 stably expressing A549 cells treated with negative control (NC) of anti-miRNA or with miR-622 inhibitor (anti-miR-622). (E) and (F) Boyden chamber assay for detection migration/invasion in miR-622 stably expressing A549 cells treated with anti-mi or with miR-622 inhibitor RNA, respectively.
(G), (H), and (I). Western blot (G) and boyden chamber assay ((H) and (I)) in A549-pLKO/miR-622 cells that were transfected with short hairpin RNA fragments of HIF-1α (shHIF-1α-1 and shHIF-1α-1). Similarly, migration (H) and invasion (I) of cells were measured in five random fields of view at 100× magnification which were counted and expressed as the average number of cells per field of view. Results represent the mean ± S.D. of three independent experiments in figure (B), (E), (F), (H), and (I). * p < 0.05 and ** p < 0.01.
Figure 4: miR-622 inhibits the growth of xenografted lung cancer tumors. (A) Mouse xenotransplantation studies were carried out in BALB/c null mice (n = 5 per group). (B) Loss of body weight as measured on day 42 following tail-vein injection with 5 × 105 A549-pLKO/miR-622 cells or A549-pLKO cells (control; p = 0.008). (C) Metastatic lung 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
tumors in mouse xenografts at 42 days after injection with A549 cells stably
overexpressing miR-622 (A549-pLKO/miR-622) or cells with stably integrated empty vector of A549-pLKO (upper panel). Number of metastatic tumor nodules in lungs of nude mice on day 42 following injection with A549-pLKO/miR-622 cells or A549-pLKO cells (control). Data represent the mean ± standard deviation for each group (lower panel). (D) Hematoxylin and eosin (H&E) staining and immunofluorescence staining detected HIF-1α of lung tissue sections bearing metastatic xenograft tumors with neovasculature formation (magnification with 200x and 400x) are shown. Arrows indicate vasculature development of the lung tumor nodule.
Figure 5: Effects of the EGF-ERK signaling pathway on the regulation of miR-622 expression in invasiveness of HCC. (A) A549-pLKO cells were treated with 5-aza-2'-deoxycytidine (5-AZA-dC; 10 or 20 μM) for 72 h. Quantitative real-time PCR was used to determine miR-622 level normalized to untreated cells. N.S., not significant. (B) Western blotting of the protein expression of HIF-1α, ERK, phosphorylated-ERK1/2, AKT kinase, and phosphorylated-AKT at threonine residue 308 (P-AKT (Thr308)) in A549-pLKO.miR-622 stably expressing cells under serum starvation or restored by treatment with either 10% fetal bovine serum (FBS) or EGF (20 nM) for 6 h in a hypoxia state as described in Materials and methods. qRT-PCR (C) detection of the miR-622 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
expression in A549-pLKO/miR-622 cells treated with epidermal growth factor (EGF, 20 nM) or 10% fetal serum albumin (FBS), respectively. RNU6B was used as an internal control. Boyden chamber assay (D) for the assessment of invading activity in A549-pLKO/miR-622 cells treated with EGF (20 nM) or 10% FBS for 12 hr. (E) Western blot of the protein expression of HIF-1α, ERK and phosphorylated-ERK1/2, (left panel) and qRT-PCR analysis of miR-622 expression (right panel) in miR-622 stably expressing A549 cells treated with ERK kinase inhibitor, U0126, for 24 hr. (F) Western blot (G) and qRT-PCR detection of the HIF-1α expression in A549-pLKO/miR-622 cells were
transfected with HIF-1α inhibitors (shHIF-1α-1 and shHIF-1α-2). The results presented in C–D are the mean ± S.D. of three independent experiments. * p < 0.05 and ** p < 0.01. N.S., non-significant.
Figure 6: Expression of the miR-622 gene is activated by FOXO3a. (A) Analysis of the miR-622 promoter. Schematic representation of the different putative miR-622 promoter regions (top panel) that were inserted upstream of the pGL4.21-basic luciferase gene, and the luciferase activity was measured. Luciferase expression was greater in the FOXO3a-positive cells transfected with the reporter gene linked to the whole promoter sequence [pGL4.21-(–845~+1)] or the partial promoter sequence [pGL4.21-(–376~+1)] compared with pGL4.21-basic vector. (B) The interaction between FOXO3a and two 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
miR-622 promoter regionsⅠ(nt –845~+1) or -II (nt –376~+1) in A549 cells was detected by chromatin immunoprecipitation with a FOXO3a-specific antibody or anti-IgG (control). (C) Western blotting of the protein expression of FOXO3a, HIF-1α, ERK1/2 and phosphorylated ERK in A549 cells treated with shFOXO3a-1 or shFOXO3a-2 in the presence of ERK inhibitor U0126. (D) miR-622 expression in A549 cells is reduced upon FOXO3a knockdown using shFOXO3a fragments. qRT-PCR analysis to determine the expression level of miR-622 in A549 cells transfected with shFOXO3a-1 or shFOXO3a-2 under treatment of the ERK inhibitor U0126. (E) Boyden chamber assay for assessment of the invasiveness of shFOXO3a-A549 cells after treatment with the U0126. Data represent the mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 7: Phosphorylated-ERK downregulates FOXO3a-miR-622 to increase HIF-1α expression in human lung cancer cell invasion. (A) Western blot detected ERK, p-ERK1/2, FOXO3a, and HIF-1α in A549-pLKO/miR-622 cells that were transfected with short hairpin RNA fragments, negative control (NC), shERK1/2, and shFOXO3a-2, respectively. (B) A549-pLKO/miR-622 cells transfected with pGL4.21 containing – 376/+1-Luc reporter construct along with shREK1/2, shFOXO-3a or shRNA negative control as indicated were incubated 24 h and then harvested for reporter gene assays. The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
relative luciferase activity is represented as mean ± S.D. (C) An interaction between FOXO3a and two miR-622 promoter regionsⅠ(nt. -845~+1) or -II (nt. -376~+1) in A549 cells was detected by chromatin immunoprecipitation with a FOXO3a-specific antibody or anti-IgG (control) as detected by transfection of shERK1/2, shFOXO3a and shRNA (NC) in A549 cells. (D) qRT-PCR analyses of HIF-1α expression levels from pLKO/miR-622-stably expressing A549 cells, (E) Representative photos of boyden chamber assay, and (F) invasion assessment of A549-pLKO/miR-622 cells transfected with different shRNAs fragments as indicated in (A) are shown. Results represent the mean ± S.D. of three independent experiments in figures (B), (D), and (E). * p < 0.05 and ** p < 0.01.
Figure 8. Schematic representation indicates the oncogenic role of hypoxia-inducible factor-1α (HIF-1α) in epithelial-mesenchymal transition via activation of Snail to promote cancer cell progression. A blockade of EGF/ERK signaling enhances FOXO3a-induced miR-622 transcription inhibits the HIF-1α-EMT axis, leading to diminished tumor invasion and metastasis in lung cancer.
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