Down-regulation of Micro-RNA-1 (miR-1) in Lung Cancer

Micro-RNAs are ∼21–25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPα, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.

Lung cancer is the leading cause of cancer-related deaths both in men and women in the United States, with an incidence of ϳ213,000 new cases/year (1). Approximately 80% of lung cancers are classified histopathologically as non-small cell lung cancers. At early stages of non-small cell lung cancer, the only treatment is surgery, with a 5-year overall survival rate of 40% (2), whereas chemotherapy is mostly employed for small cell lung cancer. The majority of patients have developed an aggressive form of the disease by the time of diagnosis, limiting the scope for therapeutic intervention. At this stage, several genetic and epigenetic changes take place, resulting in epithelial cell hyperplasia and, eventually, dysplasia. These changes are attributed to silencing of tumor suppressor genes, dysregulation of proto-oncogenes, and an up-regulation of genes that promote cell growth and transformation and ultimately tumor development (3).
In recent years, there has been a considerable interest in understanding the role of micro-RNAs (miRs) 5 in disease processes, including cancer. miRs, which encode small noncoding RNA of ϳ21 nucleotides, are now recognized as a large gene family expressed in plants, animals, and viruses as well as in unicellular algae (4). Most animal miRNAs are evolutionarily conserved and often found in clusters (5). Primary miRNAs with the stem-loop structure are predominantly transcribed by RNA polymerase II and are processed both in the nucleus and cytoplasm by RNase III-like enzymes Drosha and Dicer to generate mature miRNAs (6,7). The mature miRNA is assembled into an miRNA-induced silencing complex, which then directs its binding to the cognate sequence in the 3Ј-UTR of target mRNAs.
In animals, miRNAs are thought to regulate gene expression by imperfect base pairing with the 3Ј-UTR of target mRNAs that results in translational repression or degradation of the mRNA (4,5,8). Micro-RNAs exhibit enormous regulatory potential, since each miR can target multiple mRNAs, which, in turn, are targeted by multiple miRs in combinatorial fashion. Numerous studies to date have established the role of micro-RNAs in diverse cellular processes that include differentiation, apoptosis, cell proliferation, metabolism, immunity, and development (9 -11). Recently, it has been shown that some micro-RNAs stimulate translation of the target mRNAs under stressed conditions, such as nutrient deprivation (12) or cell cycle arrest (13), whereas translation of the same mRNAs is impeded under normal growing conditions. It is, therefore, likely that many more molecular functions of miRNAs are yet to be identified.
Aberrations in expression of micro-RNA are associated with different diseases like cardiovascular (14) and neuronal disorders (15), inflammation (16), and cancer (17). Tumor-specific micro-RNA gene expression profiling revealed distinct miR signatures for many types of cancers, including lung cancer (18 -20). Detection of miRNA genes at fragile sites that are frequently amplified, deleted, or rearranged in cancer provides further evidence of a causal role played by micro-RNAs in cancer pathogenesis (17,21). Furthermore, depending upon the cell type, several miRs deregulated in human malignancies exhibit oncogenic or tumor suppressor properties (17). Some micro-RNAs, such as let-7 and miR-29a/b/c, are down-regulated in lung cancer (20,22,23). In fact, let-7 exhibited growthinhibitory properties in lung cancer cells (24). Thus, it is conceivable that many more miRs will play a critical role in lung tumorigenesis and can potentially serve as biomarkers and targets for anticancer therapy.
Micro-RNA-1 is a muscle-enriched micro-RNA that inhibits proliferation of progenitor cells and promotes myogenesis (25)(26)(27). Several key observations prompted us to examine the antitumorigenic function of miR-1 in other tissues, such as that of the lung, which expresses miR-1, albeit at a lower level than in the muscle (28). First, comparison of genome positions of mouse tumor susceptibility loci with those of mouse miRNA genes revealed that the flanking region of miR-1-1 has six substitutions in the mouse strain susceptible to lung cancer compared with the resistant strain (29). Second, loss of function of only one of the two miR-1 genes (miR-1-2) in mice inhibits differentiation and promotes hyperplasia of cardiomyocytes (30). Third, several data bases also predicted the presence of proto-oncogene MET, which is up-regulated in many cancers, including lung cancer, as a potential miR-1 target. Here, we report that miR-1 is indeed suppressed in primary lung cancers compared with the matching normal tissues. This observation led us to explore its growth-suppressing functions in lung cancer cells as well as the underlying molecular mechanism of growth suppression.

EXPERIMENTAL PROCEDURES
Cell Culture and Tissue Procurement-Human lung cancer cell lines were obtained from ATCC. Human bronchial epithelial (BEAS-2B) cells and the lung cancer cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum, cells were harvested for RNA isolation, and whole cell extracts were subjected to Western blot analysis. Primary human lung cancer and adjacent normal tissue samples were obtained from the Cooperative Human Tissue Network at the Ohio State University James Cancer Hospital. Tissue specimens were procured in accordance with the Ohio State University Cancer Internal Review Board guidelines.
TaqMan RT-PCR for Quantification of miR-1-DNasetreated total RNA was isolated from lung cancer tissues and cell lines, and mature miR-1 and miR-191 were measured with the Applied Biosystems TaqMan micro-RNA assay protocol, as described (31). miR-1 expression was normalized to 18 S rRNA and hsa-miR-191 using the 2 Ϫ⌬CT method. The primer sequences are described in the supplemental material. Northern blot analysis was performed as described (19).
RT-PCR Analysis-MET, Pim-1, and FoxP1 mRNA and 18 S rRNA were measured in cDNA synthesized from DNasetreated total RNA using SYBR Green chemistry.
Plasmid Construction and Generation of Stable Cell Lines-miR-1 expression vector was obtained from Addgene (32). The miR-1d gene (500 bp) was excised from pTRE-TIGHT with EcoRI and SalI and cloned into the same sites of pBabe-puro. Approximately 2 ϫ 10 6 cells were plated per 100-mm dish 24 h prior to transfection. A549 cells were transfected with 20 g of miR1-pBabe or pBabe using Lipofectamine 2000 reagent (Invitrogen), following the manufacturer's protocol. After 72 h, cells were seeded into two 100-mm plates, and miR-1 expressing clones were selected with 1.5 g/ml puromycin.
The 3Ј-UTR of MET was amplified from human lymphocyte DNA using Accuprime Taq polymerase (Invitrogen) and cloned into pDrive cloning vector (Qiagen). Inserts were retrieved with MluI and NheI, cloned into the same sites of a luciferase reporter vector, pIS0, obtained from Addgene (33). Deletion of miR-1 complementary sites from 3Ј-UTR of MET was performed by PCR. The primer sequences are described in the supplemental material.
Transfection of Cells with Pre-miR-1, Antisense miR-1, or Control miR-H1299 cells were transfected with 100 nM pre-miR-1 (Applied Biosystems), and N417 cells were transiently transfected with 50 nM antisense miR-1 or negative control RNA using Lipofectamine 2000 reagent (Invitrogen), following the manufacturer's protocol. After 24 h, cells (4000/well) were suspended into complete RPMI medium and seeded into 96-well plates to monitor growth by an MTT assay. For the miR-1 assay and Western blot analysis, cells were harvested for RNA and protein, respectively, after 48 h. Cells were transfected with rat C/EBP␣ expression vector, and the miR-1 level was measured after 60 h by real time RT-PCR analysis.
In Situ Hybridization-In situ detection of miR-1 with LNAmodified anti-miR-1 (Exicon) in the lung tissue was determined as described (34,35). The negative controls consist of all reagents minus the probe and of a scrambled probe (the same sequence as the antisense miR-1 but where the nucleotides have been "scrambled" at random so that it has very low homology with the target sequence).
RT in Situ PCR of miR-1-The protocol we used has been previously described (36). Primers for the miRNA precursors were the same as used for solution RT-PCR (35).
Cell Proliferation Assay-Cell proliferation was monitored using Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science). A549 cells (3000/well) stably expressing miR-1 or pBabe were allowed to grow in 96-well plates. Cell proliferation was documented every 24 h following the manufacturer's protocol. All experiments were performed in quadruplicate. The thymidine incorporation assay was performed as described (36,37). The soft agar assay was performed as described (38).
Cell Motility Assays-Wounds were generated using a pipette in A549 cells plated at equal density in 6-well plates, grown to confluence, and rinsed with phosphate-buffered saline, and fresh culture medium was added. Wound areas were marked and photographed at different time points using a phase-contrast microscope (39).
For cell migration, A549 cells stably expressing pBabe or miR-1 were placed in a serum-free medium for 24 h. Chemotactic assays were done in 24-well trans-well inserts of 8-m pore size (Corning Costar Corp.). Cells (1 ϫ 10 4 ) were layered onto the top well in serum-free medium. The bottom chambers contained serum-supplemented medium that acted as a chemoattractant. The migration of cells was allowed to proceed for 48 h at 37°C. Cells that migrated to the bottom of the insert were fixed, stained, and counted, and the percentage of migration was determined. Filters were washed thoroughly in water and suspended in 0.5 ml of water or phosphate-buffered saline containing 5% acetic acid and 5% methanol and stained with Hema-3 (Fisher), and absorbance was measured at 595 nm. Absorbance of cells incubated in the serum-free medium in the bottom chamber was used as negative control. Each experiment was performed at least three times.
Tumor Growth in Nude Mice-Nude mice (4 -6 weeks old) were used for xenograft studies. A549 cells (2 ϫ 10 6 in phosphate-buffered saline containing 50% Matrigel) expressing miR-1 or control micro-RNA were injected subcutaneously to flanks of nude mice. After 4 weeks, tumor volume was measured using a slide caliper and was calculated using the formula, d 1 ϫ d 2 ϫ d 3 ϫ 0.5236, where d represents three orthogonal diameters.
Western Blot Analysis-Proteins extracted from cells or tumor tissues were immunoblotted with different antibodies following a published protocol (31). The catalogue number of the antibodies used is provided in the supplemental material). The signal was developed with ECL TM (GE Healthcare) or chemiluminescent peroxidase substrate (Sigma) after incubation with the appropriate secondary antibodies.
Doxorubicin-induced Apoptosis Assay-A549 cells expressing miR-1 or vector were seeded into 96-well plates (4000 cells/ well) in complete medium. Twelve h later, doxorubicin (Sigma) (1 g/ml) was added in the wells, and apoptotic cells were stained with Hoechst 33342 (5 g/ml), as described (38), to visualize cells with and without fragmented nuclei under a fluorescence microscope. Extracts made from the cells were subjected to Western blot analysis with antibodies specific for proand antiapoptotic proteins. To quantify cells with fragmented DNA upon drug treatment, the cell cycle profile was analyzed with cells fixed overnight in 70% ethanol in the FACSCalibur after PI staining in the presence of RNase A, and the data were analyzed with the program Mod Fit LT (Verity, Topsham, ME).
Protein was estimated using the Bio-Rad protein assay kit with bovine serum albumin as a standard. Kodak Imaging software was used to quantify ethidium bromide-stained gels and scanned x-ray films (Western blot data).
Statistical Analysis-Statistical significance of differences between groups was analyzed by unpaired Student's t test, and p Յ 0.05 was considered to be statistically significant.

miR-1 Is Down-regulated in Primary Human Lung Cancer-
To determine whether miR-1 is deregulated in lung cancer, we measured the mature miR-1 level in human primary lung tumors and pair-matched lung tissues by real time RT-PCR. We used both 18 S rRNA and miR-191 that is not deregulated in lung cancer (40) for normalization. The results showed that miR-1 expression in the tumors was significantly (p ϭ 0.0004) reduced in 14 lung tumors relative to their matched controls among 16 samples analyzed (Fig. 1, A and B). The normalized level of miR-1 in each sample is presented in Table S1. With the exception of two non-small cell carcinomas (numbers 11 and 15), miR-1 was down-regulated in all lung cancer samples, including non-small cell lung cancers, adenocarcinomas, squa-FIGURE 1. Expression of miR-1 is significantly down-regulated in primary human lung cancers compared with matching lung tissues. A and B, real time RT-PCR analysis of mature miR-1 in primary lung cancers and matching lung tissues. DNase I-treated total RNA was analyzed using Taqman RT-PCR assay primer and probe for miR-1, miR-191, and 18 S rRNA. Each sample was analyzed in triplicate. The results are means of three independent experiments Ϯ S.D. C, Northern blot analysis of miR-1 in lung cancer and pairmatched controls. Ten g of RNA was separated in urea-acrylamide gel, transferred to Zetaprobe, and sequentially hybridized to 32 P-labeled antisense miR-1 and 5 S rRNA deoxyoligonucleotides. The signal was captured in a PhosphorImager and quantified using ImageQuant software. D, representative in situ hybridization data showing miR-1 expression in epithelial cells lining the bronchi. Expression of miR-1 in human lung tissues was detected by in situ hybridization with LNA-modified antisense miR-1 probe and RT in situ PCR, respectively. Tissue sections were hybridized to biotin-labeled oligonucleotide (antisense miR-1 or scrambled), which was captured with alkaline phosphatase-conjugated streptavidin, and the signal (blue) was developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. The cell body was stained with Nuclear fast red.

miR-1 Suppresses Lung Tumorigenesis
mous cell carcinomas, large cell carcinoma, and bronchoalveolar cell carcinoma (Table S1). It can be noted that the miR-1 level in surrounding lung tissues varied among different individuals. Nevertheless, in primary tumor tissues, its expression was dramatically reduced irrespective of their origin. We also performed Northern blot analysis of miR-1 in seven samples having 10 g of total RNA. The results showed that miR-1 is detectable in matching lung tissues, albeit at different levels, but not in the tumors (Fig. 1C).
We next measured the miR-1 level in 16 lung cancer cell lines and in noncancerous BEAS-2B cells. The results showed that the miR-1 level was almost undetectable in these cells except in N417 cells (supplemental Table S2). Notably, miR-1 is relatively high in nontumorigenic bronchial epithelial (BEAS-2B) cells (Table S2). Taken together, these results suggest that downregulation of miR-1 occurs not only in primary lung cancer but also in lung cancer cell lines.
To identify the cell type that expresses miR-1 in the human lung, we performed in situ hybridization with the LNA-modified anti-miR-1 in the tissue section. Since miR precursors are less abundant, we used in situ RT-PCR, a newly developed technique, to amplify and detect miR-1 precursor in lung tissues. The results demonstrated that miR-1 localized to the cytoplasm of epithelial cells lining the bronchi of human lung (Fig. 1D). The absence of a signal with the scrambled LNA probe demonstrated the specificity of the reaction.
Both C/EBP␣ and Trichostatin A, an HDAC Inhibitor, Activate the Repressed miR-1 Gene in Lung Cancer Cells-Although heart-specific regulation of murine miR-1-1 and miR-1-2 genes has been studied in great detail (26,27), regulation of human miR-1 gene expression has not been explored, especially in nonmuscle cells. In muscle cells, ubiquitously expressed serum response factor (SRF) in concert with muscle-specific transcription factors activates these genes by binding to the upstream enhancer elements. To identify the molecular mechanism of down-regulation of miR-1 in lung cancer, we measured the SRF mRNA level in primary lung cancers and matching lung tissues. Like many other growth factors, SRF mRNA was elevated in the majority of primary lung cancers (Fig. S1). Further, lesser expression of SRF in N417 cells that express relatively high levels of miR-1 (Table S2) compared with A549, H1155, and H792 cells (which do not express miR-1) suggests that SRF may not play a critical role in miR-1 expression in lung cancer cells.
To identify the transcription factor that may play a role in miR-1 expression in the lung, we analyzed the upstream promoter of the intronic miR-1-1 and miR-1-2 genes using the TESS data base (41) (available on the World Wide Web), which revealed potential cognate sites for C/EBP␣, a member of the basic leucine zipper family of transcription factors. C/EBP␣ is a tumor suppressor that is frequently inactivated in different cancers due to mutation, transcriptional repression, or promoter methylation (42)(43)(44)(45). Real time RT-PCR analysis showed a significant decrease in its expression in primary lung cancer compared with matching lung tissues ( Fig.  2A). Furthermore, a higher level of miR-1 (Fig. 2D, top) in N417 cells that express C/EBP␣ (bottom) as opposed to A549 or H1299 cells, in which both C/EBP␣ and miR-1 are almost undetectable (Fig. 2, B and C) (45), suggested to us its potential role in trans-activation of miR-1 genes. To test this possibility, we measured the miR-1 level in A549 and H1299 cells expressing ectopic C/EBP␣ (Fig. 2, B and C, bottom). The result showed an increase in endogenous miR-1 level of ϳ6and ϳ5-fold (top) in A549 and H1299 cells, respectively. We also performed the reverse experiment by depleting C/EBP␣ from N417 cells and measured the miR-1 level. Indeed, transfection of N417 cells with pretroSuper harboring short hairpin RNA specific for C/EBP␣ reduced its level by 50% (Fig. 2D, bottom), with a concomitant decrease in miR-1 level by ϳ40% (top). These results suggest that the loss of C/EBP␣ expression in lung cancer cells is one of the factors causing miR-1 repression.
Next we addressed whether an epigenetic mechanism, such as DNA methylation or histone deacetylation, plays any role in the repression of miR-1 in lung cancer cells. For this purpose, we treated A549 and H1299 cells with 5-AzaC (an inhibitor of  NOVEMBER 28, 2008 • VOLUME 283 • NUMBER 48

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DNA methyltransferase) or trichostatin A (TSA; an HDAC inhibitor) for 24 h, followed by quantification of miR-1. The results showed that treatment with TSA increased miR-1 level 25-and 250-fold in A549 and H1299 cells, respectively, whereas 5-AzaC had minimal effect (Fig. 2, E and F). Prolonged treatment with 5-AzaC could not activate miR-1 expression in A549, H1155, and H792 cells (data not shown). These results suggest that hypoacetylation of nucleosomal histones but not DNA methylation plays a causal role in the suppression of miR-1 in these lung cancer cells. Since the promoter of the intronic miR-1-1 gene is embedded in CpG islands, we analyzed its methylation status by COBRA (combined bisulfite restriction analysis) in primary lung tumor and matching normal tissues. The absence of tumor-specific methylation (data not shown) suggests that DNA methylation does not play a major role in suppressing miR-1-1 in lung tumors.
Ectopic Expression of miR-1 Reduces Growth and Replication Potential of Lung Cancer Cells-Next we examined the antitumorigenic function of miR-1 in lung cancer cells. For this purpose, we generated stable A549 cell lines expressing miR-1. We used two clones (clones 1 and 2) expressing comparable levels of miR-1 (ϳ20-fold) relative to the control (vector-transfected) cells (Fig. 3A).
We first studied the effect of ectopic miR-1 on cell proliferation. The results showed that cell growth was significantly reduced in both A549 cells compared with the control (vector-transfected) cells (Fig. 3B). After 48 h, growth of miR-1-expressing clones 1 and 2 was reduced by 18 and 30%, respectively, relative to that of control cells. Replication potential of these cells was also significantly reduced, as demonstrated by the decrease in [ 3 H 1 ]thymidine incorporation into DNA in miR-1-expressing clones 1 and 2 by ϳ56 and 38%, respectively, compared with control cells (Fig. 3C). These results corroborate the growth-inhibitory property of miR-1. To test whether miR-1 also controls growth of noncancerous cells, we measured the proliferation rate of transformed bronchial epithelial cells (BEAS-2B) that expresses a relatively high miR-1 level (Table S2) after transfecting anti-miR-1, but no significant increase in cell growth was notable under this condition (Fig. S2).
Overexpression of miR-1 Reduces Migration and Motility of Lung Cancer Cells in Vitro-Invasion and migration through the basement membrane is a characteristic property of metastatic cancer cells. We used two different approaches to assess the role of miR-1 in the ability of A549 cells to migrate. In the first technique, cells were seeded in serum-free medium on the top chamber of a two-chamber trans-well cell culture plate, and the cells migrated to the lower chamber containing complete medium after 48 h were photographed (Fig. 4A) and counted. As expected, very few cells (1%) migrated to the lower chamber containing serum-free medium. Migration of miR-1-expressing clones 1 and 2 was inhibited by 27 and 35%, respectively, relative to the control cells (Fig. 4B). Colorimetric estimation of migrated cells showed a 35 and 44% decrease in clone 1 and 2, respectively, compared with the vector-transfected cells (Fig. 4C).
In the second approach, we used a "scratch wound healing" assay (39). Motility of cells at different time points after generation of the wound was monitored under a microscope. Closure of the wound was complete within 48 h in control A549 cells (Fig. 4D). In contrast, miR-1-expressing cells migrated toward the wound at a much slower rate. These results, taken together, clearly demonstrate that miR-1 expression markedly reduces the migration/motility of lung cancer cells. Stable cell lines expressing miR-1 (miR-1 #1 and #2) or the retroviral vector (pBabe) were selected with puromycin and used for the following experiments. A, total RNA (5 g) from these cells was subjected to Northern blot analysis, as described in the legend to Fig. 1B. B, cells (3000/well) were seeded in a 96-well plate, and cell growth was monitored every 24 h for 4 days using an MTT assay. Each cell type was analyzed in quadruplicate. The results are means of three independent experiments Ϯ S.D. C, cells (10,000/well) were serum-starved overnight, followed by the addition of serum and [ 3 H 1 ]thymidine, and [ 3 H 1 ]thymidine incorporated into DNA was measured in a scintillation counter. Each experiment was performed in triplicate and was repeated twice. . Ectopic expression of miR-1 in A549 cells reduces cell migration and motility. A549 cells were loaded onto the top well of a trans-well inserts for cell migration assay. A, after 48 h, cells that migrated to the bottom chamber containing serum-supplemented medium were stained with Hema-3, visualized under a phase-contrast microscope, and photographed. B, total number of cells in five fields was counted manually. C, Hema-3-stained cells were solubilized in acetic acid and methanol (1:1), and absorbance was measured at 595 nm. The results are means of three independent experiments Ϯ S.D. D, ectopic expression of miR-1 reduces cell motility in the "wound scratch assay." A uniform scratch was made in each confluent layer culture, the extent of wound closure was monitored under a phase-contrast microscope, and photographs were taken at 0, 24, and 48 h. Representative experiment was performed twice, generating similar results.

miR-1 Suppresses Lung Tumorigenesis
Oncogenic MET Is a Target of miR-1 in Lung Cancer Cells-Next, we explored the underlying molecular mechanism of the antitumorigenic property of miR-1 in lung cancer cells. Since micro-RNAs primarily mediate their biological functions in animal cells by impeding the expression of target genes, we searched different data bases (TargetScan and PicTar) for its potential targets that exhibited oncogenic properties. MET (hepatocyte growth factor receptor), which harbors two conserved miR-1 cognate sites (Fig. 5A), is a predicted target of miR-1. MET is a receptor-type tyrosine kinase, overexpressed in many human cancers (46,47). It consists of two subunits of 50 and 145 kDa processed from a 170-kDa precursor polypeptide. To determine whether MET expression is indeed regulated by miR-1, we generated pIS0-MET-3Ј-UTR, transfected the construct along with pSV40-␤-gal to A549 cells, and measured luciferase and ␤-galactosidase activities. The normalized luciferase activity was reduced by 75% in cells expressing hsa-miR-1 compared with those expressing the control (scrambled) Data were normalized to 18 S rRNA. The results are means of triplicate assays Ϯ S.D. F, luciferase activity controlled by Pim-1-3Ј-UTR is inhibited by miR-1. Cells were co-transfected with the pIS0-Pim-1-3Ј-UTR-firefly luciferase reporter, Renilla luciferase, along with hsa-miR-1. After 48 h, luciferase activities were measured. G, Pim-1 expression in miR-1 expressing A549 clones was measured by real time RT-PCR. NOVEMBER 28, 2008 • VOLUME 283 • NUMBER 48 RNA (Fig. 5A). Moreover, miR-1-dependent repression of luciferase activity was abrogated upon the deletion of both miR-1 cognate sites, whereas deletion of each site individually inhibited luciferase activity by 50%. These results showed that the MET-3Ј-UTR was instrumental in miR-1-mediated negative regulation of the reporter gene activity. Western blot analysis showed significant reduction in MET (ϳ50 and 65% in clones 1 and 2, respectively) in both miR-1-expressing clones (Fig. 5, B and C). The decrease in MET mRNA level in these cells (Fig.  5D) indicates that miR-1 also induces destabilization of its message.

miR-1 Suppresses Lung Tumorigenesis
Ectopic Expression of miR-1 Inhibits Growth, Clonogenic Survival, and Anchorage-independent Growth of H1299 Cells-To demonstrate that the antitumorigenic property of miR-1 is not restricted to A549 cells, we transfected nonexpressing H1299 cells with pre-miR-1 and measured cell growth, clonogenic survival, and anchorage-independent growth. Northern blot analysis showed a 45-fold increase in miR-1 level in these cells compared with those transfected with control RNA (Fig.  6A). Proliferation of miR-1-expressing cells was inhibited by 44 and 49% on day 3 and 4, respectively, compared with that of control cells (Fig.  6B). Similarly, clonogenic survival of H1299 cells (a pair of representative plates is shown in Fig. 6C) was inhibited 62% upon ectopic expression of miR-1 (Fig. 6D). Furthermore, anchorage-independent growth, a hallmark of cancer cells, was dramatically (80%) inhibited in miR-1-expressing H1229 cells compared with controls (Fig. 6, E and F). It is noteworthy that the sizes of the few colonies formed by miR-1expressing cells are significantly smaller than the controls. These properties of miR-1-expressing cells correlated with reduced expression of its targets MET and FoxP1 in these cells (Fig. 6G).
We also performed the reverse experiment in which growth rate and MET level were measured in N417 cells following depletion of miR-1. Transfection of antisense miR-1 resulted in a ϳ75% decrease in its expression compared with cells transfected with control RNA (Fig. 7A). Western blot analysis revealed a ϳ2-fold increase in MET in miR-1-depleted cells (Fig.  7B). Depletion of miR-1 with concomitant elevation of its oncogenic target MET was associated with an ϳ20% increase in cell growth (Fig. 7C).
Next, we measured MET mRNA level in primary lung tumors by RT-PCR analysis, which showed its up-regulation in 12 of 16 tumors compared with matching control tissues (Fig.  8A). Immunohistochemical analysis of tumors and normal lung tissues demonstrated that MET is expressed at a low level in lung tissues, localizing to less than 1% of pneumocytes and cells lining bronchi in a given section (Fig. S3). In contrast, it was markedly up-regulated in the lung cancer cells. Representative data (Fig. S3) clearly demonstrate a dramatic increase in MET level in the lung cancer. Thus, down-regulation of miR-1 is probably one of the mechanisms by which its oncogenic target MET is up-regulated in lung cancer.

miR-1 Suppresses Lung Tumorigenesis
FoxP1 and HDAC4, Targets of miR-1, Are Also Differentially Regulated in Human Lung Cancer-FoxP1, a member of the Fbox family of transcription factors, is also a predicted target of miR-1 harboring three cognate sites. It is differentially regulated in human cancers and can act as an oncoprotein or a tumor suppressor, depending upon the cellular context (48). By transfecting pIS0-FoxP1-3Ј-UTR-luciferase, we confirmed that miR-1 negatively regulates reporter activity through FoxP1-3Ј-UTR (31). Western blot analysis of A549 cell extracts showed a significant decrease in the endogenous FoxP1 level in miR-1-expressing cells (Fig. 5, B and C). The steady state level of FoxP1 was reduced by ϳ80% in A549 clones expressing miR-1 relative to the control cell. Real time RT-PCR analysis demonstrated that ectopic miR-1 reduced the mRNA levels of FoxP1 (Fig. 5E).
Since HDACs have been identified as therapeutic targets in a variety of cancers, including lung cancer (49), and HDAC4 is a validated target of miR-1 (26), we measured its level in A549 cells expressing miR-1. Western blot analysis revealed a dramatic decrease in HDAC4 following miR-1 expression (Fig. 5, B and C). FoxP1 RNA level increased in 75% of lung tumors analyzed (Fig.  8A). Immunohistochemical analysis showed that FoxP1 and HDAC4 were also up-regulated in primary lung cancer tissues (a representative figure is shown, Fig. S2).
Oncogenic Ser/Thr Kinase Pim-1, a Target of miR-1, Is Significantly Up-regulated in Lung Cancer-Pim-1, a Ser/Thr kinase, induces tumorigenesis by promoting cell cycle progression and inhibiting apoptosis (50,51). Since its 3Ј-UTR harbors one miR-1 cognate site (Fig. 5F), we explored whether miR-1 can regulate Pim-1 expression in lung cancer. Dose-dependent inhibition of firefly luciferase activity driven by Pim-1-3Ј-UTR upon ectopic expression of miR-1 suggested its potential regulation by miR-1. Significant reduction in endogenous Pim-1 protein (Fig. 5, B and C) and RNA levels (Fig.  5G) in miR-1-expressing cells indicates its negative regulation by miR-1 in A549 cells. Analysis of Pim-1 mRNA level showed its significant up-regulation in primary lung cancers compared with matching normal tissues (Fig. 8B). Pim-1 is also elevated in the majority of lung cancer cell lines tested compared with BEAS-2B cells (Table S3).
Ectopic Expression of miR-1 Reduces Tumor Growth in Nude Mice-Since miR-1 expression reversed the tumorigenic property of A549 cells in vitro, it was logical to examine whether ectopic miR-1 could regress tumor growth ex vivo. For this purpose, A549 cells expressing miR-1 and vector-transfected cells were injected subcutaneously into the left and right flanks of nude mice, respectively, and tumor formation was monitored. After 4 weeks, the animals were sacrificed, and the tumor volume and weight were measured. The results showed that the tumor growth was significantly reduced in miR-1-expressing cells compared with the control cells (Fig. 9A). The reduction in tumor volume and weight in miR-1-expressing A549 cells were 55.27 and 62%, respectively (Fig. 9, B and C). Real time RT-PCR analysis showed that the miR-1 level was ϳ9-fold higher in tumor 3 relative to that in the tumor generated by the vectortransfected cells (Fig. 9D). Western blot analysis of the tumor tissue extract confirmed that the targets of miR-1 MET and FoxP1 were indeed down-regulated (ϳ50%) in miR-1-expressing tumor 3 compared with the control (Fig. 9E). These data suggest that miR-1 reduces lung tumor cell growth by suppressing the expression of its targets.
Doxorubicin-induced Apoptosis Is Augmented in A549 Cells Expressing Ectopic miR-1-It has been shown recently that certain micro-RNAs can alter sensitivity of cancer cells to therapeutic agents (52)(53)(54). We hypothesized that ectopic miR-1   NOVEMBER 28, 2008 • VOLUME 283 • NUMBER 48 may sensitize cells to anticancer drugs, because it targets MET that protects cells against DNA damaging agents (35). We therefore investigated whether miR-1 can alter sensitivity of lung cancer cells to anticancer drugs. Doxorubicin (DOXR) is one of the major drugs used in the treatment of a variety of cancers, including lung cancer (39). Induction of apoptotic cell death is a key mechanism by which DOXR inhibits cancer cell growth (55). We treated A549 cells with DOXR (1 g/ml) and counted cells undergoing nuclear fragmentation by Hoechst staining (Fig. 10A). The results showed that 17 and 23% of the cells were apoptotic after 24 h of DOXR treatment in miR-1expressing clones 1 and 2, respectively, whereas only 2% of the vector-transfected cells were apoptotic (Fig. 10B). After 36 h, 28 and 38% of cells of clones 1 and 2 were apoptotic, as opposed to only 4% of the control cells undergoing cell death (Fig. 10C).

miR-1 Suppresses Lung Tumorigenesis
We also measured the number of cells undergoing apoptosis by fluorescence-activated cell sorting analysis of cells with fragmented DNA after propidium iodide staining. The population of cells with sub-G 0 DNA content was increased from 0% in the vector-transfected cells to 23% in miR-1-expressing cells after DOXR exposure for 24 h (Fig. 10D). The population of cells with fragmented DNA was negligible in cells that were not exposed to DOXR (data not shown). It is notable that the population of cells at different stages of the cell cycle is distinct in miR-1-expressing cells from that in control cells (Fig. 10D).
To elucidate the underlying mechanism of miR-1-mediated sensitization of A549 cells to DOXR, we measured the levels of a few critical factors that mediate apoptosis by Western blot analysis. As expected, DNA damage due to DOXR exposure resulted in significant induction of p53 (Fig. 11A) and its target gene PUMA (data not shown) at a comparable level in the control and miR-1-expressing A549 cells at all time points tested. In contrast, the basal level of cleaved caspase 9, an activator caspase, was relatively high both in the control and in miR-1expressing cells (Fig. 11A, (lanes 1, 5, and 9)). However, in the vector-transfected cells, its level gradually decreased with time of DOXR exposure (Fig. 11, A (lanes 5-8) and B), whereas it increased to some extent in miR-1-expressing cells (Fig. 11A,  lanes 1-4 and 9 -12). Robust activation of the effector caspases 3 and 7 occurred within 12 h of DOXR treatment in miR-1expressing cells, which persisted even at 24 h (Fig. 11A, lanes  1-3 and 9 -11, and B). In contrast, cleavage of these caspases was delayed and significantly reduced in control cells (Fig. 11A,  compare lanes 6 -8 with lanes 1-4 and 9 -12). Proteolysis of their substrate PARP-1 followed a similar profile as those of caspases 3 and 7 (Fig. 11, A and B). Again, the level of cleaved PARP-1 was higher in miR-1-expressing cells than in the control cells at all time points tested (Fig. 11A, compare lanes 5-8 with lanes 1-4 and 9 -12).

miR-1 Suppresses Lung Tumorigenesis
We also measured the level of antiapoptotic Bcl-2 in cell extracts by immunoblot analysis using two different antibodies, which demonstrated a low level of Bcl-2 expression only in the control cells, and DOXR did not have any significant effect on it (Fig. 11A). In contrast, the basal level of antiapoptotic Mcl-1 is relatively high and comparable in three cell lines (Fig. 11A, compare lane 5 with lanes 1 and 9). However, its level declined with time only in miR-1-expressing cells upon DOXR treatment (Fig. 11A, compare lanes 6 -8 with lanes 2-4 and 10 -12, and B), which is probably due to degradation of Mcl-1 by caspase 3 (56). Thus, facilitated activation of caspases 3 and 7 and PARP-1 and depletion of Mcl-1 probably account for the sensitivity of miR-1-expressing A549 cells to DOXR-induced apoptosis, and this process appears to be independent of p53 status.

DISCUSSION
Although micro-RNA signatures for different types of cancer have been established, elucidation of the role of altered expression of specific micro-RNAs and its consequence on oncogenic transformation remains in the early stage of development. The data presented herein shed light on the regulatory role of miR-1 in lung cancer. miR-1 is predominantly expressed in cardiac tissues and smooth and skeletal muscles, where it inhibits cell cycle progression of myoblasts and promotes their differentiation (57). In cardiac tissues, miR-1 inhibits expression of some transcription repressors, such as Hand2 (27) and HDAC4 (26).
In the present study, we showed that miR-1 expression is reduced in lung cancers and that it inhibits the tumorigenic potential of lung cancer cells by down-regulating oncogenic targets, such as MET and FoxP1. To our knowledge, this is the first report that correlates loss of miR-1 with tumorigenic potential of lung epithelial cells. This contention is based upon the following observations. First, miR-1 expression was significantly reduced in primary lung cancer. Second, almost all lung cancer cell lines tested showed either very low or undetectable levels of miR-1. Third, ectopic expression of miR-1 in the lung cancer cell line (A549) significantly reduced cell growth, replication potential, and tumor growth in nude mice, whereas depletion of miR-1 facilitated lung cancer cell (N417) growth. Further, sensitization of A549 cells to the potent anticancer drug doxorubicin by ectopic miR-1 underscores its potential therapeutic application against lung cancer.
Our study also revealed that the transcriptional regulation of miR-1 in the lung may be distinct from that in muscle. The presence of C/EBP␣ cognate sites in the miR-1 promoter raised the possibility that it could be one of the transcription factors playing a key role in the activation of the miR-1 gene. Indeed, enrichment of C/EBP␣ and miR-1 in bronchial epithelial cells suggests its potential role in miR-1 gene activation in these cells. Further, frequent loss of C/EBP␣ by genetic (11) and epigenetic mechanism (52) could explain miR-1 suppression in lung cancer cells. C/EBP␣ can up-regulate gene expression by directly binding to cognate sites or indirectly through proteinprotein interactions (36). miR-1 is likely to be one of the targets of C/EBP␣, through which it inhibits cell proliferation and induces apoptosis (59). It would be of interest to determine whether binding of C/EBP␣ to miR-1-1 and miR-1-2 gene promoters is essential for their transactivation. The miR-1 suppression in lung cancer cells is also mediated by histone hypoacetylation, since treatment of A549 cells with trichostatin A, an HDAC inhibitor, significantly induced miR-1 expression. The lack of activation of miR-1 by a DNA hypomethylating agent suggests that DNA methylation may not play a key role in silencing micro-RNA genes in lung cancer cells. In contrast, methylation-mediated silencing of miR-1 occurs in human primary hepatocellular carcinomas and cell lines (31). It therefore appears that different epigenetic mechanisms may contribute to regulation of miR-1 expression, depending upon the cell type. Suppression of miR-1 in lung cancer cells expressing a high level of SRF suggests that it may not play a key role in miR-1 expression. Activation of miR-1 by TSA suggests repressed chromatin structure may play a causal role in its silencing in lung cancer cells. The lack of activation of miR-1 or any other micro-RNA (23) by a DNA-hypomethylating agent supports the notion that DNA methylation may not play a key FIGURE 11. Enhanced activation of caspases 3 and 7, cleavage of PARP-1, and depletion of antiapoptotic Mcl-1 in miR-1-expressing cells treated with DOXR. A, Western blot analysis was performed using specific antibodies in whole cell extracts prepared from DOXR-treated and control cells. Similar results were obtained from different batches of cells treated with DOXR. B, quantitative representation of the data in A. The signal in each band was quantified using Kodak Imaging software. The results are means of two independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. NOVEMBER 28, 2008 • VOLUME 283 • NUMBER 48 role in silencing micro-RNA genes in lung cancer cells. In contrast, methylation-mediated silencing of miR-1 occurs in human primary hepatocellular carcinomas and cell lines (31). It therefore appears that different epigenetic mechanisms may contribute to regulation of miR-1 expression, depending upon the cell type.

miR-1 Suppresses Lung Tumorigenesis
Like protein-coding genes, some micro-RNAs also function as oncogenes or tumor suppressors (58,59). The global reduction of micro-RNA is associated with increased tumorigenesis (60). In fact, mRNA and miRNA interactions are tightly regulated, and even a small change between these interactions may cause severe consequences to cell physiology (61). Accordingly, alterations in the expression of target genes could lead to disease states. We further showed that this decrease in miR-1 expression is associated with an increase in oncogenic MET, Pim-1, FoxP1, and HDAC4. Pim-1 is a Ser/Thr kinase overexpressed in various human cancers and plays a causal role in lymphoma and leukemogenesis (50,51). Our study shows that it is also up-regulated in the majority of human primary lung tumors and cancer cell lines. FoxP1 is an essential transcription factor required for mammalian development (62). It is also noteworthy that it maps to chromosome 3p14.1, a region that is amplified or translocated, resulting in its up-regulation in different types of human cancers, including lung (63). Higher nuclear FoxP1 expression is associated with poor prognosis in patients with diffuse large B cell lymphoma. Thus, the FoxP1 level should be tightly regulated for normal function of cells. Our data support the notion that the loss of miR-1 is a mechanism for the up-regulation of FoxP1 level in primary lung cancer.
The present study also has shed light on the potential role of miR-1 in cancer metastasis. Metastasis, an integral part of tumor progression, is a complex process that exhibits properties such as loss of cellular adhesion, increased motility and invasiveness, entry and survival into the circulation, exit into new tissue, and eventual colonization of a distant site. We showed that ectopic expression of miR-1 not only inhibited proliferation but also reduced cell migration and motility of A549 cells. MET, a receptor for hepatocyte growth factor and a tyrosine kinase (receptor-type tyrosine kinase), supports the initial steps of invasion and metastasis of most human cancers, including lung cancer (47). The down-regulation of MET could be a possible mechanism by which miR-1 regulates growth and metastatic potential of these cells.
Finally, the role of miR-1 in sensitizing resistant A549 cells to doxorubicin merits discussion. Most chemotherapeutic agents exert anti-tumor effects by triggering apoptosis (64). Apoptosis can be mediated by the extrinsic pathway, which is initiated by transmembrane death receptors and the intrinsic pathway, which is activated due to drug-induced DNA damage. Although most drugs predominantly activate the mitochondria-dependent apoptotic pathway, there is evidence that the extrinsic pathway involving death receptors is also involved in this process and that there is cross-talk between the two pathways (64). The sensitivity of miR-1-expressing A549 cells to DOXR is probably due to enhanced activation of the effector caspases and facilitated degradation of antiapoptotic Mcl-1. miR-1 probably affects some mediators downstream of p53, since DOXR-in-duced expressions of p53 and its target PUMA were comparable in control and miR-1-expressing cells. Many pro-and antiapoptotic intermediary factors are involved in the activation of effector caspases. It is likely that the function of one or more of these factors is altered in A549 cells due to expression of ectopic miR-1. Overexpression of Mcl-1, an antiapoptotic member of the Bcl-2 family, in lung cancers probably explains resistance of A549 cells to apoptosis (65). The Mcl-1 level is up-regulated in cancer cells by multiple mechanisms involving regulation at the level of transcription, alternative splicing, phosphorylation, proteolytic cleavage by casapases, and ubiquitin-proteosomal system-mediated degradation (66). It would be of interest to elucidate the molecular mechanism by which miR-1 facilitates doxorubicin-induced depletion of Mcl-1. Studies along these lines are in progress. Nevertheless, potentiation of drug-induced cell death underscores the therapeutic potential of miR-1 against lung cancers.