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. 2008 Nov 28;283(48):33394-405.
doi: 10.1074/jbc.M804788200. Epub 2008 Sep 25.

Down-regulation of micro-RNA-1 (miR-1) in lung cancer. Suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by miR-1

Mohd W Nasser  1 , Jharna Datta, Gerard Nuovo, Huban Kutay, Tasneem Motiwala, Sarmila Majumder, Bo Wang, Saul Suster, Samson T Jacob, Kalpana Ghoshal
Affiliations

Down-regulation of micro-RNA-1 (miR-1) in lung cancer. Suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by miR-1

Mohd W Nasser et al. J Biol Chem. .

Retraction in

Abstract

Micro-RNAs are approximately 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/EBPalpha, 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.

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Figures

FIGURE 1.
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 ± S.D. C, Northern blot analysis of miR-1 in lung cancer and pair-matched controls. Ten μg of RNA was separated in urea-acrylamide gel, transferred to Zetaprobe, and sequentially hybridized to 32P-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.
FIGURE 2.
FIGURE 2.
A, C/EBPα is down-regulated in primary lung cancer. Real time RT-PCR analysis of C/EBPα and 18 S rRNA was performed with 12 pairs of tumor and matching lung tissues. B and C, ectopic C/EBPα up-regulates miR-1 expression in lung cancer cells. Top, miR-1 level in A549 and H1299 cells transfected with C/EBPα expression vector or empty vector. Bottom, Western blot analysis. D, depletion of endogenous C/EBPα reduces miR-1 level in lung cancer (N417) cells. Cells were transfected with pretroSuper harboring C/EBPα-short hairpin RNA or the vector followed by analysis of miR-1 (top) and C/EBPα (bottom) levels after 60 h. The results are means of three independent experiments ± S.D. E and F, TSA induces miR-1 expression in lung cancer cells. Cells were treated with 1 μm of 5-AzaC or 300 nm of TSA for 24 h, and DNase I-treated total RNA was subjected to real time RT-PCR. The results are means of three independent experiments ± S.D. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIGURE 3.
FIGURE 3.
Ectopic Expression of miR-1 inhibits proliferation of A549 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 μg) 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 ± S.D. C, cells (10,000/well) were serum-starved overnight, followed by the addition of serum and [3H1]thymidine, and [3H1]thymidine incorporated into DNA was measured in a scintillation counter. Each experiment was performed in triplicate and was repeated twice.
FIGURE 4.
FIGURE 4.
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 ± 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.
FIGURE 5.
FIGURE 5.
MET proto-oncogene is a target of miR-1. A, the 3′-UTR of MET harbors two miR-1 cognate sites. Luciferase activity regulated by 3′-UTR of MET is inhibited by ectopic expression of miR-1. A549 cells were co-transfected with firefly luciferase-3′-UTR (MET) and hsa-miR-1 or control RNA (60 nm) along with SV40-β-gal (as internal control). After 48 h, firefly luciferase and β-galactosidase activities were measured. MET-3′-UTR deleted of both or individual miR-1 complementary sites were transfected following the same protocol. The results are means of three independent experiments ± S.D. B, the levels of miR-1 targets are reduced in A549 cells expressing miR-1. Cell extracts were subjected to Western blot analysis with specific antibodies. C, quantification of Western blot data in B. A reproducible result was obtained in two independent experiments. D and E, real time RT-PCR analysis of MET and FoxP1 miR-1-expressing cells. Data were normalized to 18 S rRNA. The results are means of triplicate assays ± S.D. F, luciferase activity controlled by Pim-1–3′-UTR is inhibited by miR-1. Cells were co-transfected with the pIS0-Pim-1–3′-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.
FIGURE 6.
FIGURE 6.
Ectopic expression of miR-1 inhibited growth, clonogenic survival, and anchorage-independent growth and reduced expression of its target proteins in H1299 cells. Cells were transfected with pre-miR-1 (100 nm) or control RNA. After 24 h, cells were trypsinized and used for different assays. A, Northern blot analysis of total RNA (10 μg). The signal was detected by autoradiography. B, growth of cells as measured by an MTT assay. Cells (4000/well) were seeded in a 96-well plate, and cell growth was monitored every 24 h for 5 days. C and D, assay of clonogenic survival. 1 × 103 cells/60-mm dish were seeded in a 100-mm dish, and colonies were stained with 0.05% crystal violet after 2 weeks. E and F, anchorage-independent growth of H1299 cells in soft agar. 1.5 × 104 cells/60-mm dish were used for the soft agar assay. A representative section of colonies formed after 4 weeks is shown. Each sample in B–D was analyzed in triplicate, and the results are means of two independent experiments ± S.D. G, Western blot analysis of miR-1 targets in H1299 cells transfected with hsa-miR-1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIGURE 7.
FIGURE 7.
miR-1 targets are up-regulated with concomitant increase in growth rate of N417 cells depleted of endogenous miR-1. A, miR-1 level in N417 cells transfected with 50 nm anti-miR-1 or control RNA. Total RNA isolated from cells after 48 h was analyzed by real time RT-PCR. B, Western blot analysis of miR-1 targets in transfected N417 cells. Reproducible results were obtained in two independent experiments. C, growth of N417 cells transfected with anti-miR-1 (50 nm) or control RNA was measured by an MTT assay beginning 48 h post-transfection (0 h). The optical density at 570 nm at 0 h was assigned a value of 1. The results are means of three independent experiments ± S.D.
FIGURE 8.
FIGURE 8.
A, RT-PCR analysis of MET and FoxP1 in human primary lung cancers and matching lung tissues. One hundred ng (for MET), 200 ng (for FoxP1), and 10 ng (for 18 S rRNA) cDNA was amplified with gene-specific primers. The PCR products were separated in an agarose gel, stained with ethidium bromide, and photographed. The asterisks denote samples in which MET and FoxP1 were up-regulated. B, real time RT-PCR analysis of Pim-1 in human primary lung cancers and matching lung tissues. The data are shown using a box-whisker plot.A horizontal line in each box represents the median value of Pim-1 mRNA normalized to 18 S rRNA in each group. The box denotes the 25th and 75th percentile range of scores, whereas whiskers represent the highest and lowest values.
FIGURE 9.
FIGURE 9.
Ectopic Expression of miR-1 in A549 cells inhibits tumor growth in athymic nude mice. miR-1-expressing A549 cells (clone 2) and vector-transfected cells were trypsinized and counted and then mixed with 50% Matrigel, and 2 × 106 cells were injected subcutaneously to the left and right flanks, respectively, of nude mice. After 4 weeks, tumors were excised and analyzed. A, photograph of tumors developed in six mice. B, average volume of tumors developed in six nude mice ± S.D. C, average weight of tumors developed in six nude mice ± S.D. D, real time RT-PCR analysis of miR-1 in tumors isolated from mouse 3. The results are means of a triplicate assay ± S.D. E, Western blot analysis of miR-1 targets in tumor tissue extracts from mouse 3. Whole cell extracts were subjected to immunoblot analysis with specific antibodies. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIGURE 10.
FIGURE 10.
miR-1 expression enhances doxorubicin-induced apoptosis in A549 cells. miR-1-expressing or vector-transfected A549 cells were treated with doxorubicin (1 μg/ml) for different time periods. A, photograph of cells stained with Hoechst. Cells grown in coverslips were stained with Hoechst and photographed under a fluorescence microscope. Cells undergoing DNA fragmentation were counted manually. The arrows denote cells with fragmented nuclei. B and C, quantitative representation of the number of apoptotic cells after 24 and 36 h of DOXR exposure. The total number of cells (∼200) with or without fragmented nuclei was counted, and the percentage of apoptotic cells was calculated. Similar results were obtained from three independent experiments. D, a representative histogram depicting cell cycle profile in DOXR-treated cells. Cells treated with DOXR for 24 h were trypsinized on ice, washed, and incubated with propidium iodide and RNase A followed by cell cycle analysis in a flow cytometer.
FIGURE 11.
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.
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