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• W2002201015 abstract "MicroRNAs are a class of small non-coding RNAs and participate in the regulation of apoptotic program. Although miR-21 is able to inhibit apoptosis, its expression regulation and downstream targets remain to be fully elucidated. Here we report that the transcriptional factor Foxo3a initiates apoptosis by transcriptionally repressing miR-21 expression. Our results showed that doxorubicin could simultaneously induce the translocation of Foxo3a to the cell nuclei and a reduction in miR-21 expression. Knockdown of Foxo3a resulted in an elevation in miR-21 levels, whereas enforced expression of Foxo3a led to a decrease in miR-21 expression. In exploring the molecular mechanism by which Foxo3a regulates miR-21, we observed that Foxo3a bound to the promoter region of miR-21 and suppressed its promoter activity. These results indicate that Foxo3a can transcriptionally repress miR-21 expression. In searching for the downstream targets of miR-21 in apoptosis, we found that miR-21 suppressed the translation of Fas ligand (FasL), a pro-apoptotic factor. Furthermore, Foxo3a was able to up-regulate FasL expression through down-regulating miR-21. Our data suggest that Foxo3a negatively regulates miR-21 in initiating apoptosis. MicroRNAs are a class of small non-coding RNAs and participate in the regulation of apoptotic program. Although miR-21 is able to inhibit apoptosis, its expression regulation and downstream targets remain to be fully elucidated. Here we report that the transcriptional factor Foxo3a initiates apoptosis by transcriptionally repressing miR-21 expression. Our results showed that doxorubicin could simultaneously induce the translocation of Foxo3a to the cell nuclei and a reduction in miR-21 expression. Knockdown of Foxo3a resulted in an elevation in miR-21 levels, whereas enforced expression of Foxo3a led to a decrease in miR-21 expression. In exploring the molecular mechanism by which Foxo3a regulates miR-21, we observed that Foxo3a bound to the promoter region of miR-21 and suppressed its promoter activity. These results indicate that Foxo3a can transcriptionally repress miR-21 expression. In searching for the downstream targets of miR-21 in apoptosis, we found that miR-21 suppressed the translation of Fas ligand (FasL), a pro-apoptotic factor. Furthermore, Foxo3a was able to up-regulate FasL expression through down-regulating miR-21. Our data suggest that Foxo3a negatively regulates miR-21 in initiating apoptosis. miRNAs 2The abbreviations used are: miRNAmicroRNAFasLFas ligandFoxo3aForkhead bOX-containing protein O subfamily 3aNFAT4nuclear translocation of nuclear factor of activated T cells 4TUNELterminal dUTP nick-end labelingChIPchromatin immunoprecipitationqRTquantitative reverse transcriptionantagomir-NCantagomir negative controlwtwild typeUTRuntranslated region. are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. Recently, the work on miRNAs renovates our understanding about apoptotic regulation. They can be classified as either pro- or anti-apoptotic miRNAs (1Park S.M. Schickel R. Peter M.E. Curr. Opin. Cell Biol. 2005; 17: 610-616Crossref PubMed Scopus (110) Google Scholar, 2He L. He X. Lim L.P. de Stanchina E. Xuan Z. Liang Y. Xue W. Zender L. Magnus J. Ridzon D. Jackson A.L. Linsley P.S. Chen C. Lowe S.W. Cleary M.A. Hannon G.J. Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2260) Google Scholar). For example, miR-1 participates in the initiation of apoptosis (3Nasser M.W. Datta J. Nuovo G. Kutay H. Motiwala T. Majumder S. Wang B. Suster S. Jacob S.T. Ghoshal K. J. Biol. Chem. 2008; 283: 33394-33405Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), whereas miR-21 is able to inhibit apoptosis (4Löffler D. Brocke-Heidrich K. Pfeifer G. Stocsits C. Hackermüller J. Kretzschmar A.K. Burger R. Gramatzki M. Blumert C. Bauer K. Cvijic H. Ullmann A.K. Stadler P.F. Horn F. Blood. 2007; 110: 1330-1333Crossref PubMed Scopus (544) Google Scholar). miRNAs are expressed at a constant level under physiological condition, and their functions depend on their expression levels. It remains a challenging question as to how their expression is regulated in the apoptotic program. microRNA Fas ligand Forkhead bOX-containing protein O subfamily 3a nuclear translocation of nuclear factor of activated T cells 4 terminal dUTP nick-end labeling chromatin immunoprecipitation quantitative reverse transcription antagomir negative control wild type untranslated region. The forkhead family of transcription factors is characterized by the presence of a conserved 100-amino acid DNA binding domain and participate in regulating diverse cellular functions such as apoptosis, differentiation, metabolism, proliferation, and survival (5Accili D. Arden K.C. Cell. 2004; 117: 421-426Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar). Foxo3a is a substrate of protein kinase Akt, and its transcriptional output is controlled via phosphorylation. In the absence of cellular stimulation and when Akt is inactive, Foxo3a is localized within the nucleus where it performs transcription of target genes. However, upon phosphorylation by Akt at Thr-32, Ser-253, and Ser-315, it binds to 14-3-3 and cannot exert the transcriptional function (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar). Fas ligand (FasL) is a potential transcriptional target of Foxo3a, but the transcriptional output can be either activation or suppression. It has been reported that Foxo3a can stimulate FasL expression, thereby triggering apoptosis (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar). However, there is also evidence showing that Foxo3a decreases the expression of FasL (7Jonsson H. Allen P. Peng S.L. Nat. Med. 2005; 11: 666-671Crossref PubMed Scopus (172) Google Scholar). The molecular mechanism by which Foxo3a regulates FasL expression remains further elusive. miR-21 has been shown to regulate apoptosis by targeting a variety of apoptotic factors. It contributes to glioma malignancy by down-regulating matrix metalloproteinase inhibitors, thereby leading to the activation of matrix metalloproteinases and promoting invasiveness of cancer cells (8Gabriely G. Wurdinger T. Kesari S. Esau C.C. Burchard J. Linsley P.S. Krichevsky A.M. Mol. Cell. Biol. 2008; 28: 5369-5380Crossref PubMed Scopus (758) Google Scholar). It promotes cell transformation by targeting the programmed cell death 4 gene (9Lu Z. Liu M. Stribinskis V. Klinge C.M. Ramos K.S. Colburn N.H. Li Y. Oncogene. 2008; 27: 4373-4379Crossref PubMed Scopus (612) Google Scholar). miR-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and the tissue inhibitor of metalloproteinase 3 (10Selaru F.M. Olaru A.V. Kan T. David S. Cheng Y. Mori Y. Yang J. Paun B. Jin Z. Agarwal R. Hamilton J.P. Abraham J. Georgiades C. Alvarez H. Vivekanandan P. Yu W. Maitra A. Torbenson M. Thuluvath P.J. Gores G.J. LaRusso N.F. Hruban R. Meltzer S.J. Hepatology. 2009; 49: 1595-1601Crossref PubMed Scopus (233) Google Scholar). Given the important role of FasL in apoptosis, it is not yet clear whether FasL can be a target of miR-21 in the apoptotic machinery. The expression levels of miRNAs remain constant under physiological condition. Their alterations may cause the pathological disorders. miRNA expression can be regulated by transcriptional factors. For example, miR-34a causes dramatic reprogramming of gene expression and promotes apoptosis, and it is transactivated by the tumor suppression gene p53 (11Raver-Shapira N. Marciano E. Meiri E. Spector Y. Rosenfeld N. Moskovits N. Bentwich Z. Oren M. Mol. Cell. 2007; 26: 731-743Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Serum response factor can directly bind to the promoter of miR-1-1 and miR-1-2 genes and activate their expression (12Zhao Y. Samal E. Srivastava D. Nature. 2005; 436: 214-220Crossref PubMed Scopus (1331) Google Scholar). Our recent work reveals that miR-23a is a transcriptional target of nuclear factor of activated T cells c3 (13Lin Z. Murtaza I. Wang K. Jiao J. Gao J. Li P.F. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12103-12108Crossref PubMed Scopus (295) Google Scholar). Because the functions of miRNAs are closely related to their expression levels, it is important to explore the molecular mechanism governing their expression. Foxo3a and miR-21 are involved in apoptosis, but it is not yet clear whether there is an impact between these two factors in the apoptotic machinery. Lung cancer is the most common cause of cancer-related death. Chemotherapy plays an important role for the treatment of lung cancer, and doxorubicin is commonly used for lung cancer therapy. However, a major obstacle of chemotherapy is the cancer resistance. It is critically important to identify the factors that participate in the regulation of cancer cell apoptosis, so that novel approaches can be developed for cancer therapy. Our present work aimed at elucidating the molecular mechanism by which Foxo3a regulates the apoptotic program in A549 human lung cancer cells. Our results revealed that Foxo3a transcriptionally represses the expression of miR-21. Furthermore, we identified FasL as a target of miR-21. In addition, we found that Foxo3a regulates FasL through miR-21. Our data shed new light on understanding the regulation of miRNA expression and the relationship among Foxo3a, miR-21, and FasL in the apoptotic cascades. Human lung cancer cells (A549) and human neuroblastoma cells (SH-EP1) were grown in Dulbecco's modified Eagle's medium; Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. The treatment with doxorubicin (Sigma) was performed as we described (14Wang J.X. Li Q. Li P.F. Cancer Res. 2009; 69: 492-500Crossref PubMed Scopus (73) Google Scholar). The culture of cardiac fibroblasts was performed as we described (15Li P.F. Dietz R. von Harsdorf R. FEBS Lett. 1999; 448: 206-210Crossref PubMed Scopus (81) Google Scholar). Cell death was determined by trypan blue exclusion, and the numbers of trypan blue-positive and -negative cells were counted on a hemocytometer. Apoptosis was determined by the terminal deoxyribonucleotidyltransferase-mediated TUNEL using a kit from Roche Applied Science. The detection procedures were in accordance with the kit instructions. The Foxo3a RNAi sense sequence is 5′-GAGCTCTTGGTGGATCATC-3′; the antisense sequence is 5′-GATGATCCACCAAGAGCTC-3′. The scramble Foxo3a RNAi sense sequence is 5′-GGCGTAGTCGTAGTTCTCA-3′; the scramble antisense sequence is 5′-TGAGAACTACGACTACGCC-3′. FasL RNAi sense sequence is 5′-AAAGGAGCTGAGGAAAGTG-3′; the antisense sequence is 5′-CACTTTCCTCAGCTCCTTT-3′. The scramble FasL RNAi sense sequence is 5′-TGGAAGAGACAGTAGAGAG-3′; the scramble antisense sequence is 5′-CTCTCTACTGTCTCTTCCA-3′. They were cloned into pSilencer adeno 1.0-CMV vector (Ambion) according to the manufacturer's instructions. Human mir-21 promoter region was amplified from human genomic DNA using PCR to generate wild type promoter. The large fragment containing two Foxo3a potential binding sites (wild type promoter-1, wt-1) was amplified using the forward primer, 5′-AAACCAAGGCTCTTACCATAGC-3′. The short fragment containing one Foxo3a potential binding (wild type promoter-2 (wt-2)) was amplified using the forward primer, 5′-GCATGAGAGAGCCACTACCAAG-3′. Both fragments were amplified using the reverse primer, 5′-TGGTACAGCCATGGAGATGTCA-3′. The promoters were cloned into the reporter plasmid, pGL4.17 (Promega). The introduction of mutations in the putative Foxo3a binding site in wt-1 fragment (−197 to −191 wild type, 5′-ATAAACA-3′; mutant, 5′-ACGGCCA-3′) was generated using QuikChange II XL site-directed mutagenesis kit (Stratagene). Human FasL promoter region (from −3157 to +2) was amplified using the same protocol as described for miR-21. The forward primer was 5′-GTGACCTGTCCAGTTCACACAG-3′; the reverse primer was 5′-TGCATGGCAGCTGGTGAGTCAG-3′. The constructs were sequence-verified. miR-21 was synthesized by PCR using human genomic DNA as the template. The upstream primer was 5′-GCATTATGAGCATTATGTCAGA-3′; the downstream primer was 5′-CATACAGCTAGAAAAGTCCCTG-3′. The PCR fragment was finally cloned into the Adeno-XTM Expression System (Clontech) according to the manufacturer's instructions. FasL with 3′-UTR was amplified by PCR. The forward primer was 5′-GAGAAGCACTTTGGGATTCTTTC-3′. The reverse primer was 5′-CCCTACAATTGCACTGGAAATAC-3′. The PCR fragment was subcloned into the pGL3 vector (Promega) immediately downstream of the stop codon of the luciferase gene. To produce mutated 3′-UTR, the mutations (wild type 3′-UTR, 5′-ATAAGCTA-3′; mutated 3′-UTR, 5′-ATCCATTA-3′) was generated using the QuikChange II XL site-directed mutagenesis kit (Stratagene). The constructs were sequence-verified. FasL with the 3′-UTR was from Origene. To produce FasL with mutated 3′-UTR, the mutations (wild type 3′-UTR, 5′-ATAAGCTA-3′; mutated, 3′-UTR: 5′-ATCCATTA-3′) was generated using QuikChange II XL site-directed mutagenesis kit (Stratagene). The constructs were sequence-verified. They were cloned into the Adeno-XTM Expression System (Clontech) according to the manufacturer's instructions. Immunoblotting was carried out as we previously described (16Li P.F. Dietz R. von Harsdorf R. EMBO J. 1999; 18: 6027-6036Crossref PubMed Scopus (425) Google Scholar). Cells were lysed for 1 h at 4 °C in a lysis buffer (20 mm Tris, pH 7.5, 2 mm EDTA, 3 mm EGTA, 2 mm dithiothreitol, 250 mm sucrose, 0.1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100) containing a protease inhibitor mixture. Samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Equal protein loading was controlled by Ponceau Red staining of membranes. Blots were probed using the primary antibodies. The anti-Foxo3a and the anti-phospho Foxo3a antibody (Thr-32) were from Cell Signaling. The anti-FasL antibody was from Abcam. The anti-PCNA (proliferating cell nuclear antigen) antibody and the anti-actin antibody were from Santa Cruz Biotechnology. After four washes with phosphate-buffered saline, Tween 20, the horseradish peroxidase-conjugated secondary antibodies were added. Antigen-antibody complexes were visualized by enhanced chemiluminescence. Immunofluorescence was performed as we described (17Li P.F. Li J. Müller E.C. Otto A. Dietz R. von Harsdorf R. Mol. Cell. 2002; 10: 247-258Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The samples were imaged using a laser scanning confocal microscope (Zeiss LSM 510 META). Subcellular fractions were prepared as described with modifications (18Krude T. Jackman M. Pines J. Laskey R.A. Cell. 1997; 88: 109-119Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). In brief, cells were washed twice with phosphate-buffered saline, and the pellets were suspended in 0.2 ml of ice-cold buffer (20 mm potassium-HEPES, pH 7.8, 5 mm potassium acetate, 0.5 mm MgCl2, and 0.5 mm dithiothreitol) containing a protease inhibitor mixture. The cells were homogenized by 12 strokes in a Dounce homogenizer. The homogenates were centrifuged at 750 × g for 5 min at 4 °C to collect nuclei. The resulting supernatants were centrifuged at 20,000 × g for 5 min at 4 °C to collect the cytosolic fractions. ChIP was performed as we and other described (19Li Y.Z. Lu D.Y. Tan W.Q. Wang J.X. Li P.F. Mol. Cell. Biol. 2008; 28: 564-574Crossref PubMed Scopus (88) Google Scholar, 20Szak S.T. Mays D. Pietenpol J.A. Mol. Cell. Biol. 2001; 21: 3375-3386Crossref PubMed Scopus (158) Google Scholar). In brief, cells were washed with phosphate-buffered saline and incubated for 10 min with 1% formaldehyde at room temperature. The cross-linking was quenched with 0.1 m glycine for 5 min. Cells were washed twice with phosphate-buffered saline and lysed for 1 h at 4 °C in a lysis buffer. The cell lysates were sonicated into chromatin fragments with an average length of 500–800 bp as assessed by agarose gel electrophoresis. The samples were precleared with Protein A-agarose (Roche Applied Science) for 1 h at 4 °C on a rocking platform, and 5 μg of specific antibodies were added and rocked for overnight at 4 °C. The anti-NFAT4 antibody was from Santa Cruz Biotechnology. Immunoprecipitates were captured with 10% (v/v) Protein A-agarose for 4 h. Before use, Protein A-agarose was blocked twice at 4 °C with salmon sperm DNA (2 μg/ml) overnight. For the analysis of Foxo3a binding to the promoter of FasL, PCRs were performed with the primers that encompass Foxo3a BS1 or BS2 of the human FasL promoter. The oligonucleotides were as follows: BS1 (forward, 5′-GGATGGGCAGAATGTATCAGAG-3′; reverse, 5′-GCCAATAACTTCCAAGTAGTTA-3′); BS2 (forward 5′-CAAGGCAAGAGGATTGCTTGAG-3′; reverse, 5′-ACCTGCTACACCCACTTTAGAA-3′). For the analysis of Foxo3a binding to the promoter region of miR-21, the oligonucleotides were as follows: BS1 (forward 5′-AAACCAAGGCTCTTACCATAGC-3′ and reverse, 5′-CATTGCACTCCAACTTGGGCAA-3′); BS2 (forward, 5′-CTCTGGTTTCAACAGACACAAA-3′; reverse, 5′-TCTGGCCTGTTAAGATCGAACC-3′). For the analysis of NFAT4 binding to the promoter region of FasL, the oligonucleotides were as follows: forward, 5′-GGTATCCAGCGCTGATTTGCT-3′; reverse, 5′-ACCTCTCTCCAGTTCTCTTCT-3′. Luciferase assay was performed as we described (13Lin Z. Murtaza I. Wang K. Jiao J. Gao J. Li P.F. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12103-12108Crossref PubMed Scopus (295) Google Scholar, 21Tan W.Q. Wang K. Lv D.Y. Li P.F. J. Biol. Chem. 2008; 283: 29730-29739Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Briefly, for miR-21 and FasL promoters luciferase assays cells were seeded in 24-well plates. They were transfected with the plasmid constructs using the Lipofectamine 2000 (Invitrogen). Each well contained 0.2 μg of luciferase reporter plasmids and 2.5 ng of SV-Renilla luciferase plasmids as the internal control. Cells were harvested at the indicated times after transfection for the detection of luciferase activity using the Dual Luciferase Reporter Assay kit (Promega) according to the manufacturer's instructions. 20 μl of protein extracts were analyzed in a luminometer. Firefly luciferase activities were normalized to Renilla luciferase activity. For FasL 3′-UTR luciferase assay, cells were co-transfected with the plasmid constructs of 200 ng/well pGL3-FasL-3′-UTR, 400 ng/well miR-21, 20 pmol of either miR-21 antagomir or antagomir negative control (antagomir-NC) using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, cells were lysed, and luciferase activity was measured. Stem-loop qRT-PCR for mature miR-21 and miR-670 was performed as described (22Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. Lao K.Q. Livak K.J. Guegler K.J. Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (3924) Google Scholar) on an Applied Biosystems AB 7000 Real Time PCR system. Total RNA was extracted using Trizol reagent. After DNase I (Takara, Japan) treatment, RNA was reverse-transcribed with reverse transcriptase (ReverTra Ace, Toyobo). The levels of miR-21 and miR-670 analyzed by qRT-PCR were normalized to that of U6. U6 primers were: forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACGAATTTGCGT-3′. qRT-PCR analysis for FasL was standardized to control values of glyceraldehyde-3-phosphate dehydrogenase. The sequences of FasL primers were: forward, 5′-CTGAAGAAGAGAGGGAACCACA-3′; reverse, 5′-AGCTCCTTCTGTAGGTGGAAGA-3′. The sequences of glyceraldehyde-3-phosphate dehydrogenase primers were: forward primer, 5′-CCAAAAGGGTCATCATCTCTGC-3′; reverse, 5′-TGCTAAGCAGTTGGTGGTGCAG-3′. Chemically modified antagomir complementary to miR-21 designed to inhibit endogenous miR-21 expression and antagomir-NC were obtained from GenePharma Co. Ltd. The antagomir sequence was 5′-UCAACAUCAGUCUGAUAAGCUA-3′. The antagomir-NC sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′. Cells were transfected with the antagomirs or the antagomir-NC using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The results are expressed as means ± S.E. of at least three independent experiments. The statistical comparison among different groups was performed by one-way analysis of variance. Paired data were evaluated by Student's t test. p < 0.05 was considered statistically significant. We analyzed whether Foxo3a and miR-21 are involved in the apoptotic program of doxorubicin. The immunofluorescence of A549 cells revealed that Foxo3a was predominantly distributed in the cytoplasm in the control cells without treatment. In contrast, Foxo3a was accumulated in the nuclei in response to doxorubicin treatment. The immunoblotting of subcellular fractions also revealed that Foxo3a was translocated from the cytoplasm to the nuclei upon doxorubicin treatment (Fig. 1A). Because doxorubicin is a component of the standard chemotherapeutic protocol to treat neuroblastoma, we tested whether it can also influence Foxo3a in the human neuroblastoma cell line SH-EP1. A similar result was obtained in SH-EP1 cells (Fig. 1B). The subcellular distributions of Foxo3a are controlled by its phosphorylation status (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar). Accordingly, we detected the subcellular distributions of the phosphorylated and nonphosphorylated Foxo3a upon doxorubicin treatment. We observed a time-dependent elevation of nonphosphorylated Foxo3a in the nuclei and a reduction of phosphorylated Foxo3a in the cytoplasm in both A549 and SH-EP1 cells (Fig. 1C). These data suggest that doxorubicin can induce the relocalization of Foxo3a to the nuclei. We detected the levels of miR-21 in cells upon doxorubicin treatment. Doxorubicin induced a reduction in miR-21 levels in A549 (Fig. 1D) and SH-EP-1 cells (Fig. 1E). To know if the alterations of miR-21 is specific or not, we detected miR-670, and no significant alterations in miR-670 levels were observed (data not shown). Thus, it appears that miR-21 can be down-regulated by doxorubicin. The simultaneous alterations in Foxo3a and miR-21 led us to consider whether these two events are related. To address this issue, we produced Foxo3a RNAi constructs and tested whether knockdown of Foxo3a can influence miR-21 levels. The RNAi construct of Foxo3a but not its scramble form was able to suppress Foxo3a expression (Fig. 2A). Surprisingly, knockdown of Foxo3a could attenuate the reduction of miR-21 induced by doxorubicin in A549 (Fig. 2B) and SH-EP1 cells (Fig. 2C). To further understand whether Foxo3a is able to influence miR-21 expression, we detected miR-21 levels in cells expressing the constitutively active form of Foxo3a (caFoxo3a) (Fig. 2D). Enforced expression of caFoxo3a led to an elevation in miR-21 levels in A549 (Fig. 2E) and SH-EP1 cells (Fig. 2F). These data suggest that Foxo3a can negatively influence the expression of miR-21. We tested whether Foxo3a and miR-21 are functionally related in apoptosis. Enforced expression of miR-21 could attenuate apoptosis induced by caFoxo3a in A549 (Fig. 3A) and SH-EP1 cells (Fig. 3B). miR-21 also could inhibit apoptosis induced by doxorubicin (Fig. 3C). We characterized the role of endogenous miR-21 in the apoptotic program of doxorubicin. Doxorubicin at a dose of 0.2 μm led to a less amount of cells to undergoing apoptosis. However, knockdown of miR-21 resulted in a significant amount of cells undergoing apoptosis in response to the same dose of doxorubicin treatment (Fig. 3D). We tested whether Foxo3a is involved in mediating the apoptotic signal of doxorubicin. Knockdown of Foxo3a was able to attenuate apoptosis induced by doxorubicin, but this effect could be abolished by miR-21 antagomir (Fig. 3E). Collectively, Foxo3a and miR-21 are functionally related in the apoptotic cascades. The functional correlation between Foxo3a and miR-21 necessitates the elucidation of the mechanism by which miR-21 is regulated by Foxo3a. Foxo3a is a transcriptional factor. Accordingly, we tested whether the regulation of miR-21 by Foxo3a occurs through a transcriptionally dependent or independent manner. To this end, we analyzed the promoter region of miR-21. There are two optimal Foxo3a consensus binding sites (Fig. 4, A and B). ChIP assay revealed an increase in the association levels of Foxo3a with BS2 in response to doxorubicin treatment. However, an association of Foxo3a with BS1 was not detectable (Fig. 4A). We tested whether Foxo3a can influence miR-21 promoter activity. Wild type miR-21 promoter (wt-1) demonstrated a low activity in the presence of caFoxo3a. Also, the truncated form of wild type miR-21 promoter containing only the binding site-2 (wt-2) showed a low activity in the presence of caFoxo3a. However, mutations in the Foxo3a consensus binding site-2 (BS2) could abolish the inhibitory effect of Foxo3a on miR-21 promoter activity (Fig. 4C). These data suggest that BS2 is the Foxo3a binding site. Doxorubicin could induce a time-dependent reduction in miR-21 promoter activity (Fig. 4D). Concomitantly, knockdown of Foxo3a could attenuate the reduction of miR-21 promoter activity induced by doxorubicin in A549 (Fig. 4E) and SH-EP-1 cells (Fig. 4F). These data indicate that miR-21 can be transcriptionally repressed by Foxo3a. Foxo3a and miR-21 cannot directly execute apoptosis. Which molecules are their downstream mediators? Foxo3a has been shown to either activate or suppress FasL transcription depending on the cellular context (6Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar, 7Jonsson H. Allen P. Peng S.L. Nat. Med. 2005; 11: 666-671Crossref PubMed Scopus (172) Google Scholar). We detected the levels of FasL in response to doxorubicin treatment. An up-regulation of FasL could be observed upon doxorubicin treatment (Fig. 5A). Knockdown of FasL attenuated cell death (Fig. 5B), suggesting that FasL participates in conveying the death signal of doxorubicin. We tested whether the up-regulation of FasL is related to Foxo3a. Knockdown of Foxo3a attenuated FasL protein levels upon doxorubicin treatment (Fig. 5C). Surprisingly, FasL mRNA levels were not significantly altered by knockdown of Foxo3a (Fig. 5D). Human FasL promoter region contains 2 Foxo3a consensus binding sites (Fig. 5E). We performed a ChIP assay to detect whether Foxo3a binds to these sites upon doxorubicin treatment. The ChIP assay revealed that there was no detectable association between Foxo3a and the consensus binding site-1 (BS1) or binding site-2 (BS2) (Fig. 5F). We tested whether caFoxo3a can influence FasL mRNA levels and promoter activity. caFoxo3a led to no significant alterations in FasL mRNA levels (Fig. 5G) as well as FasL promoter activity (Fig. 5H). We sequenced the promoter region of FasL (3157 bp) and found no mutations in this region. We used NFAT4 as a positive control because FasL promoter contains NFAT4 binding sites (23Latinis K.M. Norian L.A. Eliason S.L. Koretzky G.A. J. Biol. Chem. 1997; 272: 31427-31434Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). We tested whether NFAT4 can bind to the promoter region of FasL in the primary cardiac fibroblasts. NFAT4 association with FasL promoter could be detectable (Fig. 5I). Taken together, these data suggest that Foxo3a does not transcriptionally regulate FasL expression upon doxorubicin treatment. The deficiency of Foxo3a to transcriptionally regulate FasL led us to search for other mechanisms that account for the up-regulation of FasL upon doxorubicin treatment. miRNAs can suppress gene expression, whereas doxorubicin can induce a reduction of miR-21 expression. These lines of evidence encouraged us to test whether miR-21 participates in the regulation of FasL. Enforced expression of miR-21 could attenuate the elevation of FasL levels induced by doxorubicin (Fig. 6A). Knockdown of endogenous miR-21 augmented FasL expression (Fig. 6B). To understand whether miR-21 can directly target FasL, we analyzed the 3′-UTR region of FasL and observed that miR-21 is a potential target of FasL (Fig. 6C). A luciferase assay revealed that miR-21 could suppress the translational activity of FasL. Introduction of mutations in the 3′-UTR region of FasL led to the failure of miR-21 to repress FasL translation (Fig. 6D). These data suggest that FasL is a direct target of miR-21. To know whether the endogenous miR-21 can regulate FasL translation, the cells were transfected with the constructs of FasL 3′-UTR and then exposed to doxorubicin. Treatment with doxorubicin led to an elevation in the luciferase activity of wild type 3′-UTR but not the mutated 3′-UTR (Fig. 6E), suggesting that endogenous miR-21 can target FasL 3′-UTR. We finally tested whether miR-21 regulates FasL expression via targeting its 3′-UTR. FasL with wild type 3′-UTR expressed at a low level in the presence of miR-21. Introduction of mutations in the miR-21 binding site abolished the inhibitory effect of miR-21 on FasL expression (Fig. 6F). Taken together, it appears that miR-21 can inhibit the translation of FasL. The growing evidence has shown that miR-21 is an oncogenic miRNA and is highly expressed in a variety of malignant tumors (24Wang Y. Lee C.G. J. Cell. Mol. Med. 2009; 13: 12-23Crossref PubMed Scopus (296) Google Scholar, 25Krichevsky A.M. Gabriely G. J. Cell. Mol. Med. 2009; 13: 39-53Crossref P" @default.
• W2002201015 created "2016-06-24" @default.
• W2002201015 creator A5021202119 @default.
• W2002201015 creator A5030691366 @default.
• W2002201015 date "2010-05-01" @default.
• W2002201015 modified "2023-09-30" @default.
• W2002201015 title "Foxo3a Regulates Apoptosis by Negatively Targeting miR-21" @default.
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