Cancer Research MicroRNA145 Targets BNIP3 and Suppresses … · Molecular and Cellular Pathobiology...

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Published OnlineFirst March 23, 2010; DOI: 10.1158/0008-5472.CAN-09-3718

Molecular and Cellular Pathobiology
Cancer
Research

MicroRNA145 Targets BNIP3 and Suppresses ProstateCancer Progression

Xueqin Chen1, Jing Gong1, Hao Zeng2, Ni Chen1, Rui Huang1, Ying Huang1, Ling Nie1, Miao Xu1, Juan Xia1,Fang Zhao4, Wentong Meng3, and Qiao Zhou1

Abstract

Authors' ABiotherapyChina Med3LaboratorChina; andHelsinki, H

CorresponMedical S610041, Chzhouqiao@

doi: 10.115

©2010 Am

Cancer R

Do

The putative tumor suppressor miR145 is transcriptionally regulated by TP53 and is downregulated in manytumors; however, its role in prostate cancer is unknown. On the other hand, BCL2/adenovirus E1B 19-kDainteracting protein 3 (BNIP3) is overexpressed in various tumors, including prostate cancer, and may tran-scriptionally repress the apoptosis-inducing factor (AIF) gene. Although BNIP3 transcription is controlled byhypoxia-inducible factor 1α (also elevated in prostate cancer), we postulated the posttranscriptional regula-tion of BNIP3 by miR145 through bioinformatics analysis, and herein we experimentally showed that miR145negatively regulated BNIP3 by targeting its 3′-untranslated region. Artificial overexpression of miR145 by usingadenoviral vectors in prostate cancer PC-3 and DU145 cells significantly downregulated BNIP3, together withthe upregulation of AIF, reduced cell growth, and increased cell death. Artificial overexpression of wild-typeTP53 in PC-3 cells (which lack TP53 protein) and DU145 cells (in which mutated nonfunctioning TP53 is ex-pressed) significantly upregulated miR145 expression with consequent effects on BNIP3 and cell behavior aswith miR145 overexpression. Analysis of prostate cancer (n = 134) and benign prostate (n = 83) tissue sampleshowed significantly decreased miR145 and increased BNIP3 expression in prostate cancer (P < 0.001), partic-ularly in those with tumor progression, and both molecular changes were associated with unfavorable out-come. Abnormalities of the miR145-BNIP3 pair as part of TP53-miR145-BNIP3-AIF network may play a majorrole in prostate cancer pathogenesis and progression. Cancer Res; 70(7); 2728–38. ©2010 AACR.

Introduction

Posttranscriptional regulation of mRNA by microRNA(miRNA) is an important mechanism controlling gene func-tion. Abnormalities in these small RNAs ∼22 nt in length hasbeen implicated in the pathogenesis of a variety of diseases,notably neoplasms (1–3). Overexpression of oncogenic miRNAs(oncomirs) or underexpression of tumor suppressor miRNAsplays pivotal roles in tumorigenesis. One major tumor sup-pressor miRNA, miR145, is downregulated in such neoplasmsas colorectal, mammary, ovarian, and B-cell tumors (4–8).Although microarray screening indicated that miR145 is alsoamong the downregulated miRNAs in prostate cancer (9, 10),its role in prostate cancer is unknown.On the other hand, BCL2/adenovirus E1B 19-kDa interact-

ing protein 3 (BNIP3), a BH3-only Bcl-2 family protein (11), isoverexpressed in many cancers, including prostate cancer

ffiliations: 1Laboratory of Pathology, State Key Laboratory ofand Department of Pathology, West China Hospital, Westical School, Sichuan University; 2Department of Urology andy of Stem Cell Research, West China Hospital, Chengdu,4Department of Pathology, Haartman Institute, University ofelsinki, Finland

ding Author: Qiao Zhou, West China Hospital, West Chinachool, Sichuan University, 37 GuoXueXiang, Chengduina. Phone: 86-28-85164027; Fax: 86-28-85164027; E-mail:mcwcums.com.

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(12). Although conventionally classified as a pro–cell deathprotein, BNIP3 has recently been found to function as a tran-scriptional corepressor of the apoptosis inducing factor geneAIF (13). Transcription of BNIP3 is regulated by hypoxia-inducible factor 1α (HIF-1α; refs. 14, 15); however, ourpreliminary data indicate significant contribution of HIF-1α–independent mechanisms, and bioinformatics analysisshows that miR145 may be a regulator of BNIP3. Herein, weshow that BNIP3 mRNA is a target regulated by miR145. Lossof miR145, a major cause of which may be dysfunction of itstranscription activator TP53, results in the overexpression ofBNIP3 in prostate cancer and may lead to the downregulationof the proapoptotic gene AIF. Aberrancy of this pathway issignificantly associated with prostate cancer progressionand a worse prognosis.

Materials and Methods

Cell lines and general reagents. Human prostate cancercells LNCaP, DU145, and PC-3 were from the AmericanType Culture Collection and were maintained in RPMI1640 with 10% FCS (Life Technologies). The adenovirus-immortalized human embryonic kidney epithelial cellHEK-293 was maintained in DMEM with 10% FCS. The phos-phatidylinositol-3 kinase (PI3K) inhibitor LY294002 was fromSigma. Tris base, Tween 20, DTT, and EDTA were fromAmresco. Phenylmethylsulfonyl fluoride, leupeptin, pepstatin,and aprotinin were from Roche Diagnostics.

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miR145 Targets BNIP3


Tissue samples and clinical data. Two hundred seven-teen archived formalin-fixed, paraffin-embedded samples, in-cluding 134 prostate adenocarcinoma (121 needle biopsies,13 transurethral resection of prostate samples) and 83 benignprostate (transurethral resection of prostate) tissues, wereused, as were 7 snap-frozen fresh tissue samples (2 cancerous,3 benign prostate hyperplasia, and 2 normal) obtained fromprostatectomy specimens. All tissue samples were from WestChina Hospital and were collected and used according to theethical guidelines and procedures approved by the institu-tional supervisory committee. The Gleason scores of the pros-tate adenocarcinomas were as follows: 5 to 6 (8 cases, 6%), 7(38 cases, 28%), and 8 to 10 (88 cases, 66%). The tumor-node-metastasis stages were as follows: stage II has 10 cases (7%),stage III has 79 cases (59%), and stage IV has 45 cases (34%).This cohort of patients ranged in age from 54 to 87 y (mean,71.6 y) and were treated by combined androgen blockade(surgical castration plus flutamide). Patients were followedby clinical and laboratory monitoring on a regular basis start-ing at definitive diagnosis. Disease-specific survival time is de-fined as the time from definitive diagnosis to disease-specificdeath, and progression-free survival time is defined as thetime from definitive diagnosis to any of the following eventsafter initial treatment: prostate-specific antigen elevation,local progression, metastasis, or disease-specific death asfailure of treatment.Stem-loop reverse transcription, conventional reverse

transcription-PCR, and genomic DNA PCR. Total RNAwas extracted by using the Trizol reagent (Invitrogen). Thestem-loop reverse transcription-PCR (RT-PCR) techniquewas used to examine mature miR145 (16). The stem-loopRT primer was designed as 5′-GTCGTATCCAGTGCAGGGT-CCGAGGTATTCGCACTGGATACGACAGGGAT-3′. RT wascarried out in 20 μL volume containing 2.5 μg of total RNA,1.6 mmol/L miR145 stem-loop primer, 2 μL 10 mmol/L de-oxynucleotide triphosphate, 1 μL 0.1 mol/L DTT, and 1 μLM-Mulv reverse transcriptase (Fermentas) at 16°C for30 min, 42°C for 30 min, and 85°C for 5 min. The PCR primersfor mature miR145 were designed as follows: sense, 5′-CGG-CGTCCAGTTTTCCCAGG-3′, 5′-GTGCAGGGTCCGAGGT-3′(product length, 62 bp).The random RT primer 5′-(dN)9-3′ (TaKaRa) was used for

other genes, the PCR primers of which were designed accord-ing to their respective cDNA sequences (Genbank) as follows:HIF-1α (5′-CCTATGACCTGCTTGGTGCTG-3′, 5′-CTGGCTC-ATATCCCATCAATTCG-3′; product length, 157 bp), BNIP3coding sequence fragment (5′-ACCAACAGGGCTTCTGAAA-C-3′, 5′-GAGGGTGGCCGTGCGC-3′, 202 bp), BNIP3 fragmentspanning CDS and 3′-untranslated region (UTR; 5′-ACCAA-CAGGGCTTCTGAAAC-3′, 5′-CTCGAGCCAGGATCTAACA-GCTCTTCAG-3′, 444 bp), AIF (5′-CTGAAAGACGGCAGGA-AGGTAG-3′, 5′-CTCCAGCCAATCTTCCACTCAC-3′, 253 bp),TP53 (5′-GTGTGGTGGTGCCCTATGAGC-3′ and 5′-ACAGG-CACAAACACGCACCTC-3′,188 bp), GADD45A (5′-GAGAGCA-GAAGACCGAAAGGA-3′, 5′-CACAACACCACGTTATCGGG-3′, 145 bp), and β-actin (5′-CTGGCACCACACCTTCTA-CAATG-3′, 5′-CCTCGTAGATGGGCACAGTGTG-3′, 248 bp).Standard PCR protocols were used; products were resolved

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by 2% agarose gel or 10% PAGE andwere visualized by stainingwith ethidium bromide or the fluorescent dye Goldview (SBS).Images were captured by scanning with Typhoon 8600 Multi-Imager (Molecular Dynamics) under fluorescence mode orwith Bio-Rad Gel Doc XR (Bio-Rad). Semiquantitative analysiswas performed with the ImageQuant 5.2 software (MolecularDynamics).The genomic sequence corresponding to pri-miR145 was

amplified from genomic DNA prepared with an extractionkit (Promega) and primers as described (17). The β-globingene (5′-GATCTGTCCACTCCTGATGCTG-3′, 5′-ATCAAGC-GTCCCATAGACTCAC-3′, 196 bp) was used as internal con-trol. The BNIP3 3′-UTR was amplified and sequenced byusing the following primers: 5′-TCCACCAGCACCTTTTGA-3′and 5′-GTAGACAACCTGCATCCATT-3′ (513 bp), and 5′-GCTACTTTAAGGGGTTTGTCC-3′ and 5′-CCTCTAGAAA-GATTTATTTTTTTTTCCATT-3′ (435 bp).Real-time quantitative PCR. Real-time quantitative PCR

(Q-PCR) was used together with stem-loop RT to quantitatemature miR145. Q-PCR was performed on Light Cycler 2.0(Roche), and data were analyzed with the Light Cycler soft-ware 4.05 (Roche) as described (18). The β-actin gene wasused as control. Copy number of target genes (relativeto β-actin) was determined by the 2−ΔΔCt method, withΔΔCt = ΔCtexp − ΔCtcon = (Ctexp-target − Ctexp-actin) − (Ctcon-target −Ctcon-actin), in which “exp” represents the experimental group,“con” the control group, and “target” the gene of interest.Locked nucleic acid in situ hybridization. Tissue sec-

tions were prepared from paraffin-embedded tissue blocks,deparaffinized, transferred into diethyl pyrocarbonate–treated water, treated with proteinase K (20 μg/mL; Roche)at 37°C for 30 min then with 0.2% glycine for 1 min, and fixedwith 4% paraformaldehyde. The sections were incubated in hy-bridization buffer (50% formamide, 5× SSC, 0.1% Tween,9.2 mmol/L citric acid for adjustment to pH 6, 50 μg/mLheparin, 500 μg/mL yeast RNA) at 37°C for 2 h. Digoxigen-in-labeled, LNA-modified miR145 probe (20 nmol/L; 5′-AGGGATTCCTGGGAAAACTGGAC-3′, Exiqon) was addedand incubated at 53°C for 18 h. Sections were washed with2× SSC once then with 2× SSC with 50% formamide at 53°Cthrice (30 min each). The anti–DIG-AP antibody (1:1,000,Roche) was added after PBST (PBS with 0.1% Tween 20) washand incubated at 37°C for 1 h and then at 4°C overnight. Sec-tions were washed five times with PBST, and nitroblue tetra-zolium chloride/5-bromo-4-chloro-3-indonyl phosphate wasused for hybridization signal detection. Two percent methylgreen (Sigma) was used for nuclear counterstain. The LNAprobe for U6 (5′-CACGAATTTGCGTGTCATCCTT-3′) wasused as control (hybridization temperature at 50°C).The locked nucleic acid in situ hybridization (LNA-ISH)

signal intensity was recorded semiquantitatively (19), with0 indicating no signal and 1 to 3 for weak, moderate, andstrong signals, respectively. The extent of LNA-ISH signalwas defined as the percentage of cells showing signal and re-corded as 0 (0%), 1 (1–30%), and 2 (>30%). An integratedscore of LNA-ISH signal (obtained by the product of the in-tensity score and the extent score) of 4 or more was desig-nated as miR145 positive.

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Western blot. The primary antibodies used were as fol-lows: BNIP3 (mouse monoclonal, 1:3,000, Sigma), HIF-1α(mouse monoclonal, 1:1,500, Chemicon, Inc.), phosphorylatedAKT (rabbit polyclonal, 1:600, Cell Signaling Technology,Inc.), AKT1/2 and pAKT (goat polyclonal, 1:800, Santa CruzBiotechnology), TP53 (mouse monoclonal, 1:1,000, Boster),AIF (rabbit monoclonal, 1:500, Epitomics, Inc.), glyceralde-hyde-3-phosphate dehydrogenase (GAPDH; mouse monoclo-nal, 1:10,000, Kangcheng), and β-tubulin (mouse monoclonal,1:1,000, Huatesheng). Horseradish peroxidase–labeled sec-ondary antibodies were from Zymed Laboratories, Inc. West-ern blotting was carried out as previously described (18).Immunohistochemistry and immunocytochemistry. The

antibodies and dilutions used for immunohistochemistry wereBNIP3 (mouse monoclonal, 1:300, Sigma), HIF-1α (mousemonoclonal,1:200, Chemicon), and cleaved caspase-3 antibody(rabbit polyclonal, 1:200, Cell Signaling Technology). Immu-nostaining was carried out as previously described (18).Recombinant adenoviral vectors for overexpression of

miR145 and TP53. The pri-miR145 sequence −179 to +287(+1 being the first base of the mature miR145) was amplifiedfrom HEK-293 cell genomic DNA with the primers described(17). PCR product was cloned into pMD18-T (TaKaRa), veri-fied by sequencing, and subcloned into shuttle plasmid pAd-Track-CMV (designated as pAdTrack-miR145; ref. 20).pAdTrack-miR145 linearized with PmeI was used to trans-form BJ5183-AD-1 cells harboring the adenoviral pAdeasy-1

Cancer Res; 70(7) April 1, 2010


vector (Stratagene) for homologous recombination. Colonieswere screened by plasmid miniprep and PacI restrictionanalysis to obtain clones with recombinant miR145 (desig-nated as pAdeasy-miR145). PacI linearized pAdeasy-miR145was used to transfect HEK-293 cells to obtain packaged re-combinant miR145 adenovirus (designated as AD-miR145).AD-miR145 was amplified by repeated infection and verifiedby PCR. The pAdTrack-CMV empty vector was used ascontrol (designated as AD-control). The titers and the multi-plicity of infection were determined according to the manu-facturer's protocols.The adenovirus vector for wild-type TP53 was constructed

in a similar fashion. The coding sequence of wild-type TP53was cloned from 293 cells by RT-PCR with the followingprimers: 5′-AAGCTTATGGAGGAGCCGCAGTC-5′ and 5′-TCTAGACAGTGGGGAACAAGAAGTG-3′.Cell viability assay. Cells were collected and stained with

trypan blue (Sigma, 200 mg/mL). The number of viable cellswas determined by microscopic examination.Flow cytometry. Collected cells were incubated with An-

nexin V-PE, 7-AAD, or both (BD Pharmingen) in 1 × AnnexinV binding buffer (BD Pharmingen) for 30 min at 4°C in thedark and then analyzed on BD FACSAria flow cytometer (BDPharmingen). Unstained and nontreated cells were used ascontrol. Data were collected and analyzed with the manufac-turer's software, and Annexin V–PE(+)/7-AAD(−) cells weregated as the apoptotic cell population.

Figure 1. miR145 and BNIP3 expression in normal prostate (NP), benign prostate hyperplasia (BPH), prostate cancer tissue (PCa), and prostate cancer celllines. A, stem-loop RT-PCR analysis (with actin as control) showing differential expression of mature miR145 in benign prostate tissue (NP and BPH) versusprostate cancer, and prostate cancer cells DU145, PC-3, and LNCaP; genomic DNA PCR analysis (with globin as control) of miR145 gene (genomic)showed no difference. B, further validation of loss of miR145 in prostate cancer by LNA-ISH (nuclear counterstain with methyl green) with U6 as control.The miR145 and U6 signals were in purple blue. C, in contrast to miR145, BNIP3 mRNA (top, RT-PCR, same actin control as for A) and protein (bottom,Western blotting, GAPDH as control) were significantly higher in prostate cancer tissue and cells than in benign prostate tissue. D, overexpression of BNIP3protein in prostate cancer, represented by cytoplasmic and nuclear (inset) brown staining, compared with benign prostate tissue, was further shownby immunohistochemistry (nuclear counterstain with hematoxylin).

Cancer Research





Terminal deoxynucleotidyltransferase–mediated bioti-nylated dUTP nick end-labeling. Terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL) wasperformed by using in situ cell death detection kit (Roche) aspreviously described (18).Luciferase reporter constructs and site-directed muta-

genesis. The two seed sequences (6–12 and 796–802 nt) ofBNIP3 3′-UTR with flanking sequences were amplified fromgenomic DNA of PC-3 cells. Site A (−27 bp to +84 bp, with +1being the first base after stop codon) was prepared with theprimers BNIP3-XbaI-P1 (5′-TCTAGACTGACAACCTCCAC-CAGCAC-3′) and BNIP3-XhoI-P2 (5′-CTCGAGCCAGGATC-TAACAGCTCTTCAG-3′), whereas site B (+658 bp to +911bp) was prepared with BNIP3-XhoI-P3 (5′-CTCGAGTCTGCT-GAAGGCACCTACTC-3′) and BNIP3-XbaI-P4 (5′-TCTAGA-TAGGGACCAGTCCTGGTTTC-3′). PCR products werecloned into pMD18-T then subcloned into pGL3-Promoter(Promega) and designated as pGL3–site A and pGL3–siteB, respectively, in which the site A and site B sequences wereinserted as the 3′-UTR downstream of the luciferase codingsequence, respectively. A construct with site A and B se-quences in tandem (pGL3–site A+B) was prepared by cloningof the ligated site A and B sequences.Overlapping PCR was used for site-directed mutagenesis of

the seed sequence (from AACTGGA to AGATCTC) in site A(designated as pGL3-Mut A) and site B (designated as pGL3-Mut B). A construct combining the two was prepared anddesignated as pGL3-Mut A+B. The PCR primers used wereas follows: BNIP3-site A-Mut1 (5′-GAAGATCTCTTCAT-CAAAAGGTGCTG-3′), BNIP3-site A-Mut2 (5′-GAAG-ATCTGTCTGACTTGGTTCGTTAG-3′), BNIP3-site B-Mut1(5′-CTACTTTAAAAAGATCTCAATGGAAAAA-3′), and BNIP3-site B-Mut2 (5′-CCATTGAGATCTTTTTAAAGTAGACAC-3′).Dual reporter gene assay. PC-3 cells were cultured in 24-

well plates and transfected with 0.4 μg of the reporterconstructs by using Lipofectamine 2000 (Invitrogen). ThepRL-CMV plasmid (Promega) containing the Renilla lucifer-ase gene (0.02 μg) was cotransfected as internal control. Cellswere infected with AD-miR145 and AD-control (multiplicityof infection, 100) 4 h after transfection, collected 24 h later,and the firefly and Renilla luciferase activities were assayedon Luminometer TD-20/20 (Turner Design).Statistical analysis. The SPSS 10 program was used

for general statistical and survival analysis. Fisher's exacttest was used for between-group comparisons, and Spearmanrank order correlation was used for correlation analysis.The Kaplan-Meier method with log-rank test was used forunivariate survival analysis, and the Cox proportional regres-sion model was used for multivariate survival analysis.

Results

miR145 was significantly downregulated in prostatecancer. Expression of mature miR145 was investigated byusing stem loop RT-PCR, which showed high expression le-vels of miR145 in benign prostate hyperplasia and normalprostate tissue but very low levels in prostate cancer tissue



(Fig. 1A). In PC-3, DU145, and LNCaP cells, no miR145 couldbe detected (Fig. 1A).Real-time PCR further showed that miR145 in prostate

cancer tissue was only 1/53.8 of that in benign prostate tissueand was barely detectable in the three prostate cancer celllines (<1/10,000 of that in benign prostate tissue; data notshown).In cancer tissue samples, the presence of stromal cells

(which may express miR145) could interfere with the evalu-ation of expression level with the stem loop RT-PCR tech-nique. To further examine the expression status in tissuesamples, the LNA-ISH technique was used, which showedthat miR145 was expressed at high levels in benign prostateepithelia but at significantly lower levels or was absent in themajority of prostate cancer parenchyma (Fig. 1B). ThemiR145 positivity rate in benign prostate tissue samples (56of 83, 67.5%) was significantly higher than in prostate cancertissue samples (33 of 106, 31.1%, P = 0.000; Table 1).Sequence analysis of the miR145 gene showed no mutation

or deletion (data not shown) in the three prostate cancer celllines.miR145 expression was inversely related to BNIP3 over-

expression in prostate cancer. BNIP3 overexpression inprostate cancer tissue samples and cell lines was shown byRT-PCR, Western blot analysis, and immunostaining (Fig. 1Cand D). The proportion of prostate adenocarcinoma cases(89 of 134, 66.4%) with moderate to strong BNIP3 immunos-taining (score 2 and 3) was significantly higher than that ofbenign prostate tissue (16 of 83, 19.3%, P = 0.000).

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Table 1. Association of miR145 and BNIP3 ex-pression levelswith prostate cancer progression

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miR145(+)

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Association for

BNIP3 (+)

BPH
56/83 (67.5)* 16/83 (19.3) PCa total 33/106 (31.1) 89/134 (66.4) PCa with progression 7/50 (14.0) 49/64 (76.6) PCa without progression 26/56 (46.4) 40/70 (57.1) P values BPH vs PCa total 0.000 0.000 BPH vs PCa with
progression
0.000 0.000
BPH vs PCa withoutprogression

0.015
0.000
PCa with vs withoutprogression

0.000
0.027
NOTE: miR145(+) was defined as having a LNA-ISH stain-ing score of 4 or above. BNIP3(+) was defined as moderateto strong immunostaining (score 2 and 3). P values weredetermined by Fisher's exact test.Abbreviations: BPH, benign prostate hyperplasia; PCa,prostate cancer.*Number of miR145(+) or BNIP3(+) cases/total number ofcases (positive rate %).

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Importantly, correlation analysis of BNIP3 protein level(determined by immunohistochemistry) and miR145 level(determined by LNA-ISH) showed inverse relationship be-tween the two (rs = −0.314, P < 0.01; Table 1; Fig. 1).Identification of potential BNIP3 3′-UTR seed sequences.

The 824 nt 3′-UTR of the BNIP3 mRNA (full-length 1,535 nt,coding sequence 127–711 nt) was analyzed by using Target-Scan 5.1 (http://www.targetscan.org/), which identified



miR145 as the major potential regulatory miRNA of BNIP3.The 6 to 12 nt and 796 to 802 nt of the BNIP3 3′-UTR weretwo potential seed sequences (Fig. 2A) that were highlyconserved across species (Fig. 2B). Sequence analysisshowed no mutation or deletion of the 3′-UTR in PC-3,DU145, and LNCaP cells.Dual reporter gene assays showed interaction of

miR145 with 3′-UTR of BNIP3. To show posttranscriptional

Cancer Research


,

.

Figure 2. Identification of miR145 seedsequences in BNIP3 3′-UTR and dual reportergene assays for miR145-BNIP3 3′-UTRinteraction. A and B, the 6 to 12 nt and 796 to802 nt of the BNIP3 3′-UTR were identified astwo potential seed sequences for miR145,designated as sites A and B, respectively (A),which were conserved across species (B).C, dual reporter gene assays were performedwith pGL3 expression constructs with BNIP33′-UTR regions containing the seed sequencesinserted downstream of the luciferase codingsequence, and the activity of the basic pGL3construct as baseline (pGL3-Promoter).With the artificial expression of miR145 (bycoinfection with AD-miR145), the reporter geneactivity, represented by relative luciferaseactivity (firefly/Renilla), was significantlydecreased when either site A or site B, or siteA + B (in tandem) was present in the constructswhereas mutations of the seed sequences(Mut A, Mut B, and Mut A + B) significantlyrestored reporter gene activity. Expression ofmiR145 alone had no effect on reporter geneactivity when no seed sequences were inserted

Figure 3. Effects on BNIP3, AIF, and cell behavior by artificial overexpression of miR145 in PC-3 and DU145 cells. A and B, the efficiency of Ad-miR145 andAd-control infection was shown by homogenous green fluorescence protein expression of the infected cells (A, top). Artificial overexpression of miR145by Ad-miR145 (A) resulted in the significant downregulation of BNIP3 protein level compared with Ad-control (B, top, Western blot with semiquantitativehistograms; bottom, immunocytochemistry with brown staining representing BNIP3 protein) but no change in the BNIP3 mRNA level (A, bottom, left,RT-PCR; right, Q-PCR) or HIF-1α mRNA (A) or protein (B) levels. AIF was simultaneously upregulated upon miR145 overexpression (A) and BNIP3downregulation (B). C, cell growth was significantly inhibited concomitant with the miR145-BNIP3-AIF expression change. D, increased cell death upon themiR145-BNIP3-AIF expression change as shown by flow cytometry analysis of percentage of Annexin V-PE–stained apoptotic cells (top) and TUNELassays (middle). The cell death was independent of caspase-3 activation, as shown by lack of immunostaining of cleaved (activated) caspase-3(bottom; inset, positive control of activated caspase-3 immunostaining in PC-3 cells irradiated with UV).



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regulation of BNIP3 mRNA by miR145, luciferase reportergene constructs were prepared in which the potential seedsequences (sites A and B) of BNIP3 3′-UTR were cloned intoluciferase reporter constructs, together with constructs inwhich the seed sequences were mutated.With artificial coexpression of miR145 by infection with

Ad-miR145, dual reporter assays showed significant downre-gulation of luciferase reporter gene activity by 63.6%, 50.7%,and 67.5% in the pGL3–site A, pGL3–site B, and pGL3–siteA+B constructs, respectively (Fig. 2C), whereas reporter con-structs lacking BNIP3 3′-UTR sequences were not affected.Moreover, mutation of the seed sequences significantly re-stored the luciferase gene activity in the constructs bearingthe mutated 3′-UTR sequences (Fig. 2C).Overexpression of miR145 by adenoviral vectors led to

downregulation of BNIP3 protein, upregulation of AIF,and increased cell death. The biological effects of BNIP3 reg-ulation by miR145 were further shown by assaying molecularand cellular changes in PC-3 and DU145 cells with artificialmiR145 overexpression (Fig. 3A–D). Concomitant with theoverexpression of mature miR145 by Ad-miR145 infection(Fig. 3A), BNIP3 protein level was significantly downregu-lated (Fig. 3B). In contrast, the BNIP3 mRNA, as well as theHIF-1αmRNA and protein, showed little change with miR145overexpression (Fig. 3A and B).Significantly, the AIF level was upregulated simultaneously

(Fig. 3A) with miR145 overexpression. Synchronous with theBNIP3-AIF change upon artificial miR145 overexpression,PC-3 and DU145 cells showed reduced cell growth (Fig. 3C)and increased cell death (as assayed by flow cytometry andTUNEL; Fig. 3D), which was independent of caspase-3 activa-tion (Fig. 3D).Artificial overexpression of wild-type TP53 in PC-3 and

DU145 cells restored miR145 expression, together withdownregulation of BNIP3 and upregulation of AIF. The tu-mor suppressor TP53 has been identified as a transcriptionalregulator of miR145 (21). Loss of TP53 may be an importantmechanism underlying miR145 downregulation because PC-3cells lack TP53 protein and the TP53 in DU145 cells is mu-tated and nonfunctional (Fig. 4A; refs. 22, 23). Indeed, treat-ment of PC-3 and DU145 cells with PI3K/AKT inhibitorLY294002 could not restore TP53 function and miR145 ex-pression despite significant downregulation of phosphorylat-ed AKT (p-AKT; Fig. 4A).To further show that the loss of TP53 function may have

led to the loss of miR145 expression, we used adenoviral vec-tors to artificially overexpress wild-type TP53 (Fig. 4B and C).As shown in Fig. 4C, this resulted in significant upregulationof miR145 and simultaneous downregulation of BNIP3 pro-tein (Fig. 4C). Importantly, the AIF gene expression was alsoupregulated upon TP53 overexpression (Fig. 4C).Figure 4D schematically summarizes the data presented in

the above sections showing BNIP3 regulation by miR145,which may result from loss of TP53 function in prostate can-cer cells.Biological significance of miR145 and BNIP3 in prostate

cancer. Clinical analysis of our data showed that miR145 andBNIP3 abnormalities were associated with prostate cancer



pathogenesis and progression. In addition to the significantlydifferent expression pattern of miR145 and BNIP3 in benignand cancerous prostate tissue samples (Table 1; Fig. 1), thepositive rate of miR145 in prostate cancer with progression(7 of 50, 14.0%) was significantly lower than prostate cancerwithout progression (26 of 56, 46.4%, P = 0.000; Table 1),whereas the positive rate of BNIP3 in prostate cancer withprogression (49 of 64, 76.6%) was significantly higher thanprostate cancer without progression (40 of 70, 57.1%, P =0.027; Table 1).Kaplan-Meier analysis showed that decreased miR145

level and increased BNIP3 level, among other clinicopatho-logic factors, were significant negative prognostic factorsfor both disease-specific and progression-free survival inprostate cancer patients (P < 0.05; Fig. 5). Cox proportionregression model incorporating classic clinicopathologic para-meters (Gleason score, prostate-specific antigen level, andtumor stage) showed that the expression of miR145 was alsoan independent favorable prognostic factor for progression-free survival (relative risk, 0.404; 95% confidence interval,0.174–0.941; P = 0.036).

Discussion

miR145 regulation of BNIP3. The present study is the firstto identify BNIP3 as a target of posttranscriptional regulationby miR145. Although BNIP3 transcription is regulated byHIF-1α (14, 15), posttranscriptional control by miR145 con-tributes significantly to BNIP3 regulation (Fig. 4D).miR145 is downregulated in various human tumor types,

including cancers of the gastrointestinal tract, liver, naso-pharynx, lungs, urinary bladder, ovaries, uterine cervix,B cells, and soft tissue (5–8, 19, 24–30). In some tumors,downregulation of miR145 is correlated with tumor size,stage, proliferative activity, or poorer prognosis (6, 8, 30, 31).Gradual decrease of miR145 is observed in mammary neopla-sia (19), whereas artificial overexpression of miR145 inhibitscell growth and tumor formation (8, 32). Downregulation ofmiR145 in various cancers has made it one of the mostnoticeable tumor suppressor miRNAs (1).Identification of miRNA targets is one of the most impor-

tant aspects in understanding the mechanisms by whichmiRNAs control cell behavior (2, 3, 33). Several genes, includ-ing IRS-1, OCT4, SOX2, nuclear Kruppel-like factor 4, C-MYC,and RTKN (rhotekin), have been identified as miR145 targetgenes (21, 32, 34, 35). Inhibition of the pro-proliferation andantiapoptosis factor IRS-1 by miR145 suppresses colonic can-cer cell growth (32). RTKN, a Rho-GTP–interacting andGTPase-inhibiting protein, is inhibited by miR145, the lossof which may promote breast cancer cell growth (35). Inhibi-tion of stem cell factors OCT4, SOX2, and Kruppel-like factor4 by miR145 represses pluripotency in human embryonicstem cells and promotes cell differentiation (34). However,the roles of miR145 in tumorigenesis of many neoplasmsare still to be elucidated.Our study identified BNIP3 as a novel posttranscriptional

target of miR145 and miR145-BNIP3 as an important pairderanged in prostate cancer. Because BNIP3 transcription

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Figure 4. Artificial overexpression of wild-type TP53 in PC-3 and DU145 cells restored miR145 expression. A, despite significant downregulation ofphosphorylated AKT (p-AKT), treatment with PI3K/AKT inhibitor LY294002 could not restore TP53 function and miR145 expression (bottom, RT-PCR)in either PC-3 cells (which lack the TP53 protein) or DU145 cells (in which mutated nonfunctional TP53 protein is expressed; top, Western blot with tubulinas control). B, artificial overexpression of wild-type TP53 by Ad-TP53 (compared with Ad-control) resulted in significant inhibition of cell growth andincreased cell death. C, miR145 expression was significantly restored upon wild-type TP53 expression (top, left, RT-PCR; right, Q-PCR), withsimultaneous downregulation of BNIP3 protein (bottom, Western blot with semiquantitative histograms). AIF was also upregulated upon wild-type TP53overexpression. The TP53 target gene GADD45 was used as a control to show TP53 overexpression effect, and actin and GAPDH were used as internalcontrol for RT-PCR and Western analysis, respectively. D, schematic representation summarizing data from the present study and earlier reports.Transcription of BNIP3 gene is activated by HIF-1α, whereas posttranscriptional regulation by miR145 controls BNIP3 mRNA translation. BNIP3 proteinrepresses transcription of AIF (the transcription of which is also activated by TP53).

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is regulated by HIF-1α, its overexpression in prostate cancerresulted from at least two fundamental molecular defects:overactivity of the transactivator HIF-1α at the transcription-al level and loss of the negative regulator miR145 at the post-transcriptional level (Fig. 4D).BNIP3 overexpression and its significance. BNIP3 is a mi-

tochondrial protein involved in cell death (11, 36) and is over-expressed in many cancers such as breast cancer, non–smallcell lung cancer, glioblastomamultiforme, ovarian cancer, anduterine cervical and endometrial cancers (12, 14, 37–40),although it is underexpressed in several tumor types (e.g.,tumors of the pancreas, stomach, and colon and rectum)through hypermethylation of CpG islands and histone H3deacetylation (41–43). BNIP3 overexpression is associatedwith advanced stage in cervical cancer, poor prognosis in en-dometrial and non–small cell lung cancer, and tumor progres-sion in breast ductal carcinoma in situ (14, 39, 40, 44).Functionally, BNIP3 has been classified as a proapoptotic

gene. Although overexpression of proapoptotic genes in canceris well documented (e.g., caspase-3 and caspase-8, which areoverexpressed in a variety of tumors; ref. 45), recent data indi-cate that they may have functions quite opposite to their con-ventionally assigned roles. For example, caspase-8, the mostimportant initiator caspase in the extrinsic apoptotic path-ways, possesses a surprising, catalytic activity–independentfunction of associating with focal adhesion molecule and cal-pain 2 to facilitate tumor cell migration and metastasis (45).A similar scenario seems to be unfolding for BNIP3, which

has been unexpectedly identified as a transcription repressorof the proapoptotic gene AIF (13). BNIP3 associates with



PTB-associating splicing factor and histone deacetylase 1,and binds to the promoter of the AIF gene, hence repressingits expression and resulting in increased resistance to apo-ptosis. Thus, the dual nature of BNIP3 may explain its highlevels in various solid tumors, including prostate cancer(present study) and glioblastoma multiforme (13), and its as-sociation with unfavorable outcome in prostate cancer (pres-ent study) and other tumors (14, 39, 40, 44).Mechanisms of miR145 downregulation. An intriguing

question is the cause(s) of miR145 downregulation in varioustumors. Most recently, the transcription factors TP53 andOCT-4 have been shown to regulate transcription ofmiR145 (21, 34). In colorectal cancer cells, overexpressionof TP53 or inhibition of the PI3K/AKT pathway byLY294002 (which upregulates TP53) results in the upregula-tion of miR145 and suppression of c-MYC (a miR145 target),through binding of TP53 to its response element in themiR145 promoter (21). Moreover, TP53 also participates inthe maturation of miRNA (including miR145), by associatingwith DEAD-box RNA helicase p68 (DDX5) and interactingwith the Drosha processing complex, facilitating the proces-sing of primary to precursor miRNAs (46).PC-3 cells are known for TP53 loss of heterozygosity and

codon 138 mutation (resulting in frameshift and nonfunc-tioning, truncated TP53 protein that could be degradedquickly; refs. 22, 23). DU145 cells harbor mutations of codon223 (CCT to CTT, Pro to Leu) and codon 274 (GTT to TTT,Val to Phe), also resulting in abnormal TP53 proteins (23, 47).Although LNCaP cells express wild-type TP53, it harborsmutation of CHK2 at codon 1160 (C to A, Thr to Asn), leading

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Figure 5. Prognostic significance of lossof miR145 and overexpression of BNIP3 inprostate cancer. Kaplan-Meier curves (withlog-rank test P values) showing significantpoorer prognosis in patients with decreasedmiR145 or increased BNIP.




to deficit of TP53 protein phosphorylation and activation(48). In prostate cancer tissue, about half harbor TP53abnormalities. Thus, loss of functional TP53 may have con-tributed significantly to impaired miR145 expression, as TP53is vital for both transcriptional activation and maturation ofmiR145. Because TP53 also regulates transcription of AIF(49), loss of TP53 function strikes double blows in prostatecancer by downregulating both miR145 (and consequentmiR145-BNIP3 abnormalities) and AIF.Alternative mechanisms for disrupted miR145-BNIP3 par-

ing, such as mutation, deletion, or epigenetic modificationsof the miR145 gene and selective loss of BNIP3 3′-UTR,may exist; however, evidence is yet to be found. It is worthnoting that inhibition of DNA methylation or histone deace-tylation in lung adenocarcinoma does not result in miR145upregulation (17). Our sequencing analysis did not show de-letion or mutation of the miR145 gene in the studied prostatecancer cells, nor of the BNIP3 3′-UTR.In summary, we identified BNIP3 as a functional target of

miR145 and showed that the abnormalities of the miR145-BNIP3 pair might play major roles in prostate cancer tumor-igenesis and progression. In light of recent elucidation of



miR145 regulation by TP53 (21) and transcriptional repres-sion of AIF by BNIP3 (13), the TP53-miR145-BNIP3-AIF axismay be an important part of the regulatory network de-ranged in prostate cancer (Fig. 4D).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Xianming Mo and other colleagues of the Stem Cell ResearchLaboratory and the Pathology and Urology Departments.

Grant Support

Natural Science Foundation of China (NSFC 30871383, 30800637, and30221001); NSFC grant 30700977 (H. Zeng); and National Basic Research Pro-gram of China grant 2007CB947802 (W. Meng).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/08/2009; revised 01/12/2010; accepted 01/21/2010; publishedOnlineFirst 03/23/2010.

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2010;70:2728-2738. Published OnlineFirst March 23, 2010.Cancer Res Xueqin Chen, Jing Gong, Hao Zeng, et al. ProgressionMicroRNA145 Targets BNIP3 and Suppresses Prostate Cancer

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