ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

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Original Articles

MicroRNA-30d-5p inhibits tumour cell proliferation and motility by directly targeting CCNE2 in non-small cell lung cancer Di Chen a,1, Weijie Guo a,b,1, Zhaoping Qiu a, Qifeng Wang b, Yan Li b, Linhui Liang b, Li Liu b, Q1 Shenglin Huang b, Yingjun Zhao b, Xianghuo He a,b,* a State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China b Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China

A R T I C L E

I N F O

Article history: Received 22 January 2015 Received in revised form 25 March 2015 Accepted 28 March 2015 Keywords: miR-30d Non-small cell lung cancer CCNE2 Proliferation Invasion

A B S T R A C T

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules that are dysregulated in many types of human cancers, although their precise functions in driving non-small cell lung cancer (NSCLC) are incompletely understood. In the present study, we found that miR-30d-5p, often downregulated in NSCLC tissues, significantly inhibited the growth, cell cycle distribution, and metastasis of NSCLC cells. Furthermore, we demonstrated that cyclin E2 (CCNE2), which was often upregulated in NSCLC tissues, was a direct target of miR-30d-5p. CCNE2 expression promoted the proliferation, invasion, and migration of NSCLC cells. In addition, the re-introduction of CCNE2 expression antagonised the inhibitory effects of miR-30d-5p on the capacity of NSCLC cells for proliferation and motility. Together, these results suggest that the miR-30d-5p/CCNE2 axis may contribute to NSCLC cell proliferation and metastasis, indicating miR-30d-5p as a potential therapeutic target for the treatment of NSCLC. © 2015 Published by Elsevier Ireland Ltd.

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Introduction Lung cancer has become the leading cause of cancer-related mortality in men and women in China [1], and non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases [2]. Despite tremendous advances in the diagnosis and treatment of this cancer, the global mortality rate of NSCLC remains high, and the 5-year overall survival rate associated with NSCLC is a dismal 11% [3]. Further elucidation of the molecular mechanisms underlying NSCLC may have a significant impact on the systematic treatment of this disease. MicroRNAs (miRNAs) are approximately 21–25-nucleotide, noncoding RNA molecules that are highly conserved in a variety of eukaryotic organisms. miRNAs act as a class of negative regulators by binding to the 3′ untranslated region (3′-UTR) of target messenger RNAs (mRNAs), causing either target mRNA degradation or inhibition of translation through assembling the RNA-induced silencing complex (RISC). Accumulating evidence suggests that miRNAs whose expression levels are deregulated in tumours can function

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Abbreviations: CCNE2, cyclin E2; NSCLC, non-small cell lung cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; TCGA, The Cancer Genome Atlas; CDS, coding DNA sequence; 3′ UTR, 3′ untranslated region. * Corresponding author. Tel.: +86 21 34777577; fax: +86 21 64172585. E-mail address: [email protected] (X. He). 1 These authors contributed equally to this work.

65 as oncogenes or tumour suppressors [4]. In lung cancer, several 66 miRNAs, including those in the let-7 and miR-34 families, miR67 126, miR-145, and miR-200 have been documented as tumour 68 suppressors [5–10]. In contrast, miR-21, miR-31, miR-221, and miR69 222 were found to promote NSCLC carcinogenesis [11–13]. 70 In a previous study, miR-30d was found upregulated and acts 71 as a metastasis promoter by targeting GNAI2 in human hepatocel72 lular carcinoma [14]. Recently, we investigated the expression of miR73 30d-5p in several major organs of C57BL/6J mice and found that miR74 30d-5p was highly expressed in normal lung tissues (Fig. S1A). 75 Interestingly, several reports showed that miR-30d-5p is 76 downregulated in both squamous cell lung carcinoma and NSCLC 77 [15,16], in agreement with results reported in the Cancer Genome Atlas (TCGA) miRNASeq database (https://tcga-data.nci.nih.gov/ Q2 78 79 tcga/; Fig. S1B), suggesting that miR-30d-5p may serve as a tumour 80 suppressor in NSCLC. However, the exact role and underlying mech81 anism whereby miR-30d-5p drives the development and progression 82 of NSCLC remains elusive. 83 In the present study, we performed gain- and loss-of-function 84 studies to determine the biological roles of miR-30d-5p. We also 85 integrated bioinformatics predictions, expression datasets, lucifer86 ase reporter assay results and RIP assay results to reveal its 87 underlying molecular mechanism in NSCLC. We found that miR88 30d-5p functions as a tumour suppressor in NSCLC. Cyclin E2 89 (CCNE2) is characterised as a direct and functional target of miR90 30d-5p in NSCLC cells. These findings indicate that the miR-30d91 5p/CCNE2 axis is an important regulator in the development and

http://dx.doi.org/10.1016/j.canlet.2015.03.041 0304-3835/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Di Chen, et al., MicroRNA-30d-5p inhibits tumour cell proliferation and motility by directly targeting CCNE2 in non-small cell lung cancer, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.03.041

ARTICLE IN PRESS D. Chen et al./Cancer Letters ■■ (2015) ■■–■■

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progression of NSCLC and provides a candidate target for NSCLC treatment.

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Materials and methods

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Cell culture

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HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, USA); A549 and H1299 cells were cultured in Ham’s F-12K (Kaighn’s) medium; and SPC-A1 cells were cultured in RPMI-1640 medium (Hyclone, Beijing, China). The media were supplemented with 10% fetal bovine serum (Gibco), 100 IU/ ml penicillin G, and 100 μg/ml streptomycin sulphate (Sigma-Aldrich, St. Louis, USA) in a humidified 37 °C incubator with 5% CO2.

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RNA extraction and quantitative real-time polymerase chain reaction (qPCR) analysis

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Total RNA was extracted using the TRIzol® reagent (Invitrogen, Carlsbad, USA). Reverse-transcribed complementary DNA was synthesised with the Prime-Script® RT Reagent Kit (TaKaRa, Tokyo, Japan). qPCR analyses were performed with LightCycler®480 SYBR Green I Master (Roche, Welwyn Garden, Swiss). The detailed primer sequences are listed in Table S1. For miRNA detection, mature miR30d-5p was reverse-transcribed and quantified with TaqMan® RT primers and probe, and normalised to U6 small nuclear RNA expression, using predesigned TaqMan assays (Applied Biosystems, Foster City, USA).

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Vector construction

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The pri-miR-30d sequence was amplified from normal human genomic DNA and cloned into the pWPXL lentiviral vector (a generous gift from Dr. Didier Trono) to generate pWPXL-miR-30d-5p. The CCNE2 expression vector pWPXL-CCNE2 was constructed by inserting the CCNE2 ORF sequence. The 3′-UTR sequence of CCNE2 was amplified and inserted into the psiCHECK2 vector (Promega, Madison, USA). The detailed sequences of primers and oligonucleotides are listed in Table S1.

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Oligonucleotide transfection

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MiR-30d-5p mimics and inhibitors were designed and synthesised by RiboBio (Guangzhou, China), as were CCNE2 small interfering RNA (siRNA) duplexes. Cells were transfected in individual wells of 6-well plates with an inhibitor, a mimic, or an siRNA pool (3 siRNAs were mixed in an equimolar ratio) targeting miR-30d-5p, using Lipofectamine® 2000 at a final concentration of 50 nM. At 48 h posttransfection, cells were harvested for the assays described below. The detailed sequences are listed in Table S1.

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Lentivirus production and cell transduction

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Lentivirus particles were harvested 48 h after pWPXL-mir-30d or pWPXLCCNE2 cotransfection with the packaging plasmid psPAX2 and the VSV-G envelope plasmid pMD2.G (psPAX2 and pMD2.G were gifts from Dr. Didier Trono) into HEK 293T cells using Lipofectamine® 2000. A549 and H1299 cells were infected with the resultant recombinant lentivirus in the presence of 6 μg/ml polybrene (Sigma-Aldrich).

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Cell proliferation, colony-formation assays and in vivo tumour formation assay

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Cell proliferation was measured with the Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) following the manufacturer’s instructions. For colony-formation assay, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet (Sigma-Aldrich). Macroscopic cell colonies were counted. For in vivo tumour formation, 2.5 × 106 control cells or the cells that stably expressed miR-30d-5p were suspended in 200 μl of serum-free DMEM and subcutaneously injected into one flank of each mouse (ten male BALB/c-nu/nu in each group for 5 weeks). After 5 weeks, the mice were sacrificed, and the parameters were measured.

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Cell cycle analysis

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Cells were collected and fixed overnight in 75% ethanol at −20 °C. The fixed cells were washed 3 times with phosphate-buffered saline (PBS) and stained in PBS for 30 min with 25 μg/ml propidium iodide (PI; Kaiji Biotech, Nanjing, China), 10 μg/ ml RNase A (Sigma-Aldrich), 0.05 mM ethylene diamine tetraacetic acid, and 0.2% Triton X-100. DNA contents were measured with a FACSCalibur flow cytometer (BD Biosciences, New Jersey, USA), and the results were analysed using ModFit software (BD Biosciences).

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In vitro migration and invasion assays

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For transwell migration assays, 5 × 104 cells were plated in the top chamber of each insert (BD Biosciences) with a non-coated membrane. For invasion assays, 1 × 105 cells were added to the upper chamber with 150 μg Matrigel (BD Biosciences). For both assay types, 800 μl of medium supplemented with 10% foetal bovine serum

was injected into the lower chambers. After harvest, the inserts were fixed and stained in a dye solution containing 0.1% crystal violet and 20% methanol. Cells adhering to the lower membrane of the inserts were imaged with an IX71 inverted microscope (Olympus, Tokyo, Japan), and the quantification of five randomly selected fields were counted.

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xCELLigence real-time cell analysis of migration

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Cell-migration experiments were performed using modified 16-well plates (CIM16, Roche Diagnostics GmbH, Mannheim, Germany), and the signals from indicated cells were monitored by sensors for over 24 hr, according to the manufacturer’s guidelines. The migratory activities of cells were evaluated by determining cell indexes.

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Luciferase assays

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HEK 293T cells were cultured in 96-well plates and co-transfected with 20 ng of the psiCHECK-2-CCNE2-3′-UTR vector and either 5 pmol of the miR-30d-5p mimics or the control mimics. After 48 h of incubation, firefly and Renilla luciferase activities of the cell lysates were measured using the Dual-Luciferase Reporter Assay System (Promega).

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RNA-binding protein immunoprecipitation (RIP) assays

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A549 cells were cultured in 10 cm-dish plates and transfected with 30 nmol miR30d-5p mimics or control mimics. After 24 h of incubation, cells were harvested and RIP assays were carried out with Argonaute antibody (Abcam, Cambridge, UK), according to the manufacturer’s guidelines (Millipore, Billerica, U.S.A). RNA was obtained and converted to cDNA for qPCR assay to detect the enrichment of CCNE2.

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Statistical analysis

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Results are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Unless otherwise stated, differences between two groups or more than two groups were determined, respectively, using Student’s t-test or one way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparisons test. Two-way ANOVA was used for analysing the data from the impedance-based xCELLigence Real-Time Cell Analysis Detection Platform. P-values of