STAT3-regulated exosomal miR-21 promotes angiogenesis and is involved in neoplastic processes of transformed human bronchial epithelial cells

Cancer Letters 370 (2016) 125–135

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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

Original Articles

STAT3-regulated exosomal miR-21 promotes angiogenesis and is involved in neoplastic processes of transformed human bronchial epithelial cells Yi Liu a,b,1, Fei Luo a,b,1, Bairu Wang a,b,1, Huiqiao Li c, Yuan Xu a,b, Xinlu Liu a,b, Le Shi a,b, Xiaolin Lu a,b, Wenchao Xu a,b, Lu Lu a,b, Yu Qin d, Quanyong Xiang d, Qizhan Liu a,b,* a

Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166, China The Key Laboratory of Modern Toxicology, Ministry of Education, Nanjing Medical University, Nanjing, Jiangsu 211166, China c Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166, China d Institute of Chronic Non-communicable Disease Control, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, Jiangsu 210009, China b

A R T I C L E

I N F O

Article history: Received 15 July 2015 Received in revised form 2 September 2015 Accepted 9 October 2015 Keywords: Exosomal miRNAs Intercellular communication Angiogenesis Molecular mechanism

A B S T R A C T

Although microRNA (miRNA) enclosed in exosomes can mediate intercellular communication, the roles of exosomal miRNA and angiogenesis in lung cancer remain unclear. We investigated functions of STAT3regulated exosomal miR-21 derived from cigarette smoke extract (CSE)-transformed human bronchial epithelial (HBE) cells in the angiogenesis of CSE-induced carcinogenesis. miR-21 levels in serum were higher in smokers than those in non-smokers. The medium from transformed HBE cells promoted miR21 levels in normal HBE cells and angiogenesis of human umbilical vein endothelial cells (HUVEC). Transformed cells transferred miR-21 into normal HBE cells via exosomes. Knockdown of STAT3 reduced miR-21 levels in exosomes derived from transformed HBE cells, which blocked the angiogenesis. Exosomes derived from transformed HBE cells elevated levels of vascular endothelial growth factor (VEGF) in HBE cells and thereby promoted angiogenesis in HUVEC cells. Inhibition of exosomal miR-21, however, decreased VEGF levels in recipient cells, which blocked exosome-induced angiogenesis. Thus, miR-21 in exosomes leads to STAT3 activation, which increases VEGF levels in recipient cells, a process involved in angiogenesis and malignant transformation of HBE cells. These results, demonstrating the function of exosomal miR-21 from transformed HBE cells, provide a new perspective for intervention strategies to prevent carcinogenesis of lung cancer. © 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction The development of human cancers is a multi-step process in which normal cells acquire characteristics that ultimately lead to their conversion into cancer cells [1]. In the cancer field, considerable interest is focused on characteristics of the tumor microenvironment and on the intercellular communication of malignant and nonmalignant cells of the host [2]. Tumor growth beyond 1–2 mm diameter becomes possible due to the hypoxic microenvironment, which activates the angiogenic network and results in the sprouting of blood vessels from the surrounding tissues into the tumor, during which transfer of angiogenic factors between the tumor and the host is necessary [3,4].

* Corresponding author. Tel.: +86 25 8686 8424; fax: +86 25 8686 8499. E-mail address: [email protected] (Q. Liu). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.canlet.2015.10.011 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

Exosomes, small membrane vesicles secreted from multivesicular endosomes by most cell types, are involved in intercellular communication. Exosomes contain mRNA and miRNA and thus carry genetic messages [5,6]. Intercellular communication within the cancer microenvironment is necessary to coordinate the assembly of multiple cell types for an amalgamated form and function of a cancer. Exosomes could function as extracellular messengers to facilitate cancer progression and metastasis [7], and tumor exosomes could advance cancer development through promotion of angiogenesis [8], stromal remodeling [9], metastasis, and drug resistance [10]. Such findings raise the possibility that transfer of genetic information via tumor exosomes affects surrounding tissues. miRNAs, with approximately 22 nucleotides, have regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression [11]. miRNAs are involved in the initiation, promotion, and progression of human cancers [12,13]. The biological significance of miRNAs secreted outside of cells is now recognized [14]. miRNAs transferred via exosomes may mediate cellto-cell communication. In exosomes, specific miRNAs are enriched in a cell type-dependent fashion [15]. However, the mechanisms

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whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear, particularly in relation to environmental carcinogens. Lung cancer is the leading cause of cancer mortality worldwide. The common interest in the field of lung cancer research is the identification of biomarkers for early diagnosis and accurate prognosis. Tobacco smoke is the predominant etiologic risk factor for lung cancer and chronic obstructive pulmonary disease. Of lung cancer patients in the United States, 80%–90% are smokers [16]. Although miRNAs are involved in lung cancer development and progression [17,18], how they are transferred from malignant cells to surrounding cells in the lung cancer microenvironment remains to be determined. In the present study, we evaluated effect of cigarette smoke extract (CSE)-transformed human bronchial epithelial (HBE) cells on malignant transformation and angiogenesis and investigated the underlying mechanisms. With a no-contact co-culture system, we determined the interaction of CSE-transformed HBE cells with normal HBE cells. We found that miRNAs transferred from transformed HBE cells into normal HBE cells are extracellular signaling molecules that affect angiogenesis. Such information contributes to an understanding of carcinogenesis caused by smoking.

umbilical vein endothelial cells (HUVECs) are used for angiogenesis assays [23]. These cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). HBE and HUVEC cells were maintained under 5% CO2 at 37 °C in Eagle’s minimum essential medium (MEM) and endothelial cell growth medium-2 (EGM-2), respectively, supplemented with 10% fetal bovine serum (FBS, Life Technologies/Gibco, Grand Island, NY), 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies/Gibco, Gaithersburg, MD). We previously established the model of CSE-transformed HBE cells [17]. 1 × 106 cells were seeded into 10-cm (diameter) dishes for 24 h and exposed to 0 or 20 μg/mL of CSE for 24–48 h per passage. This process was continued for about 20 weeks (40 passages). This process and the preparation of CSE were performed as described previously [17]. Transformed HBE cells (T-HBE) were derived by exposure of HBE cells to 20 μg/mL CSE for 40 passages. All other reagents used were of analytical grade or the highest grade available. Western blots Total cell lysates were prepared with a detergent buffer as described previously [17]. Protein concentrations were measured with the BCA Protein Assay according to the manufacturer’s manual (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts (80 μg) of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were incubated overnight at 4 °C with a 1:1000 dilution of anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma) and antibodies (Abs) for p-STAT3(Tyr705), STAT3, CD63, flotillin-1 (Cell Signaling Technology, Beverly, MA), or vascular endothelial growth factor A (VEGF-A, Abcam). After additional incubation with a 1:1000 dilution of an anti-immunoglobin horseradish peroxidase-linked Ab for 1 h, the immune complexes were detected by enhanced chemiluminescence (Cell Signaling Technology).

Materials and methods Quantitative real-time PCR Subjects and epidemiological data This study was approved by the Institutional Review Board of Nanjing Medical University. Written informed consent was obtained from each participant. On the basis of China Non-communicable Disease Surveillance in 2010, the study population in this cross-sectional study was a subset population recruited in Jiangsu Province, using a complex, multistage, probability sampling design [19]. In brief, 6 counties were first selected from 106 counties using stratified random sampling according to the population, gross domestic product, degree of urbanization, and geographic setting. Secondly, 4 towns from each county and 3 villages from each town were selected with the probability proportional to the population size, using cluster random sampling. Thirdly, 1 residential group including at least 50 households was selected from each village using simple random cluster sampling. Random digit function in Excel was applied in the selection of these households from each residential group. Finally, one family member aged 18 years or over was randomly selected from each household using the KISH Grid method [20]. A total of 1568 men were included in the cohort. The smoking history included age at smoking initiation, years of smoking, number of cigarettes smoked per day, and smoking cessation. An individual who never smoked or smoked fewer than 100 cigarettes in his lifetime was defined as a never smoker. Current smokers included those presently smoking and those who quit smoking less than 12 months before the interview. The pack•years of smoking were calculated according to the number of packs of cigarettes smoked per day and smoking duration (years). According to baseline data and previous studies [21], current smokers were separated into four groups including mild smokers (<15 pack•year), medium smokers (15-30 pack•year), and heavy smokers (>30 pack•year). From each group, 60 men were randomly selected, and their sera were used for our study. Serum isolation and storage From each participant, a venous blood sample (4–5 mL) was collected in tubes without any additives and, after at least 20 min of storage at room temperature, was centrifuged at 4000 rpm and 20 °C for 5 min. The serum samples were subsequently stored at −80 °C until further processing. For RNA isolation, 100 μL of serum was processed with mirVanaTM PARISTM Kits (Life Technologies). In brief, cel-miR39 was added in as control, then 100 μL of serum was mixed with 200 μL of acid phenol, 200 μL of chloroform, and 300 μL of RNase-free water. The mixture was vortexmixed at room temperature for 1 min and centrifuged for 5 min at 12,000 g. After phase separation, the aqueous layer was removed and mixed with 1.5 volumes of isopropyl alcohol and 0.1 volume of 3 μmol/L sodium acetate (pH 5.3), and the solution was stored at −20 °C for 1 h. The RNA pellet was collected by centrifugation at 16,000 g for 20 min at 4 °C and then washed once with ethanol and dried for 10 min at room temperature. Finally, the pellet was dissolved in 20 μL of RNase-free water and stored at −80 °C until further analysis. Cell culture and reagents HBE cells, a SV40-transformed normal human bronchial epithelial cell line, are nontumorigenic and retain features of human bronchial epithelial cells. They are useful for studies of multistage bronchial epithelial carcinogenesis [22]. Primary human

Total cellular RNA was isolated by using Trizol (Invitrogen) and extracelluar RNA by using mirVanaTM PARISTM Kits (Life Technologies) according to the manufacturers’ recommendations. For detection of miR-21, 2 μg of total RNA, miRNAspecific stem-loop reverse transcriptase (RT) primers, and MMLV RT (Promega Corp., Madison, WI, USA) were used in reverse transcription following the manufacturer’s protocol. Cel-miR-39 (RiBoBio, Guangzhou, China) and U6 were used as controls for conditioned media and cells, respectively. To control for variation in RNA isolation, 25 fmol of cel-miR-39 was added to the medium before starting the isolation procedure. The sequences of mature miRNAs were from Sanger miRBase (http://www.mirbase.org/). Forward (F) and reverse (R) primers were as follows: miR-21-F, 5′-ACACTCCAGCTGGGTAGCTTATCAGACTGA-3′; miR-21-R, 5′TGGTGTCGTGGAGTCG-3′; U6-F, 5′-CGCTTCGGCAGCACATATACTAAAATTGGAAC-3′; U6R, 5′-GCTTCACGAATTTGCGTGTCATCCTTGC-3′; cel-miR-39-F, 5′-ACACTCCAGCTGGGTCACCG GGTGTAAATC-3′; cel-miR-39-R, 5′-TGGTGTCGTGGAGTCG-3′. All of the primers were synthesized by Invitrogen. Quantitative real-time PCR was performed with an Applied Biosystems 7300HT machine and MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas, USA). The concentrations of extracellular miRNAs were calculated based on their Ct values, normalized by those of cel-miR-39, which was present in each reaction mixture at 1 nM [24]. Fold changes in the expression of each gene were calculated by a comparative threshold cycle (Ct) method using the formula 2−(△△Ct) [25]. Exosome isolation The culture medium was collected and centrifuged at 3000 g for 15 min, and the supernatant was filtered through a 0.22-mm PVDF filter (Millipore). An appropriate volume of Exoquick exosome precipitation solution (System Biosciences) was added to the filtered culture medium and mixed. After refrigeration for 24 h, the mixture was centrifuged at 1500 g for 30 min, and the supernatant was removed. Exosome pellets from 1 × 106 cells were suspended in 150 μL of serum-free medium. Transmission electron microscopy (TEM) The exosome-enriched pellets were suspended in 50 μL of PBS, fixed with 4% paraformaldehyde and 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at incubation temperature and kept at 4 °C until TEM analysis. A drop of each exosome sample was placed on a carbon-coated copper grid and immersed in 2% phosphotungstic acid solution (pH 7.0) for 30 s. The preparations were examined with a transmission electron microscope (JEM-1200EX; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 80 kV. Exosome labeling Exosomes from 1.5 × 106 cells were suspended in 180 μL of PBS with 20 μL of 1:50 diluted PKH67 (Sigma, in Diluent C). After 3 min of incubation at room temperature (RT), 3.8 mL of exosome-free medium was added to terminate the labeling reaction, and exosomes were harvested and washed twice with PBS by centrifugation (100,000 g for 1 h). Exosomes were suspended in 9.6 mL of basal medium, and 250 μL was added to a sub-confluent layer of HBE cells, which were incubated for

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3 h at 37 °C. Cells were washed twice with PBS, and fixed with 4% paraformaldehyde in PBS for 30 min at RT. To stain the nuclei, 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was added for 10 min, and stained cells were observed under a fluorescence microscope (Zeiss, LSM700B, Germany). Cell transfection HBE cells and CSE-transformed HBE cells were used in the transfection assays. Cells (5 × 105) were seeded into 6-well plates at 18–24 h prior to transfection. AntimiR-21 and anti-miR-nc (a negative control) were synthesized by RiBoBio (Guangzhou, China). Cells were transiently transfected using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. After 24 h, cells were harvested and used for experiments. Transfections of HBE cells were performed with the N-TER™ and AccuTarget TM Nanoparticle siRNA Transfection System (NFS, Sigma, BIONEER) following the manufacturer’s protocol. Briefly, 5 × 105 cells were seeded into each well of 6-well plates at 18–24 h prior to transfection. The siRNA nanoparticle preparations were made by adding target gene siRNA dilutions to N-TER or AccuTarget peptide dilutions. The preparations were incubated at room temperature for 30 min. NFS transfection medium (2 mL) containing target gene siRNA was transferred to each well of the culture plates, and, after 24 h, cells were treated and harvested for analysis. Control siRNA and STAT3 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 5 × 105 HBE cells were seeded in 12well plates prior to initiation of the experiment. After 1 day, these cells were coincubated with CSE-transformed HBE-derived exosomes (100 μg/mL) and α-amanitin (50 μg/mL) (Sigma-Aldrich, St. Louis, MO), a transcription inhibitor, or α-amanitin alone to suppress transcriptional activation caused by exosomes. To eliminate any residual T-HBE-derived exosomes, cells were washed with PBS twice and harvested after 0, 8, or 24 h for analysis. ELISA assays To determine the amounts of inflammatory cytokines produced by the cells, ELISA tests were performed according to the manufacturer’s instructions. Cells were plated at 1 × 106 per 100-cm2 dish. At confluency, culture supernatants were harvested, centrifuged, and placed at −70 °C. A human-specific VEGF from Beijing 4A Biotech Co., Ltd (Beijing) was used to determine the amount of inflammatory cytokines present in the culture supernatants. All assays were performed in duplicate and repeated three times. The lower limit of detection of VEGF was 2 pg/mL. Tube formation assay An assay for endothelial cell tube formation in three-dimensional Matrigel cultures of HUVEC cells was used to assess angiogenic potential. Growth factorreduced Matrigel (200 μL) was placed in 48-well plates. Cells were incubated with serum-free medium for 12 h. Cells (1.5 × 104) were then treated, or not treated, with T-HBE exosomes and transferred onto 48-well plates containing Matrigel. An equivalent volume of vehicle was added to the control groups. Tube formation was examined in photographs taken under a microscope. The lengths of blood vessels, an index of angiogenesis, were obtained by measuring the branches of blood vessels in 2 square millimeters using Olympus CellSens Standard digital imaging software. The measurements were made by investigators blinded to the treatment. The numbers were normalized to the values of the control groups. Statistical analyses Derived values are presented as the means ± SD. Comparison of means among multiple groups was accomplished by one-way analysis of variance (ANOVA), and a multiple-range least significant difference (LSD) was used for inter-group comparisons. Post hoc comparisons were performed when the one-way ANOVA was significant. P values < 0.05 were considered statistically significant. All statistical analyses were performed with SPSS 16.0.

Results Smoking-induced increase of miR-21 levels promotes the angiogenesis of HUVEC cells In the cancer microenvironment, cancer cells could regulate surrounding cells by miRNAs [26,27]. miR-21 is involved in angiogenesis and is related to cancer development [28,29]. Our previous data showed that miR-21 could regulate the cell microenvironment and malignancy with environmental chemical exposure [29,30]. We previously reported that HBE cells chronically exposed to 20 μg/mL of CSE acquire characteristics of transformed cells [17]. Since angiogenesis is essential for the growth of cancers [31], we hypothesized that CSE-transformed cells induce angiogenesis by transfer of miR21. To investigate the effects of smoking on miR-21 levels in sera

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of smokers, current smokers were separated into mild smokers (<15 pack•year), medium smokers (15-30 pack•year), and heavy smokers (>30 pack•year). In sera from 60 of each group, miR-21 levels were measured, and the results were compared with those for nonsmokers. The results showed that miR-21 levels in serum were higher in smokers than those in non-smokers. Moreover, there were higher levels of serum miR-21 in heavy smokers (Fig. 1A). To identify the miRNAs involved in the process of CSE-induced cell transformation, we performed real-time PCR for passagecontrol HBE cells (0 μg/mL CSE for 40 passages) and transformed HBE cells (20 μg/mL CSE for 40 passages) to detect the expression of miRNAs that are related to cigarette smoke and lung cancer pathogenesis. miR-21 was significantly increased in CSE-transformed HBE cells (Fig. S1). The measurements of miR-21 showed that levels were elevated in CSE-transformed HBE cells relative to HBE cells (Fig. 1B). In the culture supernatants, miR-21 was preferentially excreted by transformed HBE cells (Fig. 1C). Next, we determined the levels of miR-21 in HBE cells treated with basal medium, medium from normal HBE cells, or medium from CSE-transformed HBE cells. The medium from CSE-transformed HBE cells elevated expression of miR21 in HBE cells about 2.5-fold relative to the basal medium or medium from normal HBE cells (Fig. 1D). These results indicate that CSE-transformed HBE cells secrete miR-21, which increases the levels of miR-21 in recipient HBE cells. HUVEC cells and tube formation assays were used to measure angiogenesis. To determine if transformed HBE cells with longterm exposure to CSE had a pro-angiogenic effect, HUVEC cells were exposed to the basal medium, medium from normal HBE cells, or medium from CSE-transformed HBE cells. After 12 h, HUVEC cells cultured with the medium from CSE-transformed HBE cells had an elevated capacity for angiogenesis relative to cells cultured with the basal medium, but the medium from normal HBE cells had no effect on angiogenesis (Fig. 1E and F). Further, HUVEC cells were cultured in the medium from CSE-transformed HBE cells in the presence of an miR-21 inhibitor. The inhibitor reduced the angiogenic properties of these cells (Fig. 1G–I). These results indicate that smoking induces an increase of miR-21 levels and that the medium containing miR-21 from CSE-transformed HBE cells induces increases of miR-21 levels in HBE cells and angiogenesis in HUVEC cells. The transfer of miR-21 from CSE-transformed cells into normal HBE cells is via exosomes Exosomes, which are involved in cell-to-cell communication, may stimulate recipient cells by receptor-mediated interactions or by transfer of bioactive factors, including membrane receptors, proteins, mRNAs, miRNAs, and/or organelles from origin cells to recipient cells [32]. To establish their functions, exosomes were isolated from HBE cells and CSE-transformed HBE cells. TEM revealed that the size of exosomes from transformed HBE cells was similar to that from normal HBE cells (Fig. 2A). Immunoblotting was performed to assess the levels of CD63 and flotillin-1, which are exosome markers [33,34]. In the exosomes of normal HBE and CSE-transformed HBE cells, the levels of CD63 and flotillin-1 were consistent (Fig. 2B and C). As determined by qRT-PCR, the levels of miR-21 in exosomes from transformed HBE cells were higher than those from HBE cells (Fig. 2D). Furthermore, after exposure of HBE cells to 20 μg/mL CSE for 24 h, the levels of CD63 and flotillin-1 in exosomes were unchanged (Fig. 2E and F), but miR-21 levels in exosomes from HBE cells exposed to 20 μg/mL CSE for 24 h were higher than those from HBE cells (Fig. 2G). Thus, exosomes from CSE-treated HBE cells contained more miR-21 than those from HBE cells. To prove that miR-21 from CSE-transformed cells is transferred into normal HBE cells via exosomes, ultracentrifugation was performed to deplete exosomes in supernatants from HBE and CSEtransformed HBE cells. In exosome-depleted supernatants, miR-21

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Fig. 1. Smoking-induced increase of miR-21 levels promotes the angiogenesis of HUVEC cells. N.S: non-smoker; mild: mild smokers; medium: medium smokers; heavy: heavy smokers; basal: basal medium; CM: medium from normal HBE cells; T-CM: medium from CSE-transformed HBE cells. (A) miR-21 levels in sera of individuals from different groups were determined by qRT-PCR assays (mean ± SD, n = 3). HBE cells or CSE-transformed HBE cells were exposed to basal medium, medium from normal HBE cells, or medium from CSE-transformed HBE cells for 24 h. (B) The levels of miR-21 in HBE cells and CSE-transformed HBE cells were determined by qRT-PCR assays (mean ± SD, n = 3). *P < 0.05 difference from HBE cells. (C) The expression of miR-21 in supernatants of mono-culture media (supernatant of HBE cells or CSE-transformed HBE cells only) was determined by qRT-PCR assays (mean ± SD, n = 3). *P < 0.05 difference from culture supernatant of HBE cells. (D) Levels of miR-21 in HBE cells exposed to different media were determined by qRT-PCR assays (mean ± SD, n = 3). *P < 0.05 difference from cells in basal medium. HUVEC cells were incubated with basal medium, medium from normal HBE cells, or medium from CSE-transformed HBE cells and seeded onto Matrigel for 12 h. (E) Tube formation of HUVEC cells (bars = 250 μm) and (F) the relative tube lengths in HUVEC cells. *P < 0.05, difference from HUVEC cells treated with medium from untreated HBE cells. HUVEC cells were cultured with medium from normal HBE cells or medium from CSE-transformed HBE cells, together with miR-21 inhibitor or negative control (100 nM), and seeded onto Matrigel for 12 h. (G) The expression of miR-21 in conditioned supernatants was determined by qRT-PCR assays (mean ± SD, n = 3). (H) Tube formation of HUVEC cells (bars = 250 μm) and (I) the relative tube lengths in HUVEC cells. *P < 0.05, difference from HUVEC cells treated with medium from HBE cells, #P < 0.05 difference between cells treated with medium from T-CM cells and the negative control.

levels were low, and there was no difference in miR-21 levels in exosome-depleted supernatants between HBE cells and CSEtransformed HBE cells (Fig. 2H). These results indicated that CSEtransformed HBE cells secrete miR-21 to recipient cells mainly through exosomes, as verified by another report [35]. Then exosomes were labeled with a green fluorescent dye, PKH67. After HBE cells were incubated with labeled exosomes from CSE-transformed HBE cells for 3 h, PKH67 was localized in the cytoplasm (Fig. 2I). For HBE cells cultured with conditioned medium depleted of exosomes by ultracentrifugation, there was no significant increase in transfer of

miR-21 levels to neighboring cells (Fig. S2). Then HBE cells were incubated with α-amanitin, an inhibitor of RNA polymerase II [36], and exosomes from CSE-transformed HBE cells or with α-amanitin alone. The levels of miR-21 in the cells exposed to these exosomes were increased relative to levels in normal HBE cells and in cells exposed to α-amanitin alone (Fig. 2J). In the cells exposed to exosomes derived from HBE cells, however, there was no significant increase in the levels of miR-21 (Fig. S3), indicating that the transfer of miR-21 from CSE-transformed cells into normal HBE cells occurred via exosomes.

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Fig. 2. The transfer of miR-21 from CSE-transformed cells into normal HBE cells via exosomes. CM: medium from normal HBE cells, T-CM: medium from CSE-transformed HBE cells, CM-deplet: CM-exosome-depleted conditioned medium, T-CM-deplet: T-CM-exosome-depleted conditioned medium. Exosomes of HBE cells and CSEtransformed HBE cells were fractionated by Exoquick. Densities of bands were quantified by Eagle Eye II software. (A) Micrographs of exosomes isolated from HBE cells (left) and CSE-transformed HBE cells (right, bars = 100 nm). (B) Western blots of CD63 and flotillin-1 and (C) relative protein levels (means ± SD, n = 3). (D) qRT-PCR detection of miR-21 expression in exosomes (100 μg/mL) derived from HBE cells or CSE-transformed HBE cells (mean ± SD, n = 3). *P < 0.05 difference from exosomes of HBE cells. After HBE cells were exposed to CSE (0.0 or 20 μg/mL) for 24 h, the exosomes of treated cells were fractionated. (E) Western blots of CD63 and flotillin-1 and (F) relative protein levels (means ± SD, n = 3). (G) qRT-PCR detection of miR-21 expression in exosomes derived from HBE cells and from HBE cells exposed to 20 μg/mL CSE for 24 h (mean ± SD, n = 3). *P < 0.05 difference from exosomes of untreated HBE cells. HBE cells were incubated in the presence of exosomes derived from CSE-transformed HBE cells for 3 h. (H) The expression of miR-21 in supernatants of HBE cells and CSE-transformed HBE cells, or miR-21 in exosome-depleted supernatants of HBE cells and CSEtransformed HBE cells, as determined by qRT-PCR assays (mean ± SD, n = 3). *P < 0.05 difference from culture supernatant of HBE cells. (I) The exosomes of CSE-transformed cells were labeled with PKH67; green represents PKH67, blue represents nuclear DNA staining by DAPI. HBE cells were incubated with α-amanitin and exosomes derived from CSE-transformed HBE cells or α-amanitin alone. (J) Levels of miR-21 were determined by qRT-PCR (mean ± SD, n = 3). *P < 0.05 difference from HBE cells treated by α-amanitin alone. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

STAT3-regulated miR-21 in exosomes from CSE-transformed HBE cells is involved in the angiogenesis of HUVEC cells We have previously reported that CSE increases the secretion of IL-6, a cytokine that induces an inflammatory response [17], and that the secretion of IL-6 involves an autocrine signaling loop and activates STAT3 during the CSE transformation process [37]. STAT3

activation is involved in malignant transformation through upregulation of miR-21 expression [38,39]. In the present study, we determined if STAT3 regulates miR-21 levels in the exosomes of CSEtransformed cells. Consistent with our previous findings [37], phospho-STAT3 levels in CSE-transformed cells were higher relative to those in normal HBE cells (Fig. 3A). Moreover, in CSEtransformed HBE cells, knockdown of STAT3 by siRNA reduced the

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Fig. 3. STAT3-regulated miR-21 in exosomes from CSE-transformed HBE cells is involved in the angiogenesis of HUVEC cells. Abbreviations: HBE, HBE cells; T-HBE, HBE cells transformed by 40 passages in the presence of CSE. CSE-transformed HBE cells were transfected with 20 nM control siRNA or 20 nM STAT3 siRNA for 24 h. (A) The levels of p-STAT3 and STAT3 in these cells were detected by Western blots, and (B) the levels of miR-21 were detected by qRT-PCR (mean ± SD, n = 3). *P < 0.05 difference from HBE cells, #P < 0.05 difference from T-HBE cells with control siRNA. After CSE-transformed HBE cells were transfected with 20 nM of control siRNA or STAT3 siRNA for 24 h, exosomes of these cells were fractionated by Exoquick. (C) The levels of miR-21 in exosomes derived from these cells were detected by qRT-PCR (mean ± SD, n = 3). *P < 0.05 difference from HBE cells; #P < 0.05 difference from T-CM cells transfected with control siRNA. HUVEC cells, which were incubated with the exosomes derived from CSE-transformed HBE cells transfected with 20 nM of control siRNA or STAT3 siRNA, were seeded onto Matrigel for 12 h. (D) Tube formation of HUVEC cells (bars = 250 μm) and (E) the relative tube lengths of HUVEC cells. *P < 0.05 difference from HBE cells, #P < 0.05 difference from T-CM cells transfected with control siRNA.

increase in miR-21 levels (Fig. 3A and B). Thus, STAT3 regulates miR21 expression in CSE-transformed HBE cells. Further, the levels of exosomal miR-21 from culture supernatants were measured. The inhibition of STAT3 also reduced miR-21 levels in exosomes derived from CSE-transformed HBE cells (Fig. 3C). After HUVEC cells were treated with exosomes derived from different conditioned media, the exosomes derived from CSE-transformed HBE cells with STAT3 knockdown reduced tube formation (Fig. 3D and E). Levels of miR21 and VEGF in HUVEC cells exposed to exosomes derived from CSEtransformed cells (Fig. S4) and levels of VEGF in HBE cells exposed to exosomes derived from CSE-transformed cells that were transfected with STAT3 siRNA (Fig. S5) were measured. Thus, STAT3 is involved in the increases of miR-21 levels in exosomes of CSEtransformed HBE cells and VEGF levels in recipient cells, and thereby enhances the angiogenesis of HUVEC cells. CSE and exosomes derived from CSE-transformed cells elevate the expression and secretion of VEGF in normal HBE cells Since VEGF is elevated in cells exposed to CSE [40–42], we measured the expression and secretion levels of VEGF in HBE and CSEtransformed HBE cells, supernatants, and exosomes (Fig. S6). The levels of VEGF were higher in CSE-transformed HBE cells, but, in exosomes, no appreciable levels of VEGF were detected. After HBE cells were exposed to CSE (20 μg/mL), the expression and secretion levels of VEGF increased over periods ranging from 0 to 24 h; there were no appreciable changes of VEGF in HBE cells exposed

to 0 μg/mL CSE for 24 h (Fig. 4A and B). Since VEGF could hardly be detected in exosomes derived from HBE and CSE-transformed HBE cells, we considered that VEGF could be elevated in recipient cells by exosome stimulation. To determine if exosomes derived from CSE-transformed cells had an effect on the expression and secretion levels of VEGF, HBE cells were treated with different concentrations of exosomes isolated from HBE cells or CSE-transformed HBE cells. In a dose–response manner, exosomes from CSEtransformed HBE cells increased the expression and secretion levels of VEGF (Fig. 4C and D). Thus, in HBE cells, CSE and exosomes derived from CSE-transformed cells elevate the expression and secretion of VEGF.

Exosomes derived from CSE-transformed HBE cells promote angiogenesis of HUVEC cells To further establish the effect of exosomes on angiogenesis, HUVEC cells were exposed to 0, 10, 50, 100, or 200 μg/mL of exosomes derived from normal HBE cells or from CSE-transformed HBE cells for 12 h. Exosomes from CSE-transformed HBE cells enhanced the angiogenesis of HUVEC cells in a dose–response relationship (Fig. 5A and B), an effect not prominent in HUVEC cells cultured with medium depleted of exosomes by ultracentrifugation (Fig. 5C and D). We conclude that exosomes derived from CSEtransformed HBE cells are involved in the angiogenesis associated with CSE carcinogenesis.

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Fig. 4. The effects of CSE and exosomes derived from CSE-transformed cells on the expression and secretion of VEGF in HBE cells. HBE cells were exposed to CSE (20 μg/ mL) for 0, 3, 6, 12, or 24 h. (A) Protein levels of VEGF in cells were determined by Western blots, and (B) the levels of VEGF present in the medium (means ± SD, n = 3) were measured by ELISA. HBE cells were exposed for 24 h to exosomes (0, 10, 50,100, or 200 μg/mL) derived from CSE-transformed HBE cells. (C) Protein levels of VEGF in cells were determined by Western blots, and (D) the levels of VEGF present in the medium (means ± SD, n = 3) were measured by ELISA.

miR-21 in exosomes derived from CSE-transformed cells is involved in the angiogenesis of HUVEC cells via regulating VEGF Overexpression of miR-21 increases VEGF expression and induces tumor angiogenesis [43,44]. To determine if exosomal miR-21 has the capacity to regulate VEGF in recipient cells, the levels of VEGF were measured after normal HBE cells were treated with exosomes derived from transformed HBE cells with or without anti-miR-21. In HBE cells, anti-miR-21 reduced the increase of miR-21 levels induced by exosomes from CSE-transformed HBE cells (Fig. S7A). Knockdown of miR-21 blocked the increases of protein levels and reduced levels of VEGF induced by exosomes from CSE-transformed HBE (Fig. 6A and B). Further, a tube formation assay was performed to assess the effect of exosomal miR-21 on regulating angiogenesis. After the inhibition of miR-21 in CSE-transformed HBE cells (Fig. S7B), the exosomes derived from these cells showed reduced tube formation (Fig. 6C and D). Also, HUVEC cells transfected with anti-miR-21 showed decreased angiogenesis induced by exosomes from CSE-transformed HBE cells (Fig. 6E and F). These data indicate that exosomes derived from CSE-transformed HBE cells enhance angiogenesis of recipient cells by miR-21 regulation of VEGF. Discussion Lung cancer is the leading cause of cancer-related deaths around the world, among both men and women, with an incidence of over 200,000 new cases per year and a very high mortality rate [45]. Epidemiological evidence confirms that exposure to cigarette smoke increases the incidence of lung carcinogenesis, a leading cause of cancer deaths in the United States and other developed countries [46]. Angiogenesis is associated with tumor growth and metastasis, and acquisition of angiogenic capacity can be seen as a progression from neoplastic transformation to tumor growth and metastasis [47,48]. Although CSE induces a more malignant lung

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tumor phenotype by increasing angiogenesis, the underlying mechanisms remain unknown [49,50]. In the normal microenvironment, in which a tumor competes with antitumorigenic pressures, cancer cells could progress by transfer of genetic information to normal cells [51]. We previously reported that the medium from CSE-transformed HBE cells affects normal HBE cells [17,37]. The molecular and cellular mechanisms, especially how malignant cells affect surrounding cells in the process of angiogenesis, are not clear. Our data revealed that cells treated with CSE induced angiogenesis in HUVEC cells, consistent with previous studies [52]. We also established that, in HUVEC cells exposed to the medium from CSE-transformed HBE cells, more angiogenesis was evident relative to the effect of the control medium. These results preliminarily confirmed our assumption: we considered angiogenesis to be promoted by molecules in the medium that were secreted by transformed cells. The transmission of molecules in the cancer microenvironment promotes cancer development and progression [53,54]. miRNAs, which are aberrantly expressed during cancer development and are subject to regulation by signaling pathways associated with cancer development [55], are involved in the regulation of angiogenesis [29]. miRNAs are mediators of cell-to-cell communication and are involved in the regulation of tumorigenesis. In cells, miRNAs are mostly packed together with certain categories of mRNAs and proteins and are secreted in the form of exosomes, providing a mechanism through which cancer cells transfer signaling molecules to neighboring cells. These exosomes are recognized as mediators of cell-to-cell communication [56]. Tumor cells have an exacerbated exosome secretion that is linked to angiogenesis, metastatic spread, and immunosuppression [10]. Patients with non-small-cell lung cancer generally have a relatively high expression of miR-21 in serum [57] and in their tissues [58]. Although miR-21 is up-regulated in many types of human malignancies [59], the mechanism for regulation of miR-21 in CSEtransformed cells remains unknown. Our results show that miR21 levels were significantly higher in sera of smokers, and sera from heavy smokers have elevated levels of miR-21 compared with sera from milder smokers. Expression of miR-21 in CSE-transformed HBE cells is about three times that in normal HBE cells. miR-21 in HBE cells cultured with medium from transformed cells is overexpressed, and miR-21 is transferred into normal HBE cells to affect their signal pathways. In this process, a carrier is needed to deliver miR-21. Exosomes could transfer miRNAs and lead to propagation of cancer cells [32]. We have also demonstrated that arsenite-transformed cells release exosomes containing miR-21. Since there are high levels of miR-21 in CSE-transformed cells, exosomes from these cells also contain abundant miR-21. To confirm the existence of exosomal miR21 in CSE-transformed cells, we isolated exosomes and found that CSE-transformed cells contained higher levels of miR-21. However, whether exosomal miR-21 entered normal cells was not clear. When HBE cells were co-incubated with α-amanitin and T-HBE exosomes, α-amanitin suppressed transcriptional activation, proving that elevated miR-21 levels were from the T-HBE exosomes. We previously reported that IL-6/STAT3 signaling is involved in the epithelial mesenchymal transition (EMT) in CSE-induced carcinogenesis [17,37]. The medium from CSE-transformed HBE cells, which contains IL-6, induced activation of STAT3. Acting via several miRNAs, including miR-21, STAT3 mediates lung inflammation, EMT, and carcinogenesis [38,60]. However, the involvement of IL-6/ STAT3 in miR-21 expression during CSE carcinogenesis remained unclear. After blocking STAT3 by siRNA, miR-21 levels in transformed cells and exosomes were decreased. Similar to other reports [39], our results show that STAT3 signaling modulates miR-21 expression in CSE-induced carcinogenesis. The processes of inflammation and angiogenesis are linked, and VEGF is involved in the cross-talk between these processes [61]. The

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Fig. 5. Effect of exosomes derived from CSE-transformed HBE cells on the angiogenesis of HUVEC cells. CM-Exosomes: exosomes derived from normal HBE cells, T-CMExosomes: exosomes derived from CSE-transformed HBE cells, basal: basal medium, T-CM: medium from CSE-transformed HBE cells, T-CM-deplet: T-CM-exosomedepleted conditioned medium. HUVEC cells were incubated for 12 h with exosomes (0, 10, 50, 100, or 200 μg/mL) derived from CSE-transformed HBE cells and seeded onto Matrigel. (A) Tube formation in HUVEC cells (bars = 250 μm) and (B) the relative tube lengths of HUVEC cells. HUVEC cells, which were incubated with T-CM media or T-CMdeplet media, were seeded onto Matrigel for 12 h. (C) Tube formation of HUVEC cells (bars = 250 μm) and (D) the relative tube lengths of HUVEC cells. *P < 0.05 difference from HUVEC cells treated with basal medium, #P < 0.05 difference from cells treated with T-CM-exosomes.

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Fig. 6. miR-21 in exosomes derived from CSE-transformed cells is involved in the angiogenesis of HUVEC cells via regulating VEGF. HBE: normal HBE cells, T-HBE: CSEtransformed HBE cells, Basal: basal medium, T-CM-exosome: exosomes derived from CSE-transformed HBE cells. After HBE cells were treated with exosomes (100 μg/mL) derived from CSE-transformed HBE cells for 24 h, they were transfected with anti-miR-NC or anti-miR-21 (100 nM) for 24 h. (A) The protein levels of VEGF in cells were detected by Western blots. (B) The levels of VEGF present in the medium (means ± SD, n = 3) were measured by ELISA. *P < 0.05 difference from cells in basal medium, #P < 0.05 difference from cells treated with T-CM-exosomes. Exosomes were derived from normal or CSE-transformed HBE cells that were transfected with 100 nM of miR-21 inhibitor or negative control for 24 h. HUVEC cells were incubated with these exosomes and seeded onto Matrigel for 12 h. (C) Tube formation of HUVEC cells (bars = 250 μm) and (D) the relative tube lengths of HUVEC cells. *P < 0.05 difference from HBE cells, #P < 0.05 difference from T-CM cells transfected with the negative control. HUVEC cells, which were treated with 100 μg/mL exosomes derived from CSE-transformed HBE cells, and also transfected with anti-miR-NC or anti-miR-21 (100 nM), were seeded onto Matrigel for 12 h. (E) Tube formation of HUVEC cells (bars = 250 μm) and (F) the relative tube lengths of HUVEC cells. *P < 0.05 difference from HUVEC cells treated with basal medium. #P < 0.05 difference from cells treated with T-CM-exosomes.

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VEGF family of growth factors and receptors is a prototype for understanding angiogenesis and its regulation, and inhibition of VEGF-A is beneficial in the treatment of some types of cancers [62]. Increased expression of VEGF receptors in tumor cells also supports a role for VEGF-A as a paracrine factor to stimulate angiogenesis [47]. CSE promotes angiogenesis mainly through regulating VEGF levels [41,63]. We established that, with CSE stimulation of HBE cells, VEGF levels are elevated. miRNAs can serve as regulators of angiogenesis [64,65], and VEGF may act directly or indirectly in this process [65,66]. miR-21 promotes the expression of downstream VEGF [67,68]. We have established that CSE elevates miR-21 and angiogenesis. However, the regulatory mechanisms remain largely unknown, and the function of exosomes in this process is unclear. In this study, we found a dose–response relationship between exosomes from transformed cells and VEGF levels. Exosomes from normal cells had no effect on VEGF. After inhibition of exosomal miR21, levels of VEGF in cells treated with exosomes from transformed cells were reduced. The changes of tube formation were consistent with changes in VEGF, indicating that CSE-transformed cells secrete exosomes containing miR-21 that change VEGF levels in recipient cells to promote angiogenesis. To confirm the effects of exosomes on angiogenesis, we depleted exosomes from the CSEtransformed medium and showed that the pro-angiogenesis effect was partly abolished. However, the effect on angiogenesis could not be abolished, no matter if the cells were treated with exosomes derived from miR21-inhibited, CSE-transformed cells or with CSE-transformed medium after depletion of exosomes. Since exosomes contain miRNAs, mRNAs, DNA fragments, and various proteins [69], other exosomal proteins and miRNAs could affect angiogenesis mainly through regulating VEGF in recipient cells, as TGF-β1 does in prostate cancer cells and mesothelioma cells [70], EGFR in several cancer cells [71], and miR-210 in lung adenocarcinoma cells [72]. Our results support the assumption that other exosomal contents may contribute to angiogenesis induced by CSE-transformed HBE cells. Secreted VEGF may have a role in this process. Further studies related to exosomal contents would be helpful for understanding this process. Thus, we propose a new molecular determinant of cancer,

including, but not limited to, miR-21, as found in exosomes from CSE-transformed HBE cells. In conclusion, our work reveals a previously unknown mechanism in the tumor microenvironment. With the stimulation of CSE, increased miR-21 in exosomes is dependent on STAT3 activation, which changes VEGF levels in recipient cells. Such a change is involved in CSE-induced angiogenesis and malignant transformation (Fig. 7). Together, our results, which show the involvement of exosomal miR-21 in the process of CSE-induced carcinogenesis, indicate that exosomal miRNAs serve as mediators in cell–cell communication during carcinogenesis induced by environmental chemicals and provide a strategy for intervention in the process of carcinogenesis caused by smoking. Funding This work was supported by the Natural Science Foundation of China (81473011, 81573199, 81273114, 81430077), the Key Program of Educational Commission of Jiangsu Province of China (11KJA330002), the Postgraduate Innovation Project of Jiangsu province (CXZZ14_0421, CXZZ14_0951, and CXZZ13_0600), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (2010). Acknowledgment The authors wish to thank Donald L. Hill (University of Alabama at Birmingham, USA) for editing. Conflict of interest The authors declare that they have no competing financial interests. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.10.011. References

Fig. 7. A schematic representation of the proposed pathway for exosomal miR-21mediated pro-angiogenesis roles in CSE carcinogenesis. CSE activates the IL-6/ STAT3 signal pathway, which elevates miR-21 levels in both cells and exosomes. Exosomal miR-21 derived from CSE-transformed HBE cells increases VEGF levels of recipient cells, which induces angiogenesis in CSE-induced carcinogenesis.

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