MOLECULAR CARCINOGENESIS 54:E192–E204 (2015)

Functional Role and Mechanism of LncRNA LOC728228 in Malignant 16HBE Cells Transformed by AntiBenzopyrene-Trans-7,8-Dihydrodiol-9,10-Epoxide Gongcheng Hu,1 Ti Yang,1 Jingli Zheng,1 Jiabing Dai,1 Aruo Nan,1 Yandong Lai,1 Yajie Zhang,2 Chengfeng Yang,3 and Yiguo Jiang1* 1

State Key Laboratory of Respiratory Disease, Institute for Chemical Carcinogenesis, Guangzhou Medical University, Guangzhou 510182, PR China 2 Department of Pathology, Guangzhou Medical University, Guangzhou 510182, PR China 3 Department of Physiology and Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824

Lung cancer is a major health problem, and is considered one of the deadliest cancers in humans. It is refractory to current treatments, and the mechanisms of lung cancer are unknown. Long noncoding RNAs (lncRNAs) are involved in various biological processes and human diseases. However, the exact functional roles and mechanisms of lncRNAs are largely unclear. In this study, we attempted to identify lung-cancer-related lncRNAs. We found changes in lncRNA expression in the anti-benzo(a) pyrene-7,8-diol-9,10-epoxide (anti-BPDE)-transformed human bronchial epithelial cell line (16HBE-T cells) using microarrays and qRT–PCR. Of these lncRNAs, LOC728228 was upregulated relative to its expression in control untransformed16HBE (16HBE-N) cells. LOC728228 knockdown inhibited cell proliferation, caused G0/G1phase cell-cycle arrest, reduced cellular migration, suppressed colony formation in vitro, and inhibited tumor growth in a nude mouse xenograft model. LOC728228 knockdown also suppressed cyclin D1 expression, and the depletion of cyclin D1 induced G0/G1-phase cell-cycle arrest and inhibited cell proliferation, thus influencing the malignant potential of cancer cells. In summary, our results suggest that lncRNA LOC728228 has an oncogene-like function and plays a vital role in human lung cancer. © 2015 Wiley Periodicals, Inc. Key words: lung cancer; long noncoding RNA; LOC728228; 16HBE-T cells

INTRODUCTION Researchers have shown that a large number of noncoding RNAs (ncRNAs) are transcribed from the human genome [1,2]. Long noncoding RNAs (lncRNAs) have more than 200 nucleotides, with no protein-coding potential, and have recently received considerable attention due to their significant roles in diverse biological processes and human diseases [3–7]. The analysis of their roles in cancer development is focus of current research. Accumulating evidence shows that many lncRNAs have a strong association with cancer development and progression [8–12], and some are considered to be a newly emerging class of oncogenic and tumor-suppressor transcripts [13]. Lung cancer is one of the most common cancers worldwide in terms of its incidence and associated mortality. However, the molecular mechanisms involved in lung cancer are complex and are as yet incompletely understood. Recent studies have shown that some lncRNAs are critical in lung cancer. For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is upregulated in certain histological subtypes of non-small-cell lung cancer (NSCLC). MALAT1 promotes cell migration and has been identified as an oncogene [12,14,15]. Several other lncRNAs have been reported to play vital roles in lung cancer, including maternally expressed gene 3 ß 2015 WILEY PERIODICALS, INC.

(MEG3) [16], H19 [17], and smoke cancer-associated lncRNA-1 (SCAL1) [18]. However, lncRNAs and their

Abbreviations: lncRNAs, long noncoding RNAs; anti-BPDE, anti benzo(a)pyrene-7,8-diol-9,10-epoxide; MALAT1, metastasis associated lung adenocarcinoma transcript 1; NSCLC, non small-cell lung cancer; MEG3, maternally expressed gene 3; SCAL1, smoke cancer associated lncRNA-1; 16HBE, human bronchial epithelial cell line; 16HBE-T, transformed 16HBE cells; 16HBE-N, control untransformed 16HBE cells; qRT–PCR, quantitative real time reverse transcription– polymerase chain reaction; GFP, green fluorescent protein; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; GAS5, growth arrestspecific transcript 5; ncRAN, noncoding RNA expressed in aggressive neuroblastoma; PCAT-1, prostate cancer-associated transcript 1; PCGEM1, prostate specific gene 1; uc.73a, ultraconserved element 73; HOTAIR, HOX antisense intergenic RNA; lncRNA-LET, lncRNA low expression in tumor. Conflict of Interest: None. G. Hu, T. Yang and J. Zheng contributed equally to this work. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 81172633; 21277036; Grant sponsor: University Talent Program of Guangdong; Grant number: 2013-164; Grant sponsor: Innovation Team Grant of Guangzhou Municipal Education Department; Grant number: 13C06 *Correspondence to: Institute for Chemical Carcinogenesis, Guangzhou Medical University, 195 Dongfeng Road West, Guangzhou 510182, PR China. Received 9 June 2014; Revised 11 February 2015; Accepted 21 February 2015 DOI 10.1002/mc.22314 Published online 27 March 2015 in Wiley Online Library (wileyonlinelibrary.com).

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biological functions are still largely unexplored. Our research has been focused on ncRNAs and cancer research, and it was previously found that some microRNAs play important roles in lung cancer [19– 21]. In this study, we attempted to identify lungcancer-related lncRNAs. Our results show that lncRNA LOC728228 is aberrantly expressed in lung cancer cell lines and influences the malignant potential of lung cancer cells. These results indicate that lncRNA LOC728228 has an oncogene-like function, and provides new insight into the roles of lncRNAs in lung cancer. MATERIALS AND METHODS Cell Culture The human bronchial epithelial cell line (16HBE) was kindly provided by the Guangzhou Institute of Respiratory Disease (Guangzhou, China). Transformed 16HBE cells (16HBE-T) induced by antibenzo(a)pyrene -7,8-diol-9,10-epoxide (anti-BPDE) and control untransformed 16HBE cells (16HBE-N) treated with dimethyl sulfoxide were previously established in our laboratory. 16HBE-T and 16HBEN cells were cultured in minimum essential medium (Gibco, Carlsbad, CA) with 10% (v/v) calf serum and 1% (w/v) antibiotics (penicillin/streptomycin). The human lung cancer cell line A549 was purchased from the Sun Yat-Sen University Cancer Center (Guangzhou, China). A549 cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) calf serum and 1% (w/v) antibiotics (penicillin/streptomycin). The Beas-2b cell line was kindly provided by Lijin Zhu (Institute of Hygiene, Zhejiang Academy of Medical Sciences, Hangzhou, China). Beas-2b cells were cultured in H1640 medium (Invitrogen) supplemented with 10% (v/v) calf serum and 1% (w/v) antibiotics (penicillin/streptomycin). All cell lines were grown at 378C in a humidified chamber with 5% CO2, and were passaged every 2–3 days by treatment with 0.02% (w/v) EDTA and 0.25% (w/v) trypsin (Gibco BRL). Microarray Analyses Custom-designed microarrays were synthesized by Invitrogen, and the total RNA from triplicate samples of 16HBE-N and 16HBE-T cells was reverse transcribed to first-strand complementary DNA (cDNA), and then double-stranded cDNA was synthesized. Following double-stranded cDNA hybridization and washing, the processed slides were scanned using an Agilent G2565CA Microarray Scanner (Agilent Technologies, Santa Clara, CA). The Feature Extraction software (Agilent Technologies) was used to extract and analyze the signals. The thresholds for up- and down-regulated genes were set to fold changes of
2.0 or 0.05, respectively. The q-value was calculated using SAM (Significance Analysis of Microarrays) software (Stanford University, Stanford, CA). Molecular Carcinogenesis

Quantitative Real-Time PCR 1

Total RNA was extracted using TRIzol Reagent (Invitrogen), according to the manufacturer’s instructions. RNA quality and concentration were determined by measuring the absorbance at 260 nm (A260) and 280 nm (A280) with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Montchanin, DE). Quantitative real-time reverse transcription–PCR (qRT–PCR) was used to detect gene expression at the transcript level. First, cDNA was reverse transcribed from total RNA using the Prime1 Script RT Reagent Kit (TaKaRa, Dalian, China), and the SYBR Premix Ex TaqTM Kit (TaKaRa) was then used to detect gene expression with gene-specific primers, according to the manufacturer’s instructions. The reactions were performed on the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA), and the cycling conditions were: polymerase activation for 30 s at 958C, and 40 cycles at 958C for 5 s, and 608C for 34 s. 18S ribosomal RNA (rRNA) was used as the internal control. The PCR products were identified using a melting-curve analysis. The data were calculated using the 2–DDCt method and normalized to the individual internal control level [22]. All primers were synthesized by Invitrogen. The primer sets are shown in Supplementary Tables 1 and 2. RNA Interference Four small interfering RNAs (siRNAs; Supplementary Table 3) were designed to knockdown the expression of LOC728228, three siRNAs were designed to knockdown cyclin D1 expression, and three siRNAs were synthesized to silence PLK1 expression [46,47]. Based on their efficiency of interference, we decided to use siPLK1-3 as the positive control (Supplementary Figure 1A). All siRNAs were synthesized by GenePharma (GenePharma, Shanghai, China). 16HBE-T cells and A549 cells were seeded in six-well plates 24 h before transfection to ensure approximately 30% confluence on the day of transfection. The cells were TM transfected with siRNAs using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The medium was replaced 6 h after transfection. Total RNA was isolated 48 h after transfection and used for RT–PCR to measure the efficiency of siRNA-based interference. Cell Proliferation Assay A cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan) after transfection. Cells (3  103) in 100 ml of cell medium per well were plated in 96-well plates and cultured for 24 h under normal conditions, and were then transfected with siRNAs. After incubation for 24, 48, 72, or 96 h, 10 ml of CCK-8 reagent was added to each well. After incubation for 1 h, A450

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was measured for each well using a Synergy 2 microplate reader (BioTek, Winooski, VT). Cell viability (percent of control) was calculated as (ODtest–ODblank)/(ODcontrol–ODblank), where ODtest is the optical density of the siRNA-transfected cells, ODcontrol is the optical density of the negative control siRNA(siRNA-NC)-transfected cells or cells without treatment, and ODblank is the optical density of the wells without cells. Cell-Cycle Detection Cells were seeded in six-well plates 24 h before transfection. After transfection for 48 h, the cells were harvested by trypsinization and washed twice with phosphate-buffered saline (PBS). Each sample was fixed overnight in 1 mL of 70% (v/v) ice-cold ethanol at 48C, and then washed twice with 0.1% (v/v) Triton X-100 in PBS. The cells were stained with 20 mg/mL propidium iodide (PI) in PBS and treated with 200 mg/mL RNase A in PBS at 378C for 30 min, and analyzed immediately by flow cytometry (FACScan; Becton Dickinson, Franklin, NJ). The data were analyzed with FlowJo software (Tree Star Inc., Ashland, OR). Apoptosis Assay Apoptosis was evaluated using the Annexin V– FITC/PI Apoptosis Kit (KeyGen Biotech, Nanjing, China). For the annexin V/fluorescein isothiocyanate (FITC) binding assay, the cells were harvested by trypsinization 48 h after transfection, washed twice with ice-cold PBS, and resuspended in 500 ml of binding buffer. Finally 5 ml of annexin V–FITC and 5 ml of PI were added to 500 ml of cell suspension for 15 min at room temperature in the dark. Cell apoptosis was assessed by flow cytometry (FACScan). The data are expressed as the percentages of apoptotic cells in the total cells counted. Western Blot Extraction of total cellular protein and western blotting were conducted as previously described [48]. Briefly, the membrane containing both CCND1 protein and GAPDH was divided into two according to the molecular mass of prestained protein standards (Beyotime, Shanghai, China). The piece of the membrane with CCND1 proteins were incubated with a primary antibody for rabbit anti-human CCND1 (Origene, Rockville, MD) at a concentration of 1:500. The other piece of the membrane with GAPDH proteins were incubated with a primary antibody for rabbit anti-human GAPDH (Bioworld Technology, St. Louis Park, MN) at a concentration of 1:500. The proteins were visualized using enhanced chemiluminescence (Cell Signaling Technology) and scanned using the Gel Doc 1000 gel analysis system (Tanon, Shanghai, China).

Molecular Carcinogenesis

Lentiviral Vector Transfection ShRNA of human LOC728228 in a lentiviral vector carrying the GFP sequence was provided by GenePharma. The sequences of the shRNAs for LOC728228 are shown in Supplementary Table 3. Briefly, the cells were seeded in six-well plates 24 h before transduction to ensure approximately 50% confluence on the day of transduction. Complete optimal medium (2 mL) containing serum and antibiotics was added to each well and the cells were incubated overnight at 378C with 5% CO2. On the second day, a mixture of virus (10 ml) diluted in 2 mL of complete medium with Polybrene (Sigma– Aldrich, St. Louis, MO) at a final concentration of 5 mg/mL was prepared, and the cells underwent transduction following addition of the prepared virus mixture. The cells were incubated overnight at 378C with 5% CO2. On the third day, the culture medium was removed and replaced with 2 mL of complete medium without Polybrene, and the cells were subsequently divided 1:3–1:5 when confluent. The transduced cells were identified by selection with puromycin at a final concentration of 5 mg/mL, and successful transduction was determined by counting the green fluorescence emitted from GFPstained lentiviral particles under a fluorescence microscope (EVOS FL, Advanced Microscopy Group, Mill Creek, Washington) at a magnification of 20. The efficiency of LOC728228 inhibition was determined by qRT–PCR. Scratch Wound-Healing Motility and Transwell Migration Assays 16HBE-T, shRNA negative control (16HBE-T shRNA-NC) and shLOC728228 transduced (16HBE-T shLOC728228) cells were seeded in six-well plates. When the cells reached confluence, they were wounded by scraping with a sterile 200 ml plastic pipette tip. Nonadherent cells and cellular debris were removed by washing with PBS, and the cells then continued to grow in medium without FBS. The spread of cells during wound closure was monitored after 24 h, and recorded under an inverted microscope (Olympus, Japan). The area of cell migration was measured using Image J software. The relative migration area was calculated as (the area of the wound measured at 24 h)/(the area of the wound measured at 0 h). Cell migration was also examined using a Transwell migration assay. For this assay, 105 cells were added to the upper chamber of a 24-well Transwell apparatus (BD Biosciences, Becton Dickson Labware, Franklin Lakes, NJ, USA). Culture medium containing 20% FBS was added to the lower chamber as a chemoattractant. After 24 h, non-migrating cells were gently removed with a cotton swab. The migrated cells, located on the lower side of the chamber, were fixed with 4% paraformaldehyde for 30 min and then air-dried, stained with crystal violet

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for 20 min, and imaged. For the colorimetric assays, the samples were treated with 1 mL of 33% acetic acid after staining, and the A570 was measured using a Synergy 2 microplate reader (BioTek). Colony-Formation Assay The transduced and control cells were seeded in six-well plates at a density of 500 cells per well in normal culture medium and incubated at 378C with 5% CO2. The culture medium was replaced every 4 days during colony growth. After 10–14 days, the cell colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet for 30 min. A colony was counted only if it contained more than 50 cells. The efficiency of colony-formation was calculated as (the number of colonies)/(the number of seeded cells)  100%. Tumorigenicity Assays in Nude Mice Four-week-old Balb/c nude mice were purchased from the Medical Animal Experimental Center of Guangdong Province. All experimental procedures involving animals were performed in accordance with the institutional guidelines. Transduced and control 16HBE-T cells were trypsinized, washed twice, collected by centrifugation, and suspended in PBS. 106 cells in 0.2 mL of PBS were injected subcutaneously into the right or left posterior flank of each mouse. The mice remained in a pathogen-free environment and tumor formation was monitored. The tumor diameter was measured every three days and growth curves were plotted as the mean tumor volume  SD from five mice in each group. The mice were killed 20 days after injection and the tumor xenografts were excised, weighed, and fixed with 4% (v/v) formalin for pathological examination. Relative Expression of LOC728228 in the Nucleus and Cytoplasm The nuclear and cytoplasmic fractions were isolated from 16HBE-T cells using the PARIS kit (Ambion, Austin, TX). Briefly, the cells were harvested by trypsinization, washed with PBS, resuspended in cold fractionation buffer, incubated on ice, and centrifuged. The cytoplasmic fraction was collected, while the nuclear pellet was lysed with disruption buffer, and then 2  lysis/binding solution was added to each fraction. RNA was isolated from separate lysates by the addition of ethanol and filtered through a cartridge. The nuclear and cytoplasmic RNAs (800 ng) were then converted to cDNA and analyzed by qRT-PCR. Statistical Analysis Values are expressed as means  SD. All statistical analyses were performed using SPSS 13.0 software (SPSS Inc, Chicago, IL). Differences between two groups were analyzed by a two-sided Student’s t test, and multiple comparisons were made using one-way Molecular Carcinogenesis

analysis of variance (ANOVA), followed by an appropriate post hoc test. All experiments were performed at least three times, and for all tests, P < 0.05 was considered statistically significant. RESULTS Expression of LOC728228 is Upregulated in Lung Cancer Cell Lines To investigate the functional roles and mechanisms of lncRNAs in lung cancer, we previously constructed a human transformed bronchial epithelial cell line (16HBE-T) using the chemical carcinogen anti-BPDE. In the present study, lncRNA expression profiles in 16HBE-T and 16HBE-N (untransformed) cells were determined. Microarray findings showed that seven lncRNAs were upregulated more than two-fold in the16HBE-T cells compared to 16HBE-N cells (data not shown). LncRNA LOC728228, corresponding to its probe uc002wkq (data not shown), was strongly expressed and was upregulated approximately 3.17fold compared to 16HBE-N cells. We confirmed the relative expression of LOC728228 using qRT–PCR. Our data show that compared with its level in 16HBEN cells, LOC728228 was upregulated 3.19  0.19-fold in 16HBE-T cells (Figure 1A) and 8.41  0.58-fold in A549 cells compared to its expression in 16HBE-N cells (Figure 1B). We also determined the relative expression of LOC728228 in another lung cancer cell line (Beas-2bT) transformed in our laboratory [49]. The data show that LOC728228 was upregulated 146.53  29.74-fold in Beas-2bT cells compared with its expression in untransformed Beas-2b cells (Figure 1C). We selected neuroblastoma RAS viral (v-ras) oncogene homolog (NRas) as a positive control in 16HBE-T, and the expression of lncRNA-DQ786227 or MALAT1, was used as the positive control in Beas2bT and A549 cells, respectively [48–50]. Our results indicate that LncRNA LOC728228 is aberrantly expressed in transformed cell lines. LOC728228 Regulates Cell Proliferation, Perhaps by Influencing the Cell-Cycle Distribution in vitro The upregulation of lncRNA LOC728228 in 16HBE-T and A549 cells suggests that it may play a role in lung cancer. To test this, we designed four siRNAs (siLOC728228-1 to -4) and transfected 16HBE-T and A549 cells with these siRNAs at a concentration of 30 nmol/l, 50 nmol/l, or 100 nmol/l to knockdown LOC728228 expression. The efficiency of interference was evaluated using qRT–PCR. Our data showed that 50 nmol/l was the optimal concentration for the transfection of each siRNA, and siLOC728228-1 and siLOC728228-2 were more efficient than siLOC728228-3 and siLOC728228-4 in both 16HBE-T and A549 cells (Supplementary Figure 1B). The transfection of siLOC728228-1 and siLOC728228-2 inhibited the expression of LOC728228 by 87.1  1.6% and 87.8  1.9%, respectively, in 16HBE-T cells, and by

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Figure 1. LOC728228 may regulate cell proliferation by influencing the cell-cycle distribution. (A, B, C) The relative expression of LOC728228 in 16HBE-T, A549, and Beas-2bT cells was determined by qRT–PCR and normalized to 18S rRNA expression. (D, E) Cell viability was detected with the CCK-8 assay after transfection with siLOC728228-1 or siLOC728228-2 for 24 h, 48 h, or 72 h. (F) Representative flow-cytometric analysis of the cell cycle in 16HBE-T

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cells 48 h after transfection. (G) Representative flow-cytometric analysis of the cell cycle in A549 cells 48 h after transfection. (H, I) Distribution of the G0/G1, S, and G2/M phases after LOC728228 knockdown in 16HBE-T and A549 cells. The data in (A), (B), (C), (D), (E), (H), and (I) are the means  SD of three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.01, compared with the 16HBE-N group, Beas-2b group, or siRNA-NC-transfected group.

ONCOGENE-LIKE FUNCTION OF lncRNA LOC728228

73.3  13.1% and 79.4  8.5%, respectively, in A549 cells compared with the siRNA negative control (siRNA-NC; all P < 0.01; Supplementary Figure 1B). Therefore, we selected siLOC728228-1 and siLOC728228-2 at concentrations of 50 nmol/l, for subsequent biological studies. To examine the functional role of LOC728228, the effects of reduced LOC728228 expression on cell proliferation were investigated in 16HBE-T and A549 cells. We repressed the expression of LOC728228 by transfecting the cells with siLOC728228-1 and siLOC728228-2 for 24 h, 48 h, or 72 h. Cell proliferation was measured in vitro with CCK-8. The 16HBE-T and A549 cells transfected with siLOC728228s showed significant reductions in cell viability compared with those transfected with siRNA-NC or the untransfected control cells at 24 h, 48 h, and 72 h after transfection, respectively (Figure 1D and E). These results demonstrate that LOC728228 affects the proliferation of 16HBE-T and A549 cells. Because LOC728228 affects cell proliferation, we hypothesized that it may do so by affecting the cell cycle and/or apoptosis. Cell-cycle distribution and apoptosis were determined 48 h after siRNA transfection in both 16HBE-T cells and A549 cells. As seen in Figure 1F and 1H, DNA content analysis using flow cytometry revealed that siLOC728228-2-transfected 16HBE-T cells had a higher proportion of cells in G0/ G1 phase (65.82%, compared with 56.63% in control cells, P < 0.01) and a reduced proportion in S phase (16.89%, compared with 24.4% in control cells; P < 0.05). As expected, it was also shown that the downregulation of LOC728228 by transfecting A549 cells with siLOC728228-2 induced an accumulation of cells in G0/G1 phase (89.045%, compared with 75.275% in control cells, P < 0.01), and a reduction in S phase (6.25%, compared with 11.06% in control cells; P < 0.05). In the siLOC728228-1-transfected group, the cells in G0/G1 phase also increased and the difference was also statistically significant (Figure 1G and I). These results indicate that G0/G1 arrest occurred in both cell lines in response to LOC728228 downregulation. We then determined the level of apoptosis in the transfected 16HBE-T and A549 cells, however, there was no difference between the siLOC728228s-transfected cells and the siRNANC-transfected cells (data not shown). These data suggest that the growth inhibition induced by the knockdown of LOC728228 is caused by the induction of G1 arrest rather than by apoptosis. Stable Knockdown of LOC728228 Expression Suppresses Colony Formation Because siRNAs can only transiently suppress LOC728228 expression, we established a stably transduced cell line with constantly reduced LOC728228 expression for further functional analysis. 16HBE-T cells were transduced with lentivirusmediated shLOC728228 or shRNA negative control Molecular Carcinogenesis

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(shRNA-NC) vectors. The transduced cells were detected as green fluorescent protein (GFP)-positive cells following the addition of GFP together with the LOC728228 shRNAs. The successful expression of the lentivirus-mediated LOC728228 shRNAs was confirmed by fluorescence imaging (Figure 2A). The level of LOC728228 detected by qRT–PCR was significantly reduced following transduction by LOC728228 shRNAs compared with that in the 16HBE-T and shRNA-NC-transduced cell groups (Figure 2B) (P < 0.01). These findings confirmed that the lentivirus-mediated transduction of the LOC728228 shRNAs were effective. The expression levels of LOC728228 were determined by qRT–PCR weekly during all the experiments. The effect of LOC728228 downregulation on 16HBE-T cells was first investigated using a colonyformation assay. The numbers and sizes of the colonies were observed in the untransduced 16HBET, shRNA-NC-transduced and shLOC728228 -transduced cell groups. Reductions in the numbers and sizes of the colonies of the shLOC728228 -transduced cells were clearly observed by microscopy (Figure 2C and D). The numbers of colonies were counted in all the cell groups. As shown in Figure 2D, the colonyforming efficiency was significantly reduced after transduction with shLOC728228-1 (by 15.5  2.7%, P < 0.01) and shLOC728228-2 (by 16.6  9.1%, P < 0.01) compared with that in the shRNA-NCtransduced group. Collectively, these results strongly demonstrate that both the numbers and sizes of 16HBE-T cell colonies were reduced following knockdown of LOC728228 expression. Stable Knockdown of LOC728228 Expression Reduces Cell Migration and Suppresses Tumor Growth To determine the effect of LOC728228 knockdown on the migration of 16HBE-T cells, we performed a scratch wound-healing motility assay and a Transwell migration assay with stably transduced 16HBE-T cells. As shown in Figure 3A and B, cell motility was altered by the stable knockdown of LOC728228. The relative area of cell migration was measured to establish the migration rate, which was found to be reduced after transduction with shLOC728228-1 and shLOC7282282 by 36.0  3.6% and 37.1  2.2%, respectively, compared with that in the untransduced 16HBE-T and shRNA-NC-transduced cells (all P < 0.01). In the Transwell migration assay, the A570 values for cell migration were significantly reduced after transduction with shLOC728228 cells compared with those in shRNANC-transduced and untransduced cells (Figure 3C). These results demonstrated that the migration capacity was inhibited in vitro when LOC728228 expression was knocked down in 16HBE-T cells. The significant effects of reduced LOC728228 on cell growth and colony formation in vitro prompted us to investigate the possible effects of LOC728228 shRNA transduction on tumor growth in vivo. The

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Figure 2. Relationship between the expression level of LOC728228 and the colony-forming capacity of 16HBE-T cells. (A) Fluorescence micrographs of 16HBE-T cells after transduction with lentivirus containing LOC728228 shRNAs or shRNA-NC at a magnification of 20. (B) The relative expression of LOC728228 after transduction with shLOC728228-1 or shLOC728228-2. (C) Representative pictures of the

Molecular Carcinogenesis

colony numbers and sizes of 16HBE-T cells after crystal violet staining. (D) The effects of shLOC728228-1 and shLOC728228-2 transduction on colony-forming efficiency. The data in (B) and (D) are the means  SD of three independent experiments, each performed in triplicate. **P < 0.01, compared with the shRNA-NC-transduced group or the untransduced 16HBE-T group.

ONCOGENE-LIKE FUNCTION OF lncRNA LOC728228

Figure 3. Cell migration capacity in vitro and tumor growth in vivo after stable knockdown of LOC728228 expression. (A) Representative photographs of transduced 16HBE-T cells taken at 0 h and 24 h in the scratch wound-healing motility assay. (B) Relative migration areas of the shLOC728228 and shRNA-NC transduced and untransduced 16HBE-T cells. (C) The OD values for the migration of cells transduced with LOC728228 shRNAs in the Transwell migration assay. (D) Tumor growth curves were constructed after mice were injected with untransduced 16HBE-T cells or shRNA-NC-transduced, shLOC272881-transduced, or shLOC27288-2-transduced cells. The tumor volume V (in cubic millimeters) was calculated using the formula V ¼ 0.5 LW2, where L is the length of the tumor (in millimeters), W is the width of the

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tumor (in millimeters), and V is the mean tumor volume. (E) Tumor weight in injected mice. (F) Effect of LOC728228 knockdown on tumorigenesis in vivo. The photograph illustrates representative features of the tumor xenografts 20 days after injection. (G) Pathological examination of tumors with hematoxylin and eosin staining (original magnification, 100. Pathological examination showed the growth of squamous cell carcinomas. The data in (B) and (C) are the means  SD of three independent experiments, each performed in triplicate. The data in (D) and (E) are means  SD (n ¼ 5 per group). *P < 0.05, **P < 0.01, compared with the shRNA-NC group or untransduced 16HBE-T group.

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mice in each group were injected subcutaneously with shLOC72828 and shRNA-NC transduced or untransduced 16HBE-T cells and displayed tumor growth 5 days after injection. Tumor growth was measured every three days and the tumors were harvested 20 days after injection. The tumors derived from mice injected with shLOC728228-transduced cells were significantly smaller and weighed less than those in mice injected with shRNA-NC-transduced or untransduced 16HBE-T cells (Figure 3D–F). There was no difference in tumor growth or tumor weight between the shRNA-NC-transduced group and the untransduced group. A pathological examination revealed that the mice injected with the four groups of cells developed squamous cell carcinomas. Compared with the shRNA-NC-transduced and untransduced groups, the tumors derived from the shLOC728228-transduced groups showed more necrosis (Figure 3G). These results indicate that the suppression of LOC728228 reduced tumor growth. LOC728228 Transcript May Function in the Cytoplasm Following the observed effects of LOC728228 on lung cancer cells, we attempted to determine the underlying mechanism. According to the study by

Derrien [2], LOC728228 is a 480-bp intergenic noncoding RNA. Recent studies have found that some lncRNAs act in cis, therefore they can regulate the expression of one or more nearby genes on the same chromosome [23–26]. We investigated whether LOC728228 acts in cis. The expression of 20 nearby genes, SMOX, PROKR2, CDS2, PCNA, TMEM230, SLC23A2, RASSF2, PRND, PRNP, ADRA1D, GPCPD1, RNF24, PANK2, MAVS, AP5S1, CDC25B, CENPB, SPEF1, C20orf27, and HSPA12B, extending across approximately 1.50 Mb (Figure 4A), was detected by qRT–PCR after the knockdown of LOC728228 by siRNAs. When LOC728228 was significantly knocked down, the expression of some of these genes was slightly altered (data not shown). These results indicate that LOC728228 does not regulate the expression of these genes directly, thus LOC728228 may not exert its function in cis. We next determined the cytoplasmic/nuclear distributions of LOC728228. We separated the nuclear and cytoplasmic fractions of 16HBE-T cells, and determined the relative expression of LOC728228 in each fraction using three controls: glyceraldehyde 3phosphate dehydrogenase (GAPDH) mRNA, which occurs in both the nucleus and cytoplasm; U6

Figure 4. LOC728228 may function in the cytoplasm. (A) Relative locations of 20 neighboring genes on chromosome 20. (B) Relative expression of LOC728228 in the nuclear and cytoplasmic fractions of 16HBE-T cells was determined by qRT–PCR with two pairs of specific primers. The data shown here are the means  SD of three independent experiments, each performed in triplicate.

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ONCOGENE-LIKE FUNCTION OF lncRNA LOC728228

Figure 5. LOC728228 partially exerts its function by regulating cyclin D1. (A) The expression of 17 genes was determined by RT–PCR in 16HBE-T cells after transfection with siLOC728228-2. (B) Relative expression of cyclin D1 in 16HBE-T cells after transfection with siLOC728228-1. (C, D) Cyclin D1 protein levels were analyzed by western blotting. (E) Cell viability was determined using the CCK-8 assay 24 h, 48 h, and 72 h after transfection with siCCND1-1 or

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siCCND1-3 in 16HBE-T cells. (F) Distribution of the G0/G1, S, and G2/M phases after CCND1 knockdown in 16HBE-T cells. (G) Representative flow-cytometric analysis of the cell cycle in transfected and control cells 48 h after transfection. The data shown in (A), (B), (D), (E), and (F) are the mean  SD of three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.01, compared with the 16HBE-T group or siRNA-NC group.

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spliceosomal RNA, a nuclear transcript involved in RNA processing which only occurs in the nucleus; and a mitochondrial gene MT RNR1, which was used as a cytoplasmic control. The data are presented as cytoplasmic/nuclear ratios. Because GAPDH and MT RNR1 display more transcripts in the cytoplasm, their ratios were greater than 1 in our data (Figure 4B). The LOC728228 RNA content in the cytoplasm was at least twice that in the nucleus, which was verified using two pairs of specific primers (Supplementary Table 1). These results indicate that LOC728228 mainly occurs in the cytoplasm and therefore probably functions there. LOC728228 Partially Exerts its Function by Regulating Cyclin D1 We noted that the transient depletion of LOC728228 reduced cell proliferation, and this was mainly caused by G0/G1 cell-cycle arrest. To determine how LOC728228 influences the cell cycle, we examined the expression of 17 common genes required for different aspects of cell cycle activation or progression in 16HBE-T cells using qRT–PCR 48 h after the knockdown of LOC728228 by siLOC7282282. 18S rRNA and GAPDH, which are constantly expressed in 16HBE-T cells under different experimental conditions, were chosen as the reference genes, and all PCR results (below) were normalized to their expression. As shown in Figure 5A, only cyclin D1 was downregulated more than two-fold compared with the siRNA negative control (P < 0.01), whereas p15 and p16 were undetectable in 16HBE-T cells and are not shown. Several genes were identified using siLOC728228-1 (Figure 5B), and the results showed that cyclin D1 was significantly lower than that in the control (P < 0.01). The level of cyclin D1 protein (CCND1) was downregulated more than two-fold 72 h after the knockdown of LOC728228 by siLOC7282282 (Figure 5C and D). Cyclin D is important in initiating the cell cycle, and plays an important role in many tumor types. Therefore, we determined whether cyclin D1 influences the G1 phase of the cell cycle in 16HBE-T cells. To test this, we used three siRNAs directed against cyclin D1. SiCCND1-1 and siCCND1-3, which afford the most efficient interference, were selected for further experiments. 16HBE-T cells were transfected with the two siRNAs, and 48 h after transfection, cell proliferation was determined with CCK-8, and the cell-cycle distribution was determined by flow cytometry. Transfection of the siRNAs significantly reduced cell proliferation compared with that of the untransfected and negative control groups (Figure 5E) and caused G0/G1 cell-cycle arrest (Figure 5F and G). These data are consistent with the long non-coding RNA LOC728228 influencing cell cycle and cell proliferation in 16HBE-T cells, at least in part, through regulation of cyclin D1 expression.

Molecular Carcinogenesis

DISCUSSION The incidence of lung cancer is increasing, however, the molecular mechanisms involved in lung carcinogenesis remain unclear. Recent studies have revealed that a large number of lncRNAs are transcribed from the human genome, and have been ascribed broad roles across various biological processes [2,27]. Numerous studies have shown that lncRNAs are involved in various cancers, including lung, breast, prostate, colon, bladder, and liver cancers [28– 30]. This provides new avenues in cancer research and extends our understanding of carcinogenesis. In the present study, we found that the expression of lncRNA LOC728228 was significantly increased in 16HBE-T cells, A549 cells, and Beas-2bT cells relative to that in the control cells. We also found that LOC728228 influenced tumor cell growth by affecting the cell-cycle distribution, but not cell apoptosis. The stable knockdown of LOC728228 suppressed 16HBE-T cell-colony formation in vitro and tumor growth in vivo. In recent years, lncRNAs including growth-arrest-specific transcript 5 (GAS5), noncoding RNA expressed in aggressive neuroblastoma (ncRAN), prostate-cancer-associated transcript 1 (PCAT-1), prostate-specific gene 1 (PCGEM1), and ultraconserved element 73 (uc.73a) have been reported to regulate tumor growth and progression by altering the balance between cell proliferation and cell death [10,11,31-33]. Our data suggest that LOC728228 influences tumor growth by affecting cell proliferation. Metastasis is another important aspect of malignancy, and is the most challenging problem in tumor prognosis and therapy. Research into lncRNAs involved in tumor metastasis, including HOX antisense intergenic RNA (HOTAIR), MALAT-1, and lncRNA-low expression in tumor (lncRNA-LET), have also been reported [9,14,34]. Our results show that the knockdown of LOC728228 suppressed 16HBE-T cell migration, thus LOC728228 may also affect the migration of tumor cells. Taken together, our data indicate that lncRNA LOC728228 plays an important role in lung cancer cell lines and may have an oncogene-like function. Based on the results described above, we investigated the genes involved in LOC728228 function. Although the functions of lncRNAs have been intensively investigated, it is the mechanisms by which lncRNAs mediate their function that are still poorly understood. To date, lncRNAs have been implicated in such diverse processes as transcriptional regulation in cis or in trans, or at the posttranscriptional level [4,35,36]. Our results indicate that LOC728228 does not function in cis. Indeed, most cis-acting lncRNAs are imprinting-related genes, and some are very long, such as the antisense of IGF2R non-protein-coding RNA (118 kb) and KCNQ1 opposite strand/antisense transcript 1(91 kb) [37,38]. We also found that LOC728228 localizes predominantly

ONCOGENE-LIKE FUNCTION OF lncRNA LOC728228

in the cytoplasm, which suggests that LOC728228 probably functions there. Studies have shown that cytoplasmic lncRNAs can regulate protein localization [39], protein stabilization [34], mRNA translation [40], and mRNA stabilization [41–43]. This provided useful information for further exploring the mechanism of LOC728228. Because the cell cycle was arrested in G0/G1 phase in 16HBE-T cells following the transient knockdown of LOC728228, we examined the expression of cellcycle-related genes and found that the expression levels of cyclin D1 mRNA and protein were significantly downregulated after the knockdown of LOC728228 expression. Therefore, LOC728228 may regulate cyclin D1 expression levels to some extent. Cyclin D1 is known to be involved in the regulation of cell-cycle progression and has been identified as a prooncogene. Its amplification or overexpression plays a pivotal role in a subset of human cancers, and its downregulation is often linked to cell-growth inhibition [44,45]. Our experiments confirmed that the knockdown of cyclin D1 caused cell-cycle arrest at G0/ G1 and inhibited cell proliferation in 16HBE-T cells. From these data, we suggest that LOC728288 influences cyclin D1 mRNA levels, thus regulating the cell cycle, and consequently affects the proliferation of 16HBE-T cells. This extends our understanding of LOC728228, and supports its oncogene-like function. However, it is unknown how LOC728228 influences cyclin D1 mRNA levels. A possible mechanism is that LOC728228 binds an RNA-binding protein to regulate cyclin D1 mRNA, such as growth-arrested DNA damage-inducible gene 7 (GADD7) [42]. More research is required to establish these mechanisms. In conclusion, our data demonstrate that LOC728228 is a lung-cancer-related lncRNA and plays an important role in 16HBE-T cancer cells. Our findings indicate that the non-coding RNA LOC728228 may have an oncogene-like function in lung cancer ACKNOWLEDGMENTS We thank the Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation (Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University) for their support with the flow cytometry analysis. REFERENCES 1. Birney E, Stamatoyannopoulos JA, Dutta A, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 2007;447: 799–816. 2. Derrien T, Johnson R, Bussotti G, et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 2012;22:1775–1789. 3. Batista PJ, Chang HY. Long noncoding RNAs: Cellular address codes in development and disease. Cell 2013;152: 1298–1307.

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Molecular Carcinogenesis

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