Xiao Yu, Xiaofei Ye, Hongyan Lin, Nannan Feng, Sumeng Gao, Xiaohong Zhang, Yu Wang, Herbert Yu,Xiaobei Deng, Biyun Qian

1Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Faculty of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 2Cancer Epidemiology Program, University of Hawaii Cancer Center,Honolulu, HI 96813, USA

ABSTRACT Objective: Long non-coding RNAs (lncRNAs) are involved in numerous biological processes in lung cancer cells. In our previous studies, we identified a lncRNA, ENST00000439577, which is highly expressed in lung carcinomas, and termed it lung cancer progression-associated transcript 1 (LCPAT1). To characterize the role of LCPAT1 in lung cancer, we conducted the current study.Methods: Expression of LCPAT1 and autophagy-associated markers in tumor tissues and lung cancer cell lines was determined by real-time quantitative polymerase chain reaction (qPCR). Hematoxylin and eosin (HE) staining, qPCR, Western blot, and immunohistochemistry were performed to evaluate xenografted tumor tissues. Autophagy induced by rapamycin was detected by Western blot and immunofluorescence in lung cancer cell lines.Results: Expression of LCPAT1 and microtubule-associated protein 1 light chain 3 beta (LC3B) was positively correlated in lung cancer. Knockdown of LCPAT1 inhibited tumor growth and suppressed cell autophagy in vivo. Moreover, LCPAT1 knockdown in lung cancer cell lines resulted in decreased autophagy-associated gene expression and alleviated the cell autophagy induced by rapamycin.Conclusions: We speculate that LCPAT1 plays a crucial role in regulating autophagy in lung cancer.

KEYWORDS Lung cancer; autophagy; long non-coding RNA; LCAPT1; ENST00000439577

Introduction

Lung cancer is one of the leading causes of cancer deaths worldwide1. The most common type of lung cancer is nonsmall cell lung cancer (NSCLC) and its 5-year survival rate is＜ 15%2. Although extensive research has been carried out,the pathological process of lung cancer remains unclear. For the past few years, long non-coding RNAs (lncRNAs) have garnered increasing attention in lung cancer research, both as modulators of pathogenesis and as potential therapeutic targets3-5.

LncRNAs are a class of non-protein coding transcripts that are 200 nucleotides or longer in length6. Though they do not code for proteins, lncRNAs control gene expression by functioning as guides, scaffolds, and decoys of many biomolecules. Also, transcription of lncRNAs is exquisitely regulated and the expression of lncRNAs reflects different developmental stages or responds to diverse extracellular signals7. LncRNAs are now recognized as novel regulators in tumor development and progression. It is well-known that the lncRNAsH198,HOTAIR9,MEG310,MALAT111, andANRIL3are dysregulated in cancer. In our previous study,we reported that expression ofLOC146880andENST00000439577was elevated in lung cancer5.

ENST00000439577is a 311-bp transcript and its gene is located on chromosome 1 (chr1: 17, 406, 760-17, 407, 382).We previously reported thatENST00000439577expression was associated with metastasis status and disease stage in lung cancer patients. Moreover, high expression ofENST00000439577was associated with poor overall survival of lung cancer patients5. Thus, we termedENST00000439577lung cancer progression-associated transcript 1 (LCPAT1),suggesting thatLCPAT1may act as an oncogene in lung cancer.

LncRNAs participate in tumorigenesis through multiple pathways, including autophagy11-15. Autophagy is an intracellular bulk degradation process through which proteins, lipids, and organelles are delivered to lysosomes for degradation16. In this process, autophagy related 5 (ATG5),autophagy related 14 (ATG14),BECLIN1, and other autophagy-related genes play an important role.Sequestosome 1 (p62) is incorporated into mature autophagosomes and degraded during autophagy.Microtubule-associated protein 1 light chain 3 beta (LC3B)levels increase andLC3B-encoded LC3-I is converted to LC3-II which accumulates and adsorbs on the autophagosome membrane. The degradation of p62 and increase of LC3-II are commonly used as markers of autophagy17.

Autophagy acts as a double-edged sword in cell survival and cell death18. In the process of tumor development,autophagy suppresses chronic inflammatory reactions to prevent tumorigenesis, and enable tumor survival by recycling nutrients19. Genetic deletion of an autophagy protein, ATG5, accelerates the initiation of KRas (G12D)-driven lung tumors in mice, but impairs the progression of lung cancer, suggesting that autophagy may prevent oncogenesis, but promote tumor growth20.

Several lncRNAs targeting autophagy have been described.The overexpression of the lncRNABANCRresults in an increase in the ratio of LC3II-to-LC3I, which indicates the formation of an autophagosome21. Our previous study revealed that the lncRNALOC146880participates in PM2.5exposure-induced autophagy in lung cancer4. The purpose of the current study was to determine the role ofLCPAT1in regulating autophagyin vivoandin vitro. We found thatLCPAT1was positively correlated withLC3Bin lung cancer.In vitroandin vivoevidence demonstrated thatLCPAT1knockdown inhibits activation of autophagy in lung cancer.These findings indicate the pivotal role of lncRNALCPAT1in lung cancer and the possible mechanisms involved with autophagy.

Materials and methods

Patients

Tumor samples and matched adjacent normal tissues were collected with informed consent from lung cancer patients during surgeries performed between May 2006 and July 2011 at the Tianjin Medical University Cancer Institute and Hospital (TMUCIH). RNA extraction and analysis were performed as previously described4,5,22. The current study was approved by the medical ethical review committees at TMUCIH and Shanghai Jiao Tong University School of Medicine.

Cell culture

HEK-293T and lung cancer cell lines, A549 and H1975, were obtained from the American Type Culture Collection(ATCC). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Waltham, MA, USA)supplemented with 10% fetal bovine serum (Sigma-Aldrich,St. Louis, MO, USA) and 100 U penicillin-streptomycin under 5% CO2at 37 °C.

Lentiviral small hairpin (sh)RNA

Plasmids for shRNA targetingLCPAT1(sense, 5′-CAATGTTGTTGTTTATTTA-3′ and antisense, 5′-TAAAT AAACAACAACATTG-3′) and scrambled (sense, 5′-TTC TCCGAACGTGTCACGT-3′ and antisense, 5′-ACGTGACA CGTTCGGAGAA-3′) were obtained from Shanghai Integrated Biotech Solution Company and the interfering vector used to generate shRNA was pLKD-CMV-G & PRU6-shRNA. Lentiviral vector DNA and package vectors were transfected into HEK-293T cells by Lipofectamine? 2000 transfection reagent (Invitrogen). At 48 and 72 h after transfection, lentivirus supernatants were harvested and used to infect H1975 cells. Stable shRNA cell lines were generated following selection with 1 μg/mL puromycin. The efficiency of gene knockdown was detected by real-time quantitative polymerase chain reaction (qPCR).

Animals

BALB/c mice, 4–6 weeks old, were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and raised in a specific-pathogen-free grade environment. Three female and three male mice were used for a control experiment. H1975 cells transfected with scrambled shRNAs orLCPAT1-shRNA were suspended in phosphate-buffered saline with Matrigel matrix basement membrane extract(Bedford, MA, USA) in a 1:1 ratio. Then, the cell solutions(5 × 106cells/100 μL/mouse) were injected subcutaneously in the left and right hind flanks of mice. Tumor growth was evaluated by measuring tumor diameters every 2 days with a vernier caliper, and tumor volume was calculated using the following formula: V = 1/2×a×b2, where V = tumor volume,a = the larger perpendicular diameter, and b = the smaller perpendicular diameter. The animals were sacrificed on day 21 after implantation and tumors were collected for analysis.All applicable guidelines of the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine were followed.

Hematoxylin and eosin (HE) staining

Xenografted tumor tissues were fixed with 4%paraformaldehyde (BOSTER, Wuhan, China), embedded in paraffin, sectioned, and visualized at 100× and 200×magnification after HE staining.

Immunohistochemical (IHC) staining

For lung cancer patients’ tissue, the sections (5 μm) from the tissue microarray blocks were labelled with antibodies. The LC3B antibody was diluted 1:2000. An HRP- conjugated secondary antibody (Beijing Sequoia Jinqiao Biological Technology Co., Ltd.) was used. The specific target was visualized with a 3, 3’-diaminobenzidine (DAB) detection kit(Beijing Sequoia Jinqiao Biological Technology Co., Ltd.) and counterstained with hematoxylin. Microphotographs from each arrayed tissue were taken with a fixed exposure time and color balance to ensure consistency. LC3B production was quantified using ImagePro Plus9.1 (Media Cybernetic, Silver Spring, MD).

For animal tumor tissue, paraffin sections were first stained with anti-ATG5 (#ab108327, 1:50; Abcam,Cambridge, MA, USA) and anti- LC3B (#ab48394, 1:50;Abcam). Horseradish peroxidase (DAKO, Glostrup,Denmark) was then used as a secondary antibody. When an antigen-antibody-antibody complex was formed, a substrate of the peroxidase, diaminobenzidine, was added as chromogen. The staining was performed according to the manufacturer’s instructions. The pictures were taken at a magnification of 400 × and analyzed using Image Pro-plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).Five different pictures were measured for each sample.

Small interfering RNA (siRNA) transfectionss

Control andLCPAT1-specific small interfering RNA were obtained from Genepharma (Shanghai, China). Three specific siRNAs forLCPAT1were designed and mixed for transfection. The sequences ofLCPAT1siRNAs are listed in Table 1. Cells (3 × 105) were seeded into six-well plates and transfected with siRNAs after overnight incubation at 37 °C.The transfection was performed with Lipofectamine?2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol .

Real-time quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from cells and tissues using TRIzol,and cDNA was synthesized using a FastQuant RT Kit with gDNase (TIANGEN, Beijing, China). The primers used for analyzing the expression ofLCPAT1,BECLIN1,LC3B,p62,ATG3,ATG5,ATG7,ATG12,ATG14, and β-ACTIN were synthesized by Sangon Biotech (Shanghai, China) (Table 2).Quantitative RT-PCR was performed in the ABI Prism 7500 system (Applied Biosystems, Foster City, CA, USA) using the SYBR Green Supermix (Applied Biosystems). Relative gene expression was analyzed according to the formula: 2???CT.

Table 1 Sequences of siRNAs

Table 2 Sequences of PCR primers

Western blot analysis

Tissue samples and cultured cells were lysed with RIPA lysis buffer containing protease inhibitor. SDS-PAGE was used to detect the proteins of interest. Proteins were resolved on SDS gels and were electro-transferred to polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). Then, the membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with primary antibodies,including anti-LC3B(#ab48394, 1:500; Abcam), anti-p62(#ab56416, 1:500; Abcam), anti-ATG5 (#ab108327, 1:500;Abcam), anti-BECLIN1 (#ab55877, 1:500; Abcam), and antiβ-ACTIN (#A8481, 1:4000; Sigma-Aldrich). After washing,the membranes were incubated with a secondary antibody and imaged with Odyssey SA (Gene Company Limited, Hong Kong, China).

Immunofluorescence assay

Cultured cells were grown on glass coverslips overnight to detect the LC3B puncta. Cells fixed with 4%paraformaldehyde (BOSTER) were treated with 0.2% Triton X-100 and blocked with 2% albumin from bovine serum(BSA). Then, cells were incubated with a primary anti- LC3B antibody (#ab48394, 1:200; Abcam) at 4 °C overnight. After the incubation of Alexa Fluor 568-conjugated goat antirabbit IgG (#ab175694, 1:1000; Abcam), cells were stained with 0.2 mg/mL DAPI and imaged with a confocal microscope (Zeiss LSM710, Carl Zeiss, Dresden, Germany).The LC3B puncta were measured using Image Pro-plus 6.0 software (Media Cybernetics). Five different pictures were measured for each group.

Statistical analysis

All data were analyzed using the SPSS software package,version 19.0 (SPSS, Chicago, IL, USA) and graphed by GraphPad Prism 5 (La Jolla, CA, USA). The results were presented as mean ± standard error based on three independent experiments. Statistical analysis was performed using correlation analysis andttest.Pvalue ＜ 0.05 was defined as statistically significant.

Results

Correlation between LCPAT1 and LC3B in lung cancer

In lung cancer patients, tumor tissues showed higherLCPAT1andLC3Bexpression versus normal tissues at the gene expression level (Figure 1A). Also, IHC staining showed that the cytoplasmicLC3Bprotein was highly expressed in lung tumor tissues (Figure 1B). To investigate the relationship betweenLCPAT1and autophagy, we analyzed the correlation ofLCPAT1andLC3Bexpression in tumor tissues. Our results showed that the correlation coefficient between these genes was high,r= 0.783 (Figure 1C),suggesting thatLCPAT1may play a role in autophagy in lung cancer.

Suppression of tumor growth in vivo by LCPAT1 knockdown

We evaluated the involvement ofLCPAT1in tumor growthin vivo. H1975 cells transfected with scrambled shRNAs orLCPAT1-shRNA were injected subcutaneously into nude mice. Tumor growth was recorded and analyzed after the injection. We observed smaller tumor sizes in the right hind flanks of animals that were injected withLCPAT1-shRNA(Figure 2A). We also found that tumor growth was slower in theLCPAT1-shRNA tumors compared with that of the controls (Figure 2B). HE tissue staining revealed that tumors withLCPAT1-shRNA possessed less malignant morphology compared to the controls (Figure 2C). These findings suggest thatLCPAT1may promote tumor growth.

Suppression of autophagy by LCPAT1 knockdown in vivo

As shown in Figure 3A and 3B,LCPAT1expression was reduced by nearly 60% in stableLCPAT1knockdown H1975 cells and 50% inLCPAT1knockdown tumors. We found that the expression of autophagy-associated genes was significantly reduced inLCPAT1-shRNA tumors compared to that in the controls, with an approximately 50% drop inLC3B, 59% drop inATG3, 42% drop inATG5, 54% drop inATG7, 44% drop inATG12, 36% drop inATG14, and 59%drop inBECLIN1(Figure 3C). We further validated our findings using Western blot. We observed thatLCPAT1knockdown significantly suppressed the expression of ATG5(approximately 48% reduction) and LC3-II (approximately 57% reduction) and stimulated the expression of p62(approximate 2.5-fold increase; Figure 3D). Additionally, the immunohistochemical analysis of LC3B and ATG5 showed a decreasing trend in shLCPAT1xenografted tumor tissues(Figure 3E). Taken together, autophagy appeared to be suppressed afterLCPAT1knockdownin vivo.

Figure 1 Correlation between LCPAT1 and LC3B expression in lung cancer. (A) Gene expression of LCPAT1 and LC3B in tumor tissues and matched adjacent normal tissues of lung cancer patients. Data are presented in mean ± SEM; ＊＊P ＜ 0.01. (B) Representative immunohistochemical staining images (400 ×) of cytoplasmic LC3B and its density (IOD/Aera) in normal and lung cancer tissues. (C)Correlation between LCPAT1 and LC3B expression in tumor tissues of lung cancer patients.

Suppression of autophagy by LCPAT1 knockdown in vitro

Based on the abovein vivofindings, we further investigated the role ofLCPAT1in lung cancer cells. After detecting the expression ofLCPAT1in the normal bronchial epithelial cell line HBE and lung cancer cell lines H520, H1299, A549, and H1975, we found thatLCPAT1expression was higher in lung cancer cell lines than in HBE, especially in A549 and H1975(Figure S1). It was observed that whenLCPAT1-siRNA was transfected into A549 and H1975 cells (Figure 4A and 4B),mRNA expression of autophagy-associated genes was markedly decreased. For example, in H1975 cell lines,LC3B,ATG5,ATG7,ATG14, andBECLIN1were decreased by 60%–80%, andATG3andATG12were reduced by 40%. In addition, upon treating cells with rapamycin, an autophagy inducer, we observed the accumulation of ATG5, BECLIN1,and LC3-II, and the degradation of p62. However, this effect was inhibited whenLCPAT1was silenced (Figure 4C and 4D). Furthermore, the assembly of rapamycin-inducedLC3Bpuncta was inhibited inLCPAT1knockdown cells based on immunofluorescence assays (Figure 4E and 4F). These experiments showed thatLCPAT1regulated cell autophagyin vitro.

Discussion

Following our previous work on lncRNALCPAT1, we further analyzed its relationship with autophagy in lung cancer in the current study. We found a strong positive correlation betweenLCPAT1andLC3B. Since high expression ofLC3Bindicates increased autophagy17, we speculate thatLCPAT1may promote autophagy in lung cancer. We investigated the role ofLCPAT1in autophagy by knocking downLCPAT1expression in tumor cells and assessing its impact on autophagyin vitroandin vivo. Our results demonstrated thatLCPAT1could increase autophagy in lung cancer.

Figure 2 LCPAT1 knockdown xenograft models. (A) Upper: LCPAT1-shRNA (sh LCPAT1)- and control shRNA (shcontrol)-transfected H1975 cells were implanted in flanks of mice (n = 6). The red arrow indicates the tumors implanted with shLCPAT1-transfected H1975 cells on the left side, and the black arrow indicates the tumors implanted with shcontrol-transfected H1975 cells on the right side. Lower: tumors expressing shLCPAT1 or shcontrol. (B) Tumor growth curve. Data are presented as mean ± SEM; ＊P ＜ 0.05, ＊＊P ＜ 0.01, ＊＊＊P ＜ 0.001. (C) H &E staining of xenografted tumor tissues of shLCPAT1- and shcontrol-transfected H1975 cells (Left: 100 ×. Right: 200 ×).

More and more lncRNAs are being found to be involved in tumorigenesis. In lung cancer,HOTAIRis up-regulated and the up-regulation promotes tumor cell invasion and metastasis23. In contrast toHOTAIR,MEG3is significantly down-regulated in lung cancer; increasing its expression in lung cancer cells could inhibit cell proliferation and induce apoptosis24. Considering that a large number of lncRNAs are known and only a few are recognized for their involvement in lung cancer, we previously performed an expression microarray analysis to identify dysregulated lncRNAs in lung cancer5. A methylation microarray chip was also used to determine the status of DNA methylation in the same samples. Among 8500 differentially-expressed lncRNAs,expression ofLOC146880andLCPAT1was found to be elevated in tumors and the expression was negatively correlated with DNA methylation in the promoter. We further noticed that cell proliferation, invasion, and migration were inhibited when lowering the expression ofLOC146880orLCPAT1in lung cancer cells5. In the present study, our animal experiments further confirmed that suppressingLCPAT1expression by shRNA could inhibit tumor growth in a xenograft model.

Autophagy plays a complicated role in tumorigenesis.Autophagy can suppress cancer initiation during the early stage of tumorigenesis. Karantza-Wadsworth et al.25reported that a defect in autophagy led to DNA damage, genomic instability, and further promoted the activation of protooncogenes and resulted in tumorigenesis. In contrast,autophagy promotes the growth of established cancer25. In malignant glioma, autophagy can be activated by hypoxia,thereby promoting tumor cell growth through the HIF-1α/AMPK signaling pathway26. Our finding of high expression ofLC3Bin lung cancer supports the notion that autophagy promotes cancer progression. We also found thatLCPAT1could increase autophagy in lung cancer, suggesting thatLCPAT1may act as an oncogene by promoting autophagy in lung cancer.

Figure 3 Autophagy regulation by LCPAT1 in vivo. (A) qPCR analysis of the expression of LCPAT1 in stable-transfected H1975 cell line.Data are presented as mean ± SEM; ＊＊P ＜ 0.01. (B) qPCR analysis of the expression of LCPAT1 in xenografted tumor tissues. Data are presented in mean ± SEM; ＊＊＊P ＜ 0.001 (n = 6). (C) qPCR analysis of the expression of LC3B, ATG3, ATG5, ATG12, ATG14, and BECLIN1 in shLCPAT1 and shcontrol xenografted tumor tissues (n = 6). Data are presented in mean ± SEM; ＊P ＜ 0.05, ＊＊P ＜ 0.01, ＊＊＊P ＜ 0.001. (D)Representative image and quantification of Western blot assays for p62, ATG5, BECLIN1, and LC3B expression in shLCPAT1 and shcontrol xenografted tumor tissues (n = 4). Nos.1-4 represent the number of mice. Data are presented in mean ± SEM; ＊P ＜ 0.05. (E) Representative image (400 ×) and quantification of immunohistochemical assays for LC3B and ATG5 expression in shLCPAT1 and shcontrol xenografted tumor tissues using Image Pro-plus 6.0 software (n = 4). Data are presented in mean ± SEM; ＊P ＜ 0.05, ＊＊P ＜ 0.01.

Figure 4 Autophagy regulation by LCPAT1 in vitro. (A, B) mRNA expression of autophagy-related genes in A549 cells (A) and H1975 cells(B) transfected with LCPAT1 siRNA (siLCPAT1) and scrambled siRNA (sicontrol). Data are presented as mean ± SEM; ＊P ＜ 0.05, ＊＊P ＜ 0.01,＊＊＊P ＜ 0.001. (C, D) Representative images and quantification of Western blot assays for p62, ATG5, BECLIN1, and LC3B expression in siLCPAT1- and sicontrol-transfected A549 cells (C) and H1975 cells (D), after exposure to 100 μM rapamycin for 48 h. Data are presented in mean ± SEM; ＊P ＜ 0.05, ＊＊P ＜ 0.01. (E, F) Representative images and quantification of LC3 dots in siLCPAT1- and sicontrol-transfected A549 cells (E) and H1975 cells (F) after treatment with 100 μM rapamycin for 48 h. The LC3B puncta were quantified by Image Pro-plus 6.0 software. Data are presented in mean ± SEM; ＊P ＜ 0.05, ＊＊P ＜ 0.01, ＊＊＊P ＜ 0.001.

The molecular mechanisms underlying howLCPAT1regulates autophagy are unclear. LncRNAs regulate the expression of genes through various mechanisms, including interacting with DNA, mRNAs, microRNAs, or proteins. In breast cancer,linc-RORhas been shown to suppress gemcitabine-induced autophagy by silencing miR-34a27.Through binding to EZH2, lncRNAH19down-regulates DIRAS3 expression, consequently inhibiting autophagy in cardiomyocytes28. Wu et al.29has reported that the lncRNAHNF1A-AS1promotes autophagy in hepatocellular carcinoma by composing a competitive endogenous RNAs system with miR-30b, which targetedATG529. We used starBase (http://starbase.sysu.edu.cn/)30and lncRNASNP(http://bioinfo.life.hust.edu.cn/lncRNASNP/)31databases to predict whether there were miRNA binding sites forLCPAT1and autophagy-related genes. The results showed that mir-186-5p binds toLCPAT1,ATG7, andATG12. In addition,mir-206 can be combined withLCPAT1andATG14at the same time, and mir-218-5p can be combined withLCPAT1,ATG12, andATG14at the same time. Studies have shown that mir-186-5p, mir-206, and mir-218-5p are all tumor suppressors32-34. We suspect thatLCPAT1can compete with autophagy-related genes for specific anti-cancer miRNAs,thereby elevating autophagy gene levels and exerting tumorigenic effects. The current study was an initial investigation into the role played byLCPAT1in lung cancer.Further studies are warranted to explore the mechanisms ofLCPAT1in regulating autophagy in lung cancer.

In conclusion, we confirm thatLCPAT1is a tumorpromoting lncRNA in lung cancer. We also found thatLCPAT1could increase autophagy bothin vitroandin vivoin lung cancer.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (Grant No. 81401046 and No.21777099), Shanghai Jiao Tong University Interdisciplinary Research Key Grant (Grant No. YG2015ZD01) and Shanghai Jiao Tong University “New Young Teachers Startup Plan” .

Conflict of interest statement

No potential conflicts of interest are disclosed.