Background
Lung cancer is the most frequent cause of cancer-related death in worldwide [
1,
2]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer newly cases and can be categorized into two common subtypes, squamous cell carcinoma, adenocarcinoma and large cell carcinoma [
3]. Even though current advances in the chemotherapy and molecular targeting therapy for NSCLC, the overall 5-year survival rate for the disease is less than 15% due to the limited therapeutic options, tumor metastasis, and recurrence [
4]. Undoubtedly, better understanding of the carcinogenesis is critical for the advance of diagnostic markers and aid novel effective therapies for NSCLC patients.
In recent years, it has become increasingly apparent that the noncoding portion of the genome is of crucial functional importance in both normal physiology and diseases [
5]. Long non-coding RNAs (lncRNAs) are classified as a new kind of non-coding RNA (ncRNA) that is longer than 200 nucleotides in length with no or limited protein-coding potential [
6,
7]. Increasing studies have shown that lncRNAs are pervasively involved in many biological processes, including cellular development, differentiation, apoptosis, inflammation, autophagy, and cancer [
8‐
12]. In addition, an emerging paradigm of the aberrant lncRNAs has been found to participate in NSCLC development and progression. For example, the lncRNA MALAT1 is a highly conserved nuclear ncRNA and a predictive marker for metastasis development in lung cancer [
13]. Elevated LINC00473 expression correlates with poor prognosis, and elevated LINC00473 serve as a robust biomarker for LKB1-inactivated NSCLC [
14]. In this regard, identifying the associated molecular mechanisms of cancer-associated lncRNAs is necessary for understanding progression and establishing better treatment of NSCLC.
LncRNA actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), a 6.8-kb lncRNA that is located in the chromosome 4p16.1, was initially identified by Wu et al. and was extremely hypomethylated and overexpressed in Barrett esophagus (BE) and esophageal adenocarcinoma (EAC) tissues and cells [
15]. In addition, recently studies showed that increased expression of AFAP1-AS1 could participate in the progression of a variety of tumors, including nasopharyngeal carcinoma, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, colorectal cancer and gallbladder cancer [
16‐
20]. These results indicate that AFAP1-AS1 may be necessary for development, and that its dysregulation may participate in human cancer progression. Although, Zhang et al. found that knockdown of AFAP1-AS1 inhibits tumor cell growth and invasion in lung cancer [
21]. However, the biological functions of AFAP1-AS1 in the control of NSCLC tumorigenesis have not been well characterized. Especially, the molecular mechanism and global genes that were mediated by AFAP1-AS1 had not yet been established. These prompted us to explore the role of AFAP1-AS1 in human NSCLC.
In this study, we investigated the potential molecular mechanisms of AFAP1-AS1 on NSCLC progression. We found that AFAP1-AS1 was significantly upregulated in NSCLC tissues compared with normal lung tissues, and may serve as an independent predictor for the overall survival in NSCLC. In addition, AFAP1-AS1 could regulate cell proliferation both in vitro and in vivo. Further experiments demonstrated that AFAP1-AS1 was associated with EZH2 and that this association was required for the epigenetic repression of p21, which encodes a potent cyclin-dependent protein kinase inhibitor (CKI), thus contributing to the regulation of both the NSCLC cell cycle and proliferation. Taken together, the findings may provide new insights into the critical role of the lncRNA AFAP1-AS1 in human NSCLC tumorigenesis.
Methods
Tissue collection and ethics statement
We obtained paired NSCLC and adjacent normal lung tissues from 92 patients who underwent primary surgical resection in the Department of Thoracic Surgery, zhongda Hospital, Southeast University School of Medicine, China. No patient had received local or systemic treatment before any operation. All collected tissue samples were immersed in RNA Later stabilization solution (Qiagen) and were immediately frozen in liquid nitrogen and stored at − 80 °C until RNA isolation. The clinicopathologic characteristics of the patients with NSCLC were summarized in Table
1. Our study protocol was approved by the Institutional Review Board of Southeast University, and all of the participants signed an informed consent form.
Table 1
The clinic-pathological factors of 92 NSCLC patients
Sex | | | | 0.402 |
male | 42 | 23 | 19 | |
female | 50 | 23 | 27 | |
Age | | | | 0.835 |
≤60 | 45 | 22 | 23 | |
> 60 | 47 | 24 | 23 | |
Histological grade | | | | 0.179 |
Middle or low | 75 | 35 | 40 | |
high | 17 | 11 | 6 | |
Histological classification | | | | 0.532 |
SCC (Squamous cell carcinoma) | 47 | 25 | 22 | |
AD (adenocarcinoma or other) | 45 | 21 | 24 | |
TNM stage | | | | 0.182 |
Iand II | 62 | 34 | 28 | |
III and IV | 30 | 12 | 18 | |
Lymph node metastasis | | | | 0.529 |
negative | 41 | 22 | 19 | |
positive | 51 | 24 | 27 | |
Tumor size | | | | < 0.001** |
≤3 cm | 36 | 28 | 8 | |
> 3 cm | 56 | 18 | 38 | |
History of smoking | | | | 0.144 |
Ever | 49 | 28 | 21 | |
Never | 43 | 18 | 25 | |
Cell culture
Cell lines (16HBE, A549, SPC-A1 and H1299) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI Medium 1640 basic media (GIBCOBRL, Invitrogen, Carlsbad, CA), and (DMEM; GIBCO-BRL, Invitrogen) in a humidified incubator at 37 °C with 5% CO2. All of the media used was supplemented with heat-inactivated 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Invitrogen).
RNA extraction and qRT-PCR analyses
The total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen), according to the manufacturer’s protocol. One microgram total RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). cDNA was used for subsequent qRT-PCR reactions (SYBR, TaKaRa) according to the manufacturer’s instructions. The results were normalized to the expression of GAPDH. The qRT-PCR and data collection were carried out on ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences are summarized in Additional file
1: Table S1.
Subcellular fractionation location
The separation of the nuclear and cytosolic fractions was performed using the PARIS Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Transfection of cell lines
NSCLC cell lines were transfected with specific siRNA oligonucleotides. Typically, cells were seeded at six-well plates and then transfected the next day with specific siRNA (100 nM) and control siRNA (100 nM) by using Lipofectamine 2000, according to the manufacturer’s protocol (Invitrogen). The sequences for siRNAs were listed in Additional file
1: Table S1.
Cell proliferation assays
Cell proliferation was monitored using Cell Proliferation Reagent Kit I (MTT) (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) (Roche, Basel, Switzerland). The transfected cells were plated in 96-well plates (3000 cells/well). Cell proliferation was determined every 24 h following the manufacturer’s protocol. For the colony-formation assay, a certain number of transfected cells were placed into each well of a six-well plate and maintained in media containing 10% FBS for 2 weeks, replacing the medium every 4 days. Colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min. The colony formation was determined by counting the number of stained colonies. Triplicate wells were measured in each treatment group. BrdU experiments were performed using a BrdU Cell Proliferation Assay Kit (Millipore, Cat.No.2750) according to the manufacturer’s instructions. The higher OD reading represents the higher BrdU concentration in the sample.
Flow cytometric analysis
Cells for cell-cycle analysis were stained with propidium oxide by the CycleTEST PLUS DNA Reagent Kit (BD Biosciences) following the protocol and analyzed by FACScan. The percentage of the cells in G0–G1, S, and G2–M phase were counted and compared.
Xenograft study
A549 cells were stably transfected with shRNA and empty vector and harvested from cell culture plates, then cells were xenografted into BALB/c male nude mice. The tumor volumes and weights were measured every three days in mice; the tumor volumes were measured as length×width
2 × 0.5. Twenty-one days after injection, the mice were killed and tumor weights were measured and used for further analysis. Immunohistochemistry was performed as previously described [
22]. Anti-Ki-67 was from Abcam.
Whole transcriptome deep sequencing
Total RNA from the A549 cells with AFAP1-AS1 knockdown and control A549 cells were isolated and quantified. The complementary DNA (cDNA) libraries for single-end sequencing were prepared using Ion Total RNA-Seq Kit v2.0 (Life Technologies) according to the manufacturer’s instructions. The cDNA libraries were then processed for the Proton Sequencing process according to the commercially available protocols. Samples were diluted and mixed, the mixture was processed on a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) for preparing the template-positive Ion PI™ Ion Sphere™ Particles (Life Technologies) according to Ion PI™ Template OT2 200 Kit v2.0 (Life Technologies). After enrichment, the mixed template-positive Ion PI™ Ion Sphere™ Particles of samples was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies) by NovelBio Corp. Laboratory, Shanghai. Data are available in Additional file
2: Table S2.
Western blot assay
The cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche) and PMSF (Roche). Fifty micrograms of the protein extractions were separated by 10% SDS-PAGE transferred to 0.22 mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies.The autoradiograms were quantified by densitometry (Quantity One software; Anti-p21 was from Abcam. Results were normalized to the expression of GAPDH.
RNA immunoprecipitation
RNA immunoprecipitation (RIP) experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Antibodies for RIP assays against EZH2 and SUZ12 were purchased from Abcam.
ChIP assays
The ChIP assays were performed using the EZ-CHIP KIT according to the manufacturer’s instructions (Millipore, Billerica, MA, USA). EZH2 and SUZ12 antibodies were obtained from Abcam (Hercules, CA, USA). H3 trimethyl Lys 27 antibody was purchased from Millipore. Quantification of immunoprecipitated DNA was performed using qPCR with SYBR Green Mix (Takara). The ChIP data were calculated as a percentage relative to the input DNA using the eq. 2[Input Ct- Target Ct] × 100 (%).
Statistical analysis
Statistical analysis was performed using the SPSS software package (version 17.0, SPSS Inc., Armonk, NY, USA) and GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Statistical significance was tested by a Student’s t-test or a Chi-square test as appropriate. Survival analysis was performed using the Kaplan–Meier method, and the log-rank test was used to compare the differences between patient groups. Statistics with P-value < 0.05 were considered as statistically significant.
Discussion
Recently, it is becoming clear that mammalian genomes encode thousands of lncRNAs [
13,
31]. To date, increasing evidence links dysregulation of lncRNAs to diverse human diseases especially in tumorigenesis [
14,
32]. Our previous studies also revealed that lncRNA CCAT1 could regulate cell proliferation and migration in esophageal squamous cell carcinoma [
33]. And TUG1 play an important role in NSCLC [
34]. In this study, we discovered an lncRNA AFAP1-AS1 whose elevated expression in NSCLC tissues was highly associated with the tumor suppressor p21. Moreover, we showed that increased AFAP1-AS1 expression was associated with a poor prognosis and shorter survival time in NSCLC patients. Our gene expression and functional data strongly support the potential utility of AFAP1-AS1 as a biomarker and as a therapeutic target for NSCLC.
AFAP1-AS1 is the first identified lncRNA that is overexpressed in primary BE and EAC tissues as well as in EAC cell lines [
15]. Besides, knockdown of LncRNA AFAP1-AS1 in GBC cells inhibited EMT by upregulating the E-cadherin and downregulating the transcription factor Twist1 and Vimentin [
20]. Furthermore, AFAP1-AS1 can promote HCC development through upregulation of RhoA/Rac2 signaling and provide a potential therapeutic target for HCC [
18]. Recently, Deng J et al. found that AFAP1-AS1 may be an independent prognostic marker for NSCLC [
35]. Collectively, these studies have revealed that AFAP1-AS1 has an important role in tumor carcinogenesis and acts as an oncogenic lncRNA. But, the biological functions of AFAP1-AS1 in the control of NSCLC tumorigenesis have not been well characterized. Especially, the molecular mechanism and global genes that were mediated by AFAP1-AS1 remain unknown. Our study suggested that AFAP1-AS may have an oncogenic function in NSCLC progression.
Generally, lncRNAs exert their function through interacting with various RNA binding proteins (RBPs) and leading to inactivation or activation of gene expression via chromosome reprogramming, DNA methylation, RNA decay and histone protein modification [
36,
37]. HOTAIR is one of the most studied lncRNAs involved in chromatin modification, which can recruit PRC2 genome-wide to alter H3K27 methylation and gene expression patterns [
28,
38]. It is evident that more than 20% of lncRNAs are bound by PRC2 in various cells, and silence downstream target genes [
25]. It is well documented that PRC2 is a critical regulator of histone modification, which catalyzes the trimethylation of H3K27 to mediate gene silencing. Recent findings implicate that PRC2 is an important driver of tumor development and progression by suppressing various key genes. EZH2 is a core subunit of PRC2 complex that could catalyze the trimethylation of lysine residue 27 of histone 3 (H3K27me3), which is over-expressed in multiple cancers [
39]. In this study, RIP assays confirmed that AFAP1-AS1 could bind to EZH2 in NSCLC cells.
RNA-Seq data showed that p21 might be downstream mediators of AFAP1-AS1. P21, is one of the most crucial member of the cyclin-dependent kinase (CDK) inhibitor family for G1/S transition [
40‐
42]. Additionally, substantial evidence from biochemical and genetic studies indicates that p21 acts as a master molecule of multiple tumor suppressor pathways for promoting anti-proliferative activities in cancer cells [
43,
44]. Notably, Our RNA-seq data found that p21 was significantly upregulated after AFAP1-AS1 knockdown. In addition, ChIP assays validated that knockdown of AFAP1-AS1 resulted in the loss of H3K27 trimethylation and EZH2 binding to the genomic loci of p21, confirming that p21 was a bona target of AFAP1-AS1/EZH2-regulated genes. Our findings demonstrated that AFAP1-AS1 promotes lung cancer cell proliferation by epigenetically repressing p21 expression.
Conclusions
In summary, we had evidenced that expression of the lncRNA AFAP1-AS1 is upregulated in the NSCLC tissues and cells, and increased AFAP1-AS1 is associated with poor prognosis of NSCLC patients. The loss of AFAP1-AS1 inhibited NSCLC cell proliferation in vitro and suppressed tumor growth in vivo. Furthermore, AFAP1-AS1-mediated oncogenic effects occurred partially through epigenetic suppression of p21 expression by binding to EZH2. Our findings increase our understanding of the pathogenesis and progression of NSCLC and may facilitate the development of lncRNA-directed diagnostics and therapeutics in NSCLC.