Background
In the past decades, lung cancer has been the leading cause of cancer-related death worldwide [
1]. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which consists of two most common histological types, squamous cell carcinoma and adenocarcinoma. To date, the improvements in the treatment of lung cancer have been achieved by the development of combined treatments, such as surgical resection, systemic chemotherapy and targeted drugs. However, the overall five-year survival rate of NSCLC remains unsatisfactory [
2]. Therefore, to develop more effective treatment methods, it is urgent to fully discover the genetic and molecular features of NSCLC.
Recently, evidence has shown that at least 90 % of the total mammalian genome is actively transcribed [
3]. However, only approximately 1.5 % of the genome sequence comprise protein-coding genes [
4]. Non-protein-coding RNA (ncRNA) transcripts consist of >98 % of the mammalian transcriptome and were once thought to be “junk” or “transcription noise”. Recent evidence has proven that ncRNAs—for example microRNAs—play significant roles in various biological processes [
4‐
6]. Long noncoding RNAs (lncRNAs), ncRNAs larger than 200 nucleotides, play a crucial role in diverse cellular processes such as cell growth [
7], differentiation [
8], the immune response [
9], and cancer metastasis [
10‐
12].
By analyzing a published lncRNA microarray dataset of NSCLC [
13], we found that the novel lncRNA SBF2 antisense RNA 1 (SBF2-AS1) was significantly upregulated in NSCLC tissues compared with the corresponding non-tumor tissues. SBF2-AS1 is a 2708 nt antisense RNA to SBF2, which is located at the 11p15.1 locus. However, the expression profile and potential function of SBF2-AS1 in NSCLC remain unknown. In the present study, we validated the upregulation of SBF2-AS1 in NSCLC and found that a high expression level of SBF2-AS1 was correlated with advanced TNM stage. Using small interfering RNA (siRNA)-mediated silencing of SBF2-AS1, NSCLC cell proliferation was inhibited both in vivo and in vitro.
Methods
Patients and tissue samples
Primary NSCLC tissues and adjoining normal tissues were collected from patients received surgical resection of NSCLC from 2012 to 2014 at the Department of Thoracic Surgery, Cancer Institute of Jiangsu Province. All patients did not receive radiotherapy or chemotherapy before surgical resection. All tumor specimens and adjoining normal specimens were snap-frozen immediately after resection, and stored in liquid nitrogen until total RNA extraction. All tumor and paired normal tissues were verified by experienced pathologists. Clinical characteristics were also collected for each patient, and informed written consents were obtained from all patients included in this research. This study was approved by the Ethics Boards of the Cancer Institute of Jiangsu Province.
Cell lines and culture conditions
All cell lines (A549, NCI-H1975, NCI-H358, NCI-H1299, SPC-A1, and human bronchial epithelial cell (HBE)) were purchased from Shanghai Institutes for Biological Science, China. NCI-H1975, A549 and NCI-H1299 cells were cultured in RPMI 1640 medium (KeyGene, Nanjing, China), NCI-H358, SPC-A1, and HBE cells were cultured in DMEM medium (KeyGene, Nanjing, China), supplemented with 10 % fetal bovine serum with 100U/ml penicillin and 100 mg/ml streptomycin included. All cell lines were grown in humidified air at 37 °C with 5 % CO2.
RNA extraction and qRT-PCR analyses
Total RNA was isolated with TRIzol reagent (Life Technologies, Scotland, UK) according to the manufacturer’s protocol. About 1.0ug total RNA was reverse transcribed in a final volume of 20ul using the PrimeScript RT Master Mix (Takara, Cat.#RR036a) according to the manufacturer’s protocol. After reverse transcription, the quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using the SYBR Select Master Mix (Applied Biosystems, cat: 4472908) with 0.5ul cDNA on ABI 7900 system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The relative levels of SBF2-AS1 were confirmed by qRT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were measured as internal controls. The qRT-PCR reaction was implemented under the following conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The fold changes of individual genes were calculated by 2-ΔΔCt methods [
14]. QRT-PCR primers used were: 5′-GGACTAGTGGAGAAGGTGCG-3′ (Forward) and 5′-GGGCGCTGCCCATCATCATG-3′ (Reverse) for P15, 5′-AAACTTGGAAATCCCGAGATTGC-3′ (Forward) and 5′-CGAAACCAGTTCGGTCTTTCAA-3′ (Reverse) for P18, 5′-AGACCATGTGGACCTGTCACTG-3′ (Forward) and 5′-GTTTGGAGTGGTAGAAATCTGTC-3′ (Reverse) for P21, 5′-TGCAACCGACGATTCTTCTACTCAA-3′ (Forward) and 5′-CAAGCAGTGATGTATCTGATAAACAAGG-3′ (Reverse) for P27, 5′-CACCGAATAGTTACGGTCGG-3′ (Forward) and 5′-GCACGGGTCGGGTGAGAGTG-3′ (Reverse) for P16, 5′-AGGCTGACCACGAGCTTTTC-3′ (Forward) and 5′-GGTGCTATGAGATTCCGAGTTC-3′ (Reverse) for SUZ12, 5′-GAAATCGTGCGTGACATTAA-3′ (Forward) and 5′-AAGGAAGGCTGGAAGAGTG-3′ (Reverse) for β-actin, and 5′-CCACATCGCTCAGACACCAT -3′ (Forward) and 5′-ACCAGGCGCCCAATACG -3′ (Reverse) for GAPDH.
Western blot assay
Cells were harvested, and protein was extracted from transfected cells and quantified as previously described49 using 12 % or 4 ~ 20 % polyacrylamide gradient SDS gel. Anti-β-actin and anti-SUZ12 were from Abcam (Hong Kong, China). Anti-P21 and anti-Cyclin D1 were from Cell Signaling Technology (Boston, MA, USA).
siRNA and plasmid transfection of NSCLC cells
A549 and H1975 cells were planted in six-well plate 24 h before transfection. When they were about 70 % confluent, cells were transfected with siRNA targeting specific genes or negative control (RealGene, Nanjing, China) by using the Lipofectamine RNAimax reagent (Invitrogen, USA) according to the protocol provided by the manufacturer. The siRNA sequences for SBF2-AS1 were 5′-CAGAAGGAGUCUACUGCUAAG-3′ (Sense) and 5′-UAGCAGUAGACUCCUUCUGGG-3′ (Antisense) for 204 site and 5′-GCAAGCCUGCAUGGUACAUTT -3′ (Sense) and 5′-AUGUACCAUGCAGGCUUGCTT -3′ (Antisense) for 1021 site. The siRNA sequences for SUZ12 were 5′-GUCGCAACGGACCAGUUAATT-3′ (Sense) and 5′-UUAACUGGUCCGUUGCGACTT-3′ (Antisense).
The SBF2-AS1 sequence was synthesized according to the full-length SBF2-AS1 sequence (based on the GAS5 sequence, NR_002578, in NCBI) and then subcloned into a pCDNA3.1 vector (Invitrogen, Shanghai, China). The pCDNA-GAS5 or empty vector was transfected into SPC-A1 cells using Lipofectamine 3000 reagent (Invitrogen, USA), according to the manufacturer’s instructions. The empty pcDNA3.1 vector was used as the control.
Cell proliferation assay
Cell proliferation was assayed by Cell Counting Kit-8 (CCK8) assay (Promega). The transfected cells were plated in 96-well plates (4000cells per well) 24 h after transfection and cultured at 37 °C and 5 % CO2 atmosphere. CCK8 assay was used to detect the relative cell growth every 24 h according to the instructions of manufacturer. Simply, 20ul of CCK8 solution was added to each well, and each well was measured spectrophotometrically at 450 nm after incubating for 2 h.
Cell migration and invasion assays
For migration assay, transfected cells (3 * 105) were plated in the upper chamber of transwell assay inserts (8 mm pores, Millipore, Billerica, MA) containing 200ul of serum-free 1640 medium. The lower chambers were filled with 1640 containing 10 % FBS. After 24 h of incubation, the cells on the filter surface were fixed with methanol, stained with crystal violet, and photographed. Migration was assessed by counting the number of stained cell nuclei from 5 random fields per filter in each group.
For invasion assay, transfected cells (5 * 105) were plated in the top chamber with a matrigel-coated membrane (BD Biosciences) in 500ul serum-free 1640. Also, the bottom chambers were filled with conditioned 1640. The invasion function was determined after incubating 48 h as mentioned previously in migration.
Flow-cytometric analysis
Transfected cells were harvested after transfection by trypsinization. After the double staining with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide was done by the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s recommendations. The cells were analyzed with a flow cytometry (FACScan; BD Biosciences) equipped with a Cell Quest software (BD Biosciences). Cells for cell-cycle analysis were stained with propidium oxide by the Cycle TEST PLUS DNA Reagent Kit (BD Biosciences) following the protocol and analyzed by FACScan. The percentage of the cells in G1, S, and G2–M phase were counted and compared.
RNA immunoprecipitation (RIP)
RIP experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Antibodies of EZH2 and SUZ12 were from Abcam.
Chromatin immunoprecipitation (ChIP) assays
The ChIP assays were performed using EZ-CHIP KIT according to the manufacturer’s instruction (Millipore, Billerica, MA, USA). H3K27 antibody was from Millipore. The ChIP primers for the promoter region of P21 were as follows: 5′-GCCTTCCTCACATCCTCC-3′ (Forward) and 5′-CAAGAGTGCCCAGTCCAG-3′ (Reverse).
Xenograft experiment
Transient transfection was performed in A549 cells with shLUADT1 or scrambled control sequence using Lipofectamine 2000 (Invitrogen). After 48 h of transfection, the cells were collected and injected into either side of the posterior flank of the same male BALB/c nude mouse. The tumor volumes and weights were measured every 2 days in the mice; the tumor volumes were measured as length × width2 × 0.5. Sixteen days after injection, the mice were sacrificed, the tumor weights were measured, and the tumors were collected for further analysis. The LUADT1 levels were determined by qRT-PCR.
Immunohistochemistry
Xenograft tumor tissue samples were immunostained for p27 and Ki67. Anti-Ki67 was from Santa Cruz Biotechnology.
Statistical analysis
Student’s t-test, one-way ANOVA analysis, and Spearman test were performed to analyze the data using SPSS 18.0 software. P < 0.05 was considered statistically significant.
Discussion
In the current study, we characterized the expression profile of SBF2-AS1 in NSCLC and found that high expression of SBF2-AS1 was associated with advanced TNM stage. By the silencing of SBF2-AS1, the cell proliferation and metastasis ability was inhibited in vitro. The silencing of SBF2-AS1 also inhibited xenograft tumor growth in vivo. Thus, SBF2-AS1 could potentially function as an oncogenic gene in NSCLC.
The mortality rate of lung cancer is higher in males than in females [
17]. However, the precise molecular mechanism involved in lung carcinogenesis remains elusive. It has been widely accepted that many lncRNAs transcribed from the human genome have play broad roles in lung cancer [
18]. Numerous studies have suggested that lncRNAs are involved in various malignant tumors, such as those of the brain [
19,
20], lung [
11,
21], breast [
22], pancreas [
23,
24] and liver [
25]. This extends our knowledge concerning carcinogenesis and provides a new pathway in cancer research. For lung cancer, various lung cancer-specific lncRNAs have been identified, such as MALAT1 [
26,
27], TARID [
28], and LUADT1 [
29].
SBF2-AS1 is a novel lncRNA transcribed from chromosome 11p15.4, a finding that has not been reported previously. Firstly, we explored the expression of SBF2-AS1 in NSCLC tissues and analyzed the relationship between the SBF2-AS1 expression level and clinical characteristics, such as gender, tumor size, and TNM stage. As indicated by the microarray data, SBF2-AS1 was upregulated in NSCLC tissues compared with adjacent non-tumor tissues, and the expression was very homogeneous. Similarly, SBF2-AS1 was overexpressed in the 4 NSCLC cell lines analyzed. Statistical analyses revealed that the overexpression of SBF2-AS1 was associated with lymph node metastasis and advanced TNM stage, indicating that SBF2-AS1 could be a biomarker for NSCLC and might be a prognostic factor for survival. The predictive value of SBF2-AS1 should be validated by more experimental evidence.
After siRNA-mediated silencing of SBF2-AS1, we found that SBF2-AS1 could modulate cell proliferation and overexpression of SBF2-AS1 increased proliferation ability of NSCLC cells. In recent years, some lncRNAs, including growth-arrest-specific transcript 5 [
30] (GAS5), prostate-specific gene 1 (PCGEM1) [
31], prostate-cancer-associated transcript 1 (PCAT-1) [
32], and colon cancer-associated transcript 2 (CCAT2) [
33], have been reported to regulate tumor cell growth and progression by altering the balance between cell proliferation and apoptosis. In the present study, we also found that SBF2-AS1 influenced tumor cell proliferation by affecting cell cycle distribution. The flow cytometric analysis suggested that the cell cycle was arrested at the G1 phase after transfection with siRNA. According to Khalil et al., most lncRNAs could bind to the PRC2 complex and negatively regulate gene expression at the transcription level [
16], such as the lncRNAs HOTAIR [
34,
35] and TUG1 [
36]. We found that SBF2-AS1 could also bind to the PRC2 complex, particularly the subunit SUZ12. ChIP assay revealed that the silencing of SBF2-AS1 decreased the enrichment of SUZ12 and H3K27 me3 at the promoter region of P21. Thus, these experiments demonstrated that SBF2-AS1 could modulate the cell cycle through epigenetic inhibition of P21.
Metastasis is another important malignant behavior of cancer and is the most troublesome problem in tumor prognosis and therapy. lncRNAs involved in the regulation of tumor metastasis have also been reported, and include MALAT-1 [
26] and HOX antisense intergenic RNA (HOTAIR) [
35]. Our study suggested that the migration and invasion ability of NSCLC cells were significantly decreased by the silencing of SBF2-AS1, suggesting SBF2-AS1 is also an important regulator of metastasis.
Conclusion
In this study, we identified a novel lncRNA SBF2-AS1 in NSCLC that is upregulated and correlated with advanced TNM stage. SBF2-AS1 could promote proliferation of NSCLC cells in vitro and in vivo, suggesting that SBF2-AS1 might be an oncogenic lncRNA in NSCLC.
Ethics approval
This study was approved by the Ethics Boards of the Cancer Institute of Jiangsu Province. Informed written consents were obtained from all patients included in this research.
Competing interests
The authors declared that they have no competing interests.
Authors’ contributions
MTQ, LX, KPX, and RY conceived the study. MTQ and RY designed the study. MTQ, JW, and RY coordinated the study. JL, CL, WJX, YTX, JW, and XCL performed most experiments and the statistical analyses. SH, RZ, MZ, and FQJ obtained the clinical parameters. MTQ and RY drafted the manuscript. LX, RY, JW, and MTQ provided funds. All authors read and approved the final manuscript.