Microarray profiles reveal that circular RNA hsa_circ_0007385 functions as an oncogene in non-small cell lung cancer tumorigenesis
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Circular RNAs (circRNAs) are a novel class of non-protein-coding RNA. Emerging evidence indicates that circRNAs participate in the regulation of many pathophysiological processes. This study aims to explore the expression profiles and pathological effects of circRNAs in non-small cell lung cancer (NSCLC).
Human circRNAs microarray analysis was performed to screen the expression profile of circRNAs in NSCLC tissue. Expressions of circRNA and miRNA in NSCLC tissues and cells were quantified by qRTPCR. Functional experiments were performed to investigate the biological functions of circRNA, including CCK-8 assay, colony formation assay, transwell assay and xenograft in vivo assay.
Human circRNAs microarray revealed a total 957 abnormally expressed circRNAs (> twofold, P < 0.05) in NSCLC tissue compared with adjacent normal tissue. In further studies, hsa_circ_0007385 was significantly up regulated in NSCLC tissue and cells. In vitro experiments with hsa_circ_0007385 knockdown resulted in significant suppression of the proliferation, migration and invasion of NSCLC cells. In vivo xenograft assay using hsa_circ_0007385 knockdown, significantly reduced tumor growth. Bioinformatics analysis and luciferase reporter assay verified the potential target miR-181, suggesting a possible regulatory pathway for hsa_circ_0007385.
In summary, results suggest hsa_circ_0007385 plays a role in NSCLC tumorigenesis, providing a potential therapeutic target for NSCLC.
KeywordsNon-small cell lung cancer Circular RNA Hsa_circ_0007385 Oncogene MiR-181
Non-small cell lung cancer (NSCLC) is a pulmonary malignant tumor that accounts for nearly 80% of lung cancers and is a leading cause of cancer-related death in China (Feng et al. 2017; Lee et al. 2017; Ma et al. 2017). With ongoing environmental degradation and pollution, the morbidity and mortality rate of NSCLC is increasing (Zhang et al. 2017b). Although a considerable amount of research has been done to advance clinical therapy, the prognosis and 5-year overall survival rates of NSCLC remain poor (Mei et al. 2017). Recent research examining molecular mechanisms involved in NSCLC has allowed for a novel understanding of NSCLC progression.
Circular RNAs (circRNAs) are a novel type of endogenous RNAs, characterized by covalently closed continuous loops (Greene et al. 2017; Weiser-Evans 2017). circRNAs are a result of splicing errors in the cell nucleus. With the rapid development of RNA sequencing (RNA-seq) and bioinformatics tools, the value of circRNAs in research have been renewed (Yang et al. 2017). Research shows that circRNAs are generated from back splicing and form into a loop structure, unlike linear transcripts and have the characteristics of tissue specificity, allowing them to be pathological diagnostic markers and therapeutic targets. Similar to long non-coding RNA, circRNAs can function as a miRNAs’sponge’ and play a role in the pathological environment (Shan et al. 2017). For example, circular RNA GLI2 is up-regulated in osteosarcoma tissue and cells, and promotes osteosarcoma cell proliferation, migration, and invasion by targeting miR-125b-5p (Li and Song 2017). In bladder cancer cells, circHIPK3 was significantly down-regulated, and targeted miR-558 suppressing the expression of heparanase (Li et al. 2017b).
This study investigated the expression profiles of circRNAs in NSCLC tissue and identified the oncogenic role of hsa_circ_0007385 in NSCLC cell progression.
Materials and methods
Eight patients with NSCLC, who underwent surgery without chemotherapy or radiotherapy at Cangzhou Central Hospital from Sep 2015 to Dec 2016, were enrolled in the study. Pairs of NSCLC tissue and adjacent non-cancerous lung tissue was excised from patients and immediately frozen for further analysis. The Ethics Committee of Cangzhou Central Hospital approved this study. Informed consent was obtained from all patients upon study enrollment.
Human circular RNA microarray
Human circRNA expression analysis was performed on three pairs of NSCLC tissue and adjacent non-cancerous lung tissue. Total RNA was extracted, and digested with Rnase R kit (Epicentre, Inc. Madison, WI, USA) to remove linear RNA. Human circRNA microarray hybridization was performed according to Arraystar standard protocols. The enriched circRNA was amplified to cDNA, and transcribed into cRNA using Arraystar Super RNA Labeling Kit (Arraystar, Rockville, MD, USA). Labeled cRNAs were then hybridized using Arraystar Human circRNA Array (8 × 15 K, Arraystar) and scanned by the Agilent Scanner G2505C.
Cell line and culture
The NSCLC cell lines (A549, SK-MES-1, H1299, and Calu-3) were purchased from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA). The normal human bronchial epithelial cells (NHBE) and HEK-293T cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% FBS (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA). All cells were cultured in a 5% CO2 humidified atmosphere at 37 °C.
RNA extraction and quantitative real-time PCR
Total RNA from the tissue samples and cells were collected using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using the ABI PRISM 7000 Fluorescent Quantitative PCR System according to the manufacturer’s instructions. The primer sequences in the study were shown as follows: divergent primers for hsa_circ_0007385, 5′-CGTGACCCAGAAGTGCGTTCACA-3′ (sense), 5′-TGGGGGTGTATCAGTCTTTGGTT-3′ (antisense); GAPDH, 5′-CCACATCGCTCAGACACCAT-3′ (sense) and 5′-ACCAGGCGCCCAATACG-3′ (antisense). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured as an internal control for the cell line. The Ct-value for each sample was calculated with the ∆∆Ct-method.
The small interfering RNAs were synthesized and purchased from GenePharma (Shanghai, China), including si-hsa_circ_0007385 and control. The sequences were as follows: si-has_circ_0007385, 5′-AAUAGAACACUUACUACUAAUCUGdTdT-3′ (sense) and 3′-dTdTAUUCCCUAGCCCUUCGAGUGTT-5′ (antisense). Small interfering RNAs (20 nmol/L) were transfected into the NSCLC cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s information.
Proliferation of NSCLC cells was performed using CCK-8 assay kit (Dojindo, Japan) according to manufacturer’s instructions. H1229 AND A549 cells were seeded in 96-well plate at density of 1 × 103 per well. Cells were then added to 10 μl CCK-8 solution at 37 °C for 90 min and incubated at 37 °C. The absorbance was measured at 450 nm. All experiments were repeated at three times.
Colony formation assay
Proliferation of NSCLC cells was performed using colony formation assay. H1229 AND A549 cells (1 × 103 cells per well) transfected with si-hsa_circ_0007385 were plated in 6-well culture plates and incubated at 37 °C for 14 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min, and the number of clones was manually counted.
Migration and invasion assay
Transwell assay was performed to measure migration and invasion. NSCLC cells (5 × 103) were added to the upper chamber and coated with 50 μL Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) 100 μL of serum-free medium for 24 h. The lower chamber was added with medium containing 10% FBS. After incubation, the migrated and invaded cells on the lower membrane surface were removed with a cotton swab, and fixed with 95% ethanol and stained with 0.2% crystal violet solution (Sigma) and counted.
The putative binding sites of miR-181 and hsa_circ_0007385 were subcloned into pGL3 luciferase promoter plasmid (Promega, Madison, WI, USA). HEK-293T cells were co-transfected with firefly luciferase reporter vector and the control vector contained Renilla luciferase using Lipofectamine 2000 (Invitrogen). Luciferase and Renilla signal was measured 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega). All experiments were performed in triplicate.
Xenograft assay in vivo
Athymic BALB/C mice (4–6-weeks-old) were purchased from the Chinese Academy of Sciences (Shanghai, China), and maintained in a specific pathogen-free facility. H1229 and A549 cells, stably transfected with lentivirus-mediated sh-hsa_circ_0007385 or vectors, were subcutaneously injected into the flanks of nude mice. Tumour size was measured every 4 days starting at the sixth day of injection. Tumour volume was harvested after sacrifice and calculated using a simplified Eq. (0.5 × length × width2).
Data were calculated from three independent experiments using SPSS 19.0 and presented as mean ± standard deviation (SD). Difference within two groups was analyzed using Student’s t test or one-way ANOVA or Pearson Chi-square test. Difference (P < 0.05) was considered to be statistically significant.
Human circular RNA microarray revealed the abnormally expressed circRNAs in NSCLC tissue
To investigate the abnormal expression of circRNAs, human circular RNA microarray analysis was performed in three pairs of NSCLC tissue and normal tissue. Results revealed there a total of 957 abnormally expressed circRNAs (> twofold, P < 0.05) in NSCLC tissue compared with normal tissue. CircRNAs were normalized and the most abnormal expressions were shown in a heat map (Fig. 1a). Three circRNAs, including hsa_circ_0020123, hsa_circ_001-235 and hsa_circ_0007385, were identified and their expression levels measured in five pairs of NSCLC tissue and normal tissue. Results showed expression of hsa_circ_0020123, hsa_circ_001-235 andhsa_circ_0007385 were significantly up-regulated in NSCLC tissue. Human circular RNA microarray revealed the abnormal circRNA expression profiles in NSCLC tissue, providing potential functional circRNAs in carcinogenesis.
Hsa_circ_0007385 knockdown suppressed the proliferation of NSCLC cells in vitro
Hsa_circ_0007385 was significantly up-regulated in NSCLC tissue; therefore, it was used as the target to investigate the role of circRNAs on NSCLC tumorigenesis (Fig. 2a). In vitro, the specifically synthesized siRNA targeting hsa_circ_0007385 was transfected into H1229 and A549 cells, and the expression of hsa_circ_0007385 was markedly down-regulated compared to control cells (Fig. 2b). CCK-8 and colony formation assay were performed in H1229 and A549 cells to determine NSCLC cell proliferation. Results showed that hsa_circ_0007385 knockdown suppresses the proliferation activity and number of clones in H1229 and A549 cells (Fig. 2c, d). The above in vitro experiments indicate that hsa_circ_0007385 knockdown suppresses the proliferation of NSCLC cells suggesting hsa_circ_0007385 plays a role in NSCLC tumorigenesis.
Hsa_circ_0007385 knockdown suppressed the migration, invasion of NSCLC cells in vitro
To further investigate the oncogenic role of hsa_circ_0007385 on NSCLC cells, transwell assay was performed in H1229 and A549 cells. Results revealed that hsa_circ_0007385 knockdown suppressed the migration and invasion of NSCLC cells compared with control groups (Fig. 3a–d). Thus, the transwell assay confirmed the oncogenic role of hsa_circ_0007385 on NSCLC cells in vitro.
Hsa_circ_0007385 knockdown inhibited the tumor growth of NSCLC in vivo
Previous studies have validated the oncogenic role of hsa_circ_0007385 in vitro. To further investigate this, the role of hsa_circ_0007385 on NSCLC tumor growth in vivo was measured using xenograft mice assay (Fig. 4a). H1229 cells and A549 cells transfected with lentivirus-mediated shRNA or control vectors were subcutaneously injected into the flanks of mice. Results showed that hsa_circ_0007385 knockdown significantly reduced NSCLC tumor volume (Fig. 4b, d) and tumour weight compared to control groups (Fig. 4c, e). In summary, results indicate that hsa_circ_0007385 knockdown inhibited the tumor growth of NSCLC in vivo.
MiR-181 acted as one of the target of hsa_circ_0007385
Presently, the well-known regulatory mechanism for circular RNAs is the miRNA ‘sponge’ (Hsiao et al. 2017). Using Arraystar software, it was predicted that hsa_circ_0007385 shared complementary binding sites with miR-181 (Fig. 5a). Previous studies report that miR-181 acts as tumor suppressor in NSCLC (Cao et al. 2017; Huang et al. 2015; Tian et al. 2016). Luciferase reporter assay revealed that miR-181 targeted hsa_circ_0007385 at a molecular level (Fig. 5b). In A549 cells transfected with si-hsa_circ_0007385, miR-181 expression was up-regulated compared with the control group (Fig. 5c). In 5 pairs of NSCLC tissue and control samples, miR-181 expression was down-regulated and negatively correlated with hsa_circ_0007385 expression (Fig. 5d). Schematic diagrams revealed the oncogenic pathway of hsa_circ_0007385 and miR-181 on NSCLC tumorigenesis. Taken together, bioinformatics analysis indicates that miR-181 acts as a target of hsa_circ_0007385, which may be the regulatory pathway for hsa_circ_0007385.
CircRNAs are a novel type of non-coding RNA closely associated with cancer development, progression, and metastasis (Yang et al. 2018, 2017). Studies have demonstrated the role circRNAs play in cancer tumorigenesis (Tang et al. 2017). The present study examined circRNA expression profiles in NSCLC tissue and validated the biologic function of hsa_circ_0007385 in NSCLC tumorigenesis.
With the rapid development of high-throughput sequencing technology, increasing numbers of new circRNAs that play a role in tumors, cardiovascular disease and metabolic disease are appearing (Dang et al. 2017; Liu et al. 2017). Human circRNA microarray analysis was used to screen circRNA expression profiles in NSCLC oncogenesis. Results found a total of 957 abnormally expressed circRNAs in NSCLC tissue compared with normal tissue. From these candidate circRNAs, hsa_circ_0007385 was chosen as the target of further research. Previously, high-throughput sequencing or human circRNAs microarray analysis has help researchers discover a large number of new circRNAs involved in other cancers, including hepatocellular carcinoma, glioma, and breast cancer (Chen et al. 2017; Gao et al. 2017). For example, in radiation-induced liver fibrosis, circRNA microarray revealed that 179 circRNAs were up-regulated and 630 circRNAs were down-regulated in irradiated hepatic stellate cells (Chen et al. 2017).
Given this, abnormally expressed circRNAs may take part in NSCLC tumorigenesis. A series of functional validation assays were performed to verify the hypothesis. Results showed that hsa_circ_0007385 knockdown suppressed the proliferation, migration and invasion of NSCLC cells in vitro, and reduced NSCLC tumor growth in vivo. This suggests that hsa_circ_0007385 plays a role in NSCLC genesis.
The role of circRNAs in carcinogenesis has been investigated in a number of other cancers and chronic diseases. In gastric cancer, circular RNA_LARP4 suppresses cell proliferation and invasion by sponging miR-424-5p/LATS1 expression, acting as tumor suppressive factor and a potential biomarker in gastric cancer (Zhang et al. 2017a). In vascular endothelial cells induced by oxLDL, hsa_circ_0003575 was significantly up-regulated and hsa_circ_0003575 silencing promoted the proliferation and angiogenesis of HUVECs (Li et al. 2017a). Finally, circ-SMARCA5 was up-regulated in prostate cancer samples compared to normal tissue and acted as an oncogene in prostate cancer by promoting cell cycle and inhibiting cell apoptosis (Kong et al. 2017).
Bioinformatic analysis, including gene ontology (GO), and KEGG pathway have indicated the circRNA/microRNA interaction network, revealing the potential functional mechanism of circRNAs on pathogenesis. For example, in colorectal cancer, hsa_circ_0020397 is up-regulated and dual-luciferase reporter assay confirms the negative correlation with miR-138, suggesting that hsa_circ_0020397 can regulate CRC cell viability, apoptosis and invasion by promoting the expression of miR-138 target genes(Zhang et al. 2017c). In the present study, bioinformatics analysis indicates that miR-181 acts as a target of hsa_circ_0007385, and may be the regulatory pathway for hsa_circ_0007385. Previous studies have reported that miR-181 acts as tumor suppressor on NSCLC (Cao et al. 2017; Huang et al. 2015). Results of our study suggest that hsa_circ_0007385 functions as an oncogenic circRNA for NSCLC, and the deep-going pathway might partly via miR-181. It has been reported that the target protein of miR-181 contains CDK1(Shi et al. 2017), Bcl-2(Huang et al. 2015). Therefore, the physiological process involved the hsa_circ_0007385 regulation might be cell cycle modulation and cellular apoptosis, which will be further explored in our follow-up research.
The present study detected the expression profiles of circular RNA in NSCLC tissue and identified the oncogenic role of hsa_circ_0007385 on NSCLC cells progression, revealing a novel functional mechanism for circRNAs on NSCLC.
This work was supported by Central Laboratory of Cangzhou Central Hospital.
Compliance with ethical standards
Conflict of interest
All the authors declare that they have no competing interests.
Research involving human participants and/or animals
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Cangzhou Central Hospital. This article does not contain any studies with animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
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