Linc00659, a long noncoding RNA, acts as novel oncogene in regulating cancer cell growth in colorectal cancer
© The Author(s). 2018
Received: 18 September 2017
Accepted: 1 March 2018
Published: 10 March 2018
Colorectal cancer (CRC) is one of the most common cancers and causes of cancer-related death worldwide. In patients with CRC, metastasis is a crucial problem that leads to treatment failure and is the primary cause of the lethality of colon cancer. Long noncoding RNAs (lncRNAs) have recently emerged as critical molecules in the development, cell growth, apoptosis, and metastasis of CRC.
We investigated the transcriptome profiles of human lncRNAs in the primary tumor tissues and in the corresponding normal mucosa of two patients with CRC by using a microarray approach. The expression levels of lncRNAs were verified in colon cancer by real-time PCR. Using bioinformatics approach to illustrate putative biological function of Linc00659 in colon cancer. The effects of Linc00659 on cell growth, proliferation, cell cycle and apoptosis were studies by in vitro assays.
Our data revealed that compared with adjacent normal tissues, 201 lncRNAs were deregulated (fold change ≥ 4 or ≤ 0.25) in CRC tissues. Among them, the expression levels of Linc00659 were significantly increased in colon cancer, and high expression levels were correlated with poor survival in patients with CRC. Bioinformatics analysis results indicated that Linc00659 was significantly coexpressed with cycle-related genes in CRC. Linc00659 expression knockdown could significantly suppress colon cancer cell growth by impairing cell cycle progression. In addition, our results showed that Linc00659 expression knockdown could accelerate cell apoptosis in colon cancer cells treated with chemotherapy drugs. Meanwhile, our results also demonstrated that silencing of Linc00659 expression leads to cell growth inhibition and induced apoptosis, possibly by suppressing PI3K-AKT signaling in colon cancer.
Linc00659 is a novel oncogenic lncRNA involved in colon cancer cell growth by modulating the cell cycle. Our findings give an insight into lncRNA regulation and provide an application for colon cancer therapy.
Colorectal cancer (CRC) is one of the most common cancers and causes of cancer-related death worldwide [1, 2]. The major causes of the lethality of colon cancer are distant metastasis and drug resistance, which usually lead to a poor survival rate . In patients diagnosed as having advanced-stage CRC, distant metastasis is frequently observed, leading to poor therapeutic effects of chemotherapy, radiotherapy, and surgery . Therefore, developing a diagnostic biomarker for early detection of CRC or as a therapeutic target will improve the survival rate of patients with CRC.
High-throughput genome-scale approaches have revealed that a large amount of transcripts are produced from more than 80% of human genomic DNA, and only 1–2% of the human genome is composed of protein-coding genes . Therefore, the human genome comprehensively harbors numerous transcripts with no protein-coding function. Recent studies have shown that numerous noncoding RNAs (ncRNAs) play a critical role in regulating various cellular processes . Long noncoding RNAs (lncRNAs) are a group of long RNA transcripts that are more than 200 nucleotides in length; however, they lack protein translation ability. An increasing body of evidence indicates that lncRNAs play a critical role in regulating various cellular processes, including cell development and growth, the cell cycle, and cancer metastasis, by (1) regulating protein-coding gene expression, (2) modifying epigenetic regulation, (3) modulating the alternative splicing process, and (4) acting as decoys that titrate microRNAs [7, 8]. To date, some studies have reported that numerous lncRNAs, including HOTAIR, PCAT-1, PVT-1, H19, BANCR, 91H, CCAT1, and MALAT1, were dysregulated in colon cancer [9–21]. These dysfunctional lncRNAs have been reported to have tumor-suppressive or oncogenic roles in regulating cell growth, the cell cycle, and cell migration in CRC. However, the function of most lncRNAs remains largely unknown in CRC. In this study, our objective was to identify dysregulated lncRNAs in CRC. For this purpose, we compared the transcriptome profiles of primary tumor with adjacent normal tissue from two patients by using a microarray approach. We further evaluated the expression levels and biological role of Linc00659 in CRC through bioinformatics and an experimental approach. Our finding revealed that Linc00659 is a novel oncogenic lncRNA that regulates CRC cell growth by impairing cell cycle progression, and thus may be a potential therapeutic target for gene therapy.
Eight human CRC cell lines, namely DLD-1, colo205, colo320DM, LS174T, HCT116, SW620, LOVO, and HT29, were obtained from the American Type Culture Collection. Cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum (Hyclone Laboratories, Inc., South Logan, UT, USA) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) in plastic tissue culture plates in a humidified atmosphere containing 5% CO2 at 37 °C.
Colon cancer tissue and the corresponding adjacent normal mucosa samples were obtained from 91 patients with CRC who underwent surgery at the Department of Surgery, Taipei Veterans General Hospital, Taiwan. Informed consent was obtained from all patients.
Extraction of RNA
Total RNA from tissue was extracted using TRIzol reagent (Invitrogen, Grand Island, NY, USA), according to instructions in the user’s manual. In brief, tissue samples were homogenized in 1 mL TRIzol reagent and mixed with 0.2 mL chloroform to extract protein, before RNA was precipitated using 0.5 mL isopropanol. The concentration, purity, and amount of total RNA were determined using a Nanodrop 1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA).
LncRNA profiling and pathway enrichment analysis
Total RNAs of four clinical samples were extracted, involving two primary CRC tissues and two corresponding adjacent normal tissues obtained from two patients with CRC, respectively. The microarray experiments and data analysis were performed by Welgene Biotech (Taipei, Taiwan) using the Agilent Oligo Chip (Agilent SurePrint G3 Human V2 GE; including protein-coding genes and lncRNAs). Differential expression protein-coding genes (tumor versus normal ≥ 2 and ≤ 0.5) were selected from microarray data and candidate genes were mapped onto the Kyoto Encyclopedia of Genes and Genomes pathways based on the Enzyme Commission numbers by using the R package SubpathwayMiner v.3.1. Subsequently, a hypergeometric test was performed to identify significantly enriched pathways and calculate the false-positive discovery rate in the false discovery rate-corrected q value.
Expression data from the cancer genome atlas
The transcriptome expression profiles of colon cancer were downloaded from The Cancer Genome Atlas (TCGA) data portal (https://cancergenome.nih.gov). The expression profiles of 616 colon cancer tissues and 51 adjacent normal tissues were obtained from TCGA data portal. In this study, the transcriptome profiles of 29 N-T pairs were used for coexpression analysis and 616 cases were included in the survival analysis.
Reverse transcription and real-time polymerase chain reaction
Small interfering RNA transfection
Small interfering RNA (siRNA) oligonucleotides targeting Linc00659 (si-Linc00659, sense: 3′-TTGGGAGGAACACGAAGUCUU-5′ and antisense: 5′-UUCUGAAGCACAAGGAGGGTT-3′) and a scrambled oligo as a negative control were designed and synthesized by GenDiscovery Biotechnology (Taipei, Taiwan). Cells were transfected with a final concentration (10 mM) of individual siRNA or control using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA). After transfection for 24 h, the RNA was extracted and knockdown efficiency was evaluated by real-time PCR.
Stable Linc00659 knockdown with short hairpin RNA
For cell proliferation analysis, 2500 living cells were transfected with either siLinc00659 or a scrambled control and plated onto 96-well plates. Cell growth was determined at 0, 1, 2, 3, and 4 days. Cell viability was determined using a CellTiter-Glo One Solution cell proliferation assay (Promega Corporation, Madison, WI, USA), which was performed according to the manufacturer’s instructions.
Soft agar assay
In brief, this assay was performed as follows: The base agar layer was prepared with 1% agar (Laboratorios CONDA, Torrejón de Ardoz, Madrid, Spain) dissolved in 1.5 mL phosphate-buffered saline on 6-well plates; subsequently, 1.5 mL of 0.7% agarose (Invitrogen, Grand Island, NY, USA) solution containing the cells (10,000 cells per well) was inoculated on top of the base agar layer. After allowing the solution to harden, 0.5 mL of fresh medium was added to the top of the hard agar layer. After 3–4 weeks, agar plates were stained with iodonitrotetrazolium chloride (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and colonies were counted.
Colony formation assay
A total of 4000 cells were seeded on each well of a 6-well plate, and the cells were transfected with individual siRNA oligonucleotides by using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA) in the 6-well plate. After incubation at 37 °C for 3 days, the cultured medium was replaced with a new medium. The cells were allowed to incubate at 37 °C for 10 days. Cell culture plates containing colonies were fixed with 4% formaldehyde for 2 min and colonies were stained with crystal violet solution (containing 0.5% crystal violet, 5% formaldehyde, 50% ethanol, and 0.85% sodium chloride) for 2 h. Wells were rinsed with H2O after air drying. The crystal violet staining of cells from each well was solubilized using 1 mL per well of 10% acetic acid, and the absorbance (optical density) of the solution was measured on a spectrophotometer at a wavelength of 595 nm.
Cell cycle analysis
A total of 1 × 106 cells were collected and mixed with 70% ethanol in a fixative at − 20 °C overnight. Cells were then stained with 4′,6-diamidino-2-phenylindole (ChemoMetec, Gydevang, Lillerød, Denmark) and detected using NucleoView NC-3000 software (ChemoMetec, Gydevang, Lillerød, Denmark).
Apoptotic cells stained with Annexin V, propidium iodide, and Hoechst 33,342
For the image flow cytometry assay, CF488A Annexin V and PI Apoptosis kit (Biotium, Fremont, CA, USA) and Hoechst 33,342 (ChemoMetec, Gydevang, Lillerød, Denmark) were used to detect cells in early apoptotic and late apoptotic stages. After staining, the cells were analyzed in NucleoCounter NC-3000 (ChemoMetec, Gydevang, Lillerød, Denmark) and NucleoView NC-3000 for automated image flow cytometry analysis.
The cells were harvested 24 h after transient transfection, washed with phosphate-buffered saline, and subjected to lysis in radioimmunoprecipitation assay buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate) at 4 °C for 30 min. Lysed cells were collected and centrifuged to remove cell debris. Protein assays were performed with the Bio-Rad Protein Assay kit based on the Bradford dye-binding procedure (Bio-Rad). Protein samples (40 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 10% or 12% resolving gel using a Mighty Small II Deluxe Mini Vertical Electrophoresis Unit (Hoefer, Inc., Holliston, MA, USA). Proteins were then electrotransferred to polyvinylidene difluoride membranes (PerkinElmer, Inc., Waltham, MA, USA) by Mighty Small Transfer Tank (Hoefer, Inc., Holliston, MA, USA). Subsequently, the membranes were blocked with a blocking buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20, 5% nonfat dry milk, 0.05% sodium azide) for 1 h at room temperature and incubated with the following primary antibodies overnight at 4 °C: CCNA2 (1:1000; 18,202–1-AP, Proteintech Group, Inc., Rosemont, IL, USA), CCNB1(1:1000; 55,004–1-AP, Proteintech Group, Inc., Rosemont, IL, USA), CCND1 (1:1000; RM-9104-S, Thermo Fisher Scientific Inc., Waltham, MA, USA), CDK4 (1:1000; MS-299-P, Thermo Fisher Scientific Inc., Waltham, MA, USA), CDKN1B (1:1000; #3686, Cell Signaling Technology, Inc., Beverly, MA, USA), CDKN1A (1:1000; #2947, Cell Signaling Technology, Inc., Beverly, MA, USA), PARP (1:1000; sc-8007, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), CASP3 (1:1000; #9662, Cell Signaling Technology, Inc., Beverly, MA, USA), E2F1 (1:200, sc-251, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), PI3K p85 (1:1000; #4292, Cell Signaling Technology, Inc., MA, USA), AKT (1:1000; #4691, Cell Signaling Technology, Inc., MA, USA), Phospho-AKT (Thr308) (1:1000; #4056, Cell Signaling Technology, Inc., MA, USA), Phospho-AKT (Ser473) (1:1000; #4060, Cell Signaling Technology, Inc., MA, USA), GSK3β (1:1000; #9315, Cell Signaling Technology, Inc., MA, USA), Bcl-2 (1:1000; #4223, Cell Signaling Technology, Inc., MA, USA), BAX (1:1000; #5023, Cell Signaling Technology, Inc., MA, USA), Bad (1:1000; #9239, Cell Signaling Technology, Inc., MA, USA), Phospho-Bad(Ser136) (1:1000; #4366, Cell Signaling Technology, Inc., MA, USA), β-catenin (1:4000; C2206, Sigma-Aldrich, Inc., Beverly, MO, USA) and ACTB (1:2000, MAB1501, EMD Millipore, Billerica, MA, USA). The membranes were then incubated with anti-rabbit (sc-2004) or anti-mouse (sc-2005) immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (1:10000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature. After three washes with Tris-buffered saline with Tween-20 buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20), immunoreactive bands were detected using a WesternBright ECL (Advansta, Menlo Park, CA, USA).
The expression levels of Linc00659 in colon cancer from TCGA database were analyzed using Student t tests. The correlation of Linc00659 with the protein-coding genes in colon cancer was determined through Pearson coefficient analysis, with r and p values as indicated. Cumulative survival curves were estimated using the Kaplan–Meier method, and comparison between survival curves was performed using the log-rank test. The difference was considered significant when p < 0.05. The data on the expression levels of lncRNAs in paired colon tissues were analyzed using a paired t test. Cell proliferation, colony formation, and soft agar assay experiments were performed in triplicate. Histograms present the mean values, and the error bars indicate the standard deviation. These data were analyzed using Student t tests.
Generation of lncRNA expression profiles in CRC
LncRNA candidates were confirmed in colon cancer
Microarray analysis of the 20 most aberrantly regulated lncRNAs in two pairs of CRC
Linc00659 coexpressed with cycle-related genes
Linc00659 was involved in colon cancer growth
Knockdown of Linc00659 suppressed cell growth by impairing cell cycle progression and inducing cell apoptosis
Linc00659 knockdown confers drug sensitivity to colon cancer cells
Effects of Linc00659 knockdown on PI3K-AKT-GSK3β signaling
Increasing evidence reveals that numerous lncRNAs play a critical role in the pathogenesis and progression of CRC. In the present study, transcriptome profiles were analyzed to identify differentially expressed lncRNA in colon cancer by using a microarray approach, which revealed that the expression levels of 201 lncRNAs were significantly different between colon cancer and the corresponding adjacent normal tissues. As expected, our microarray profiles identified several lncRNAs, which were aberrantly expressed in human cancer, such as LOC100190940, LOC100506178, H19, PVT1, VPS9D1-AS1, Linc00520, Linc00675, GNG12-AS1 CDKN2B-AS1, and CCAT1 [19, 27–36].
Chen et al. systematically examined global lncRNA dysregulation by analyzing RNA-seq data of colon cancer . They identified that LOC100190940 was upregulated in primary tumor tissues compared with adjacent normal tissues, and that high LOC100506178 expression levels were correlated with poor survival in patients with colon cancer. In addition, they reported that Linc00659 was highly expressed in primary tumor tissues with a higher H3K4me3 signal in its promoter region, compared with normal tissues. This result implies that the expression levels of Linc00659 might be regulated by the epigenetic mechanism in CRC . A previous study indicated that c-Myc could promote CCAT1 transcription by activating its promoter activity, and that ectopic CCAT1 expression could accelerate growth and invasion in colon cancer . Furthermore, high CCAT1 expression levels were significantly associated with poor prognosis and survival in patients with colon cancer [19, 29]. In this study, we identified hundreds of novel abnormal lncRNA genes to provide valuable information for lncRNA research in colon cancer.
The mechanisms through which lncRNAs participate in carcinogenesis and CRC progression are various and complex. To date, the mechanism underlying lncRNA function remains largely unknown, and the molecules with which they interact or how their expression influences signaling is unclear. Although studies have attempted to predict the putative mechanism of dysfunctional lncRNAs by using the bioinformatics approach, the lack of conserved sequences and the absence of functional motifs have made it difficult to determine the function of lncRNAs by analyzing their sequence or secondary structures. Using transcriptome data to determine which types of protein-coding genes are coexpressed with lncRNAs may facilitate exploring lncRNA function . In this study, we used TCGA database to determine coexpressed protein-coding genes and demonstrate the biological function of Linc00659 involved in modulating cancer cell growth. Using an experimental approach, we further confirmed that Linc00659 knockdown could significantly suppress colon cancer growth by impairing cell cycle progression and inducing cell apoptosis. Moreover, our data reveal that Linc00659 conferred drug sensitivity to colon cancer cells. Studies have increasingly indicated that some lncRNAs were associated with drug resistance in human cancer [38–40]. Lee et al. identified differentially expressed long intergenic noncoding RNAs (lincRNAs) in 5-FU-resistant colon cancer cells . A total of 42 lincRNA candidates were identified with differential expression in 5-FU-resistant cells. Among them, colon cancer cells with snaR loss could decrease in vitro sensitivity to 5-FU . In addition, 46 lncRNAs were identified with differential expression in vincristine-resistant colon cancer cells, compared with control cells, using next-generation sequencing profiling . The expression levels of Linc00152 conferred colon cancer cell resistance to oxaliplatin by modulating the miR-193a-3p/ERBB4/AKT signaling axis . In our study, we demonstrated that Linc00659 knockdown could accelerate cell apoptosis in colon cancer cells following oxaliplatin treatment. An analysis of the Gene Expression Omnibus database showed that the expression levels of Linc00659 were increased in drug-resistant breast and colon cancer cells [42, 43]. According to our present study, we determined a putative mechanism by which Linc00659 may play a novel oncogenic role in cell growth and drug resistance by modulating PI3K-AKT signaling.
Unlike protein-coding genes, whose transcripts are mainly exported to the cytoplasm for translation, lncRNAs are distributed in either the cytoplasm or the nucleus, depending on their function. Therefore, further examination of subcellular localization of lncRNA would facilitate determining the putative mechanism. We analyzed the subcellular localization of Linc00659, revealing that Linc00659 expression occurred both in the nucleus and cytoplasm in most colon cancer cells (Additional file 3: Figure S3). According to its location, Linc00659 might regulate colon cancer cell growth by binding with microRNA sponge, regulate transcription factors, or modulate epigenetic modification to prevent tumor suppressor gene expression. Notably, we observed that Linc00659 expression predominantly enriches in the nucleus compared with the cytoplasm in HT29 cells (Additional file 3: Figure S3). Studies have reported that the siRNA approach was less efficient for knockdown nuclear-lncRNA expression . In the present study, we were unable to explore the role of Linc00659 in HT29 cells using the same siRNA oligo. According to the varying localization of Linc00659, we suggest that Linc00659 might play an oncogenic role through the difference mechanism in HT29 cells. However, the detailed mechanism of Linc00659 remains unclear.
In this study, we identified lncRNAs with differential expression in colon cancer. We first reported that the expression levels of Linc00659 were significantly increased in colon cancer and that Linc00659 loss suppressed colon cancer cell growth by impairing cell cycle progression. Our finding also establishes Linc00659 as a prognostic biomarker for indicating CRC survival and to be used as adjuvant therapy for chemotherapy.
We acknowledge Wallace Academic Editing for editing this manuscript.
This work was supported by funding from the Ministry of Science and Technology (MOST105–2314-B-010-051), Kaohsiung Veterans General Hospital (VGHKS 105–135 and VGHKS105–092) and Zuoying Branch of Kaohsiung Armed Forces General Hospital (grant no. ZBH-105-11).
TKW and LYH executed this study and drafted the manuscript. LH performed the bioinformatics analysis. YCY and CYZ did the assay in vitro. HCW assisted in manuscript proofreading. CWS and WJH supervised the study and edited the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the ethics committee of the Taipei Veterans General Hospital and written informed consents were obtained from all patients (IRB number: 2015–06-012CC).
The authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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