Glioblastoma multiforme (GBM) is the most common malignant tumor with high morbidity and mortality. This study investigated the role of long non-coding RNAs (lncRNAs) in glioblastomagenesis progression. Using the GSE2223 and GSE59612 datasets, and RNA sequencing data of GBM from The Cancer Genome Atlas, differentially expressed (DE) genes including DE messenger RNAs (DEmRNAs) and DElncRNAs between GBM and normal controls were identified. Based on the competing endogenous RNA hypothesis, DElncRNA-micro RNA (miRNA)-DEmRNA interactions were obtained by target gene prediction. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes pathway analysis of DEmRNAs in the DElncRNA-miRNA-DEmRNA network was performed. Expression and function analyses of DElncRNAs were performed by reverse transcription-polymerase chain reaction (RT-PCR) and an established viability assay, respectively. In total, 712 DE genes were identified. Significant upregulation of lncRNA
Gliomas are the most common primary brain tumors. They arise from cancerous brain and spinal cord glial cells (
Long non-coding RNA (lncRNA) is a type of RNA that contains over 200 nucleotides and that does not encode protein (
In this study, the gene expression profile in GBM was analyzed using a microarray dataset and RNA-sequencing (RNA-Seq) datasets. Key lncRNAs in GBM were screened. Based on the ceRNA hypothesis (
Gene expression profile datasets GSE2223 (
A total of 10 GBM tissues were collected from GBM patients who were treated surgically at the People's Hospital of Zhangjiajie (Zhangjiajie, China) between April 2015 and March 2017. The 10 GBM adjacent normal brain tissues were obtained from patients with head trauma who underwent surgical treatment in the same hospital. The age of the participants was 30–60 years old, with a mean age of 49.6±6.7 years, and the male:female ratio was 8:6. All samples were collected immediately following surgical resection and frozen rapidly in liquid nitrogen at −70°C. The experimental study was approved by the hospital's ethics committee and written informed consent was obtained from all participants.
The SHG-44 and U251 GBM cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (GE Healthcare Life Sciences, Logan, UT, USA), penicillin (100 units/ml), and streptomycin (100 ug/ml) and incubated in a 5% CO2 incubator at 37°C.
Small interfering si-NC, (si)-DLEU1 and si-tumor necrosis factor receptor-associated factor 4 (TRAF4) was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The si-NC, si-DLEU1, si-TRAF4 and the empty vectors (10 nM) were transfected into SHG-44 and U251 cell line using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Silencing was confirmed 12 h after transfection, by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The specific primer sequences are presented in
Differentially expressed genes (DEGs) including differentially expressed mRNAs (DEmRNAs) and differentially expressed lncRNAs (DElncRNAs) between GBM and GBM adjacent normal brain tissues of the GSE2223 and GSE59612 datasets with normalized expression were analyzed using the Limma package (version 3.34.6;
Gene Expression Profiling Interactive Analysis (GEPIA;
The integrated miRNA-DEmRNA and miRNA-DElncRNA pairs were simultaneously predicted by miRanda (
To investigate the underlying functional role of lncRNAs, GO biological processes and KEGG pathway analysis were performed for the mRNAs in lncRNA-miRNA-mRNA interactions with DAVID 6.8 software (
Total RNA from GBM and normal brain samples was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After DNase digestion, RNA quantification and purity were measured by the ratio of 260/280 nm. RNA integrity was measured by 1.2% agarose gel electrophoresis. The reverse transcriptase reaction kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to reverse-transcribe RNA samples at 50°C for 60 min. RT-PCR was performed in triplicate according to the manufacturer's protocol of SYBR Green PCR Master Mix and reactions were carried using in a PCR Thermal Cycler (Takara Bio, Inc., Otsu, Japan). The conditions were: Initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 5 sec, and annealing and extension at 55–58°C for 30 sec. The relative expression levels of genes were calculated as relative quantification, calculated as 2−∆∆Cq (
Total proteins were extracted from cells using Radio Immunoprecipitation Assay Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were quantified using the bicinchoninic acid protein assay kit (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China). Cell lysates were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes, and membranes were blocked at room temperature with 5% skimmed milk in TSB Tween-20 (0.05% v/v; TBS-T) for 1 h and incubated with specific antibodies at 4°C overnight. The primary antibody was a 1:1,000 dilution of rabbit anti-TRAF4 (cat. no. Ab108991; Abcam, Cambridge, UK) or mouse anti-GAPDH (cat. no. AG019; Beyotime Institute of Biotechnology). The membranes were washed three times for 10 min every time with TBS-T, followed by incubation with secondary antibodies goat anti-mouse immunoglobulin (Ig)G (1:1,000; cat. no. A0216; Beyotime Institute of Biotechnology) and goat anti-rabbit IgG (1:2,000; cat. no. Ab6721; Abcam) for 1 h at room temperature. The intensities of the immunoreactivity were detected with an enhanced chemiluminescence kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The images were developed on X-ray film. The experiments were repeated ≥3 times.
A MTT kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to analyze cell proliferation according to the manufacturer's protocol. All the cells were cultured in 96-well plates in the dark, the formazan was dissolved with dimethyl sulfoxide (DMSO) and the absorbance value at 570 nm was detected every 24 h.
SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used to analyze statistical significance. All the experiments were independently performed three times. The data are expressed as the mean ± standard deviation. The difference between the groups was analyzed with an analysis of variance (ANOVA) or Student's t test. Post hoc tests were performed using a Tukey test following the ANOVA. * and ** refer to the statistically significant difference of expression (P<0.05) and extremely significant difference of expression (P<0.01), respectively. P<0.005 was considered to indicate a statistically significant difference.
A total of 730 overlapping DEGs were screened from the GSE2223, GSE59612 and TCGA-GBM datasets (
Among the DElncRNAs, the expression of DLEU1 was upregulated in GBM and a number of other tumor types, including cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, brain lower grade glioma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, thymoma, and uterine carcinosarcoma (
The integrated DLEU1-miRNA-DEmRNA interactions were identified with the miRanda and RNAhybrid methods. The network was constructed using Cytoscape software (
To investigate the functional role of genes in the DLEU1-miRNA-DEmRNA network, GO and pathway analysis of mRNAs were performed with DAVID 6.8 software (
TRAF4 serves an important role in other cancers according to previous studies (
The effects of silencing lncRNA were studied. Following silencing DLEU1 (
Similarly, the proliferation of cells was studied by silencing TRAF. Compared with the negative control transfected with the empty vector. After silencing
In the present study, microarray data profiling and RNA-Seq profiles of GBM were integrated and re-analyzed. A total of 712 DEGs (673 DEmRNAs, 36 miscellaneous RNA and 3 DElncRNAs) were identified between GBM, and GBM adjacent normal brain tissues. Among the three lncRNAs (DLEU1, PART1 and miR7-3HG), DLEU1 was more expressed in a number of types of cancer, including GBM. The DLEU1-miRNA-DEmRNAs network was constructed. Several miRNAs in the DLEU1-miRNA-mRNAs network have been reported in the progression of tumorigenesis in GBM. For example, miR-107 is downregulated in glioma tissues and cell lines (
DEmRNAs in the DLEU1-miRNA-mRNAs network were mainly enriched in the pathway terms of the Hippo signaling pathway, pathways in cancer and Wnt signaling pathway. The Hippo signaling pathway is reportedly a major signaling pathway that regulates cell proliferation and growth, and is important role in the tumorigenesis of GBM (
TRAF4 is overexpressed in tissues or cells in osteosarcoma (
In conclusion, DLEU1 was intensified in GBM tissues and silencing of DLEU1 inhibited cell proliferation. Furthermore, miRNAs and mRNAs in the DLEU1-mediated ceRNA network are involved in cell differentiation, proliferation, migration, and invasive growth of GBM cells. These collective observations support the idea that DLEU1 may serve a pivotal role in the tumorigenesis of GBM. This study identifies a novel lncRNA that may act as a ceRNA in GBM. Further studies are needed to understand the molecular role of DLEU1 in GBM progression.
Not applicable.
The present study was funded by the Natural Science Foundation of Hunan Province (grant no. C2013-225).
All data generated or analyzed during this study are included in this published article.
GH and JW were responsible for the concept and design of the present study. JW and XQ acquired the data. JW and DP performed data analysis and experiments. JW drafted the article. All authors read and approved the final manuscript.
The experimental study was approved by the ethics committee of People's Hospital of Zhangjiajie and written informed consent was obtained from all participants.
Written informed consent was obtained from all participants.
The authors declare that they have no competing interests.
Overlapping DEGs of three datasets (GSE2223, GSE59612 and TCGA GBM RNA-Sequence). (A) Overlapping DEGs of three databases between normal and tumor samples. (B) The regulation association of the DEGs in the three datasets and the gene types of DEGs with a consistent regulation association. Inconsistent regulation is evident as inconsistent upregulation or downregulation of the DEGS in the three databases. DEG, differentially expressed genes, DEGs; TCGA, the cancer genome atlas; GBM, glioblastoma; lnc, long non-coding RNA; misc, miscellaneous.
Expression level of lncRNA DLEU1 in multiple types of cancer. *P<0.05. CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; GBM, glioblastoma multiforme; LGG, brain lower grade glioma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; READ, rectum adenocarcinoma; STAD, stomach adenocarcinoma; THYM, thymoma; UCS, uterine carcinosarcoma; num, number; T, tumour; N, normal.
LncRNA DLEU1 mediates the lncRNA-miRNA-mRNA network. Diamonds represent lnRNA, round rectangles denote miRNA, and ovals represent mRNA. Red and gray denote upregulation and unknown regulation, respectively. miRNA/miR, microRNA; lnc, long non-coding RNA.
Gene Ontology and pathway analysis. (A) Gene Ontology and (B) pathway analysis of differentially expressed mRNAs in the lncRNA-miRNA-mRNA network. Red and blue denote P<0.05 and P>0.05, respectively. miRNA, microRNA; lnc, long non-coding RNA; TGF, transforming growth factor; ECM, extracellular matrix.
DLEU1 is positively associated with TRAF4. (A) Correlation analysis and relative expression analysis of lncRNA (B) DLEU1 and (C) TRAF4 in glioblastoma tissues. **P<0.01, n=3. Lnc, long noncoding RNA; TRAF4, TNF receptor associated factor 4; DLEU1, deleted in lymphocytic leukemia 1.
Silencing of lncRNA DLEU1 downregulates TRAF4 and inhibits cell proliferation. Silencing of lncRNA DLEU1 inhibited cell proliferation by MTT assays in (A) SHG-44 and (B) U251 cell. Silencing of lncRNA DLEU1 downregulates TRAF4 in SHG-44 and U251 cell transfected with si-DLEU1 at (C) the transcription and the (D) protein level. Silencing of
Sequences for si-NC, si-DLEU1#1, si-DLEU1#2, si-TRAF4#1 and si-TRAF4#2.
Name | Guide | Passenger |
---|---|---|
si-NC | 5′-UUCUCCGAACGUGUCACGUTT-3′ | 5′-ACGUGACACGUUCGGAGAATT-3′ |
si-DLEU1#1 | 5′-UUUUUUGUGCAGUUUCAGCAA-3′ | 5′GCUGAAACUGCACAAAAAAUC-3′ |
si-DLEU1#2 | 5′-UUCCUUUUUGAUAGUAUUCAA-3′ | 5′GAAUACUAUCAAAAAGGAAAA-3′ |
si-TRAF4#1 | 5′-UUAAUAAAUACAAUUCCGGAU-3′ | 5′CCGGAAUUGUAUUUAUUAAUU-3′ |
si-TRAF4#2 | 5′-UUUCAUAGGUGAAACGUGGAU-3′ | 5′CCACGUUUCACCUAUGAAACA-3′ |
TRAF4, TNF receptor associated factor 4; DLEU1, deleted in lymphocytic leukemia 1; si, small interfering.
Significant GO and pathway analysis of differentially expressed mRNAs in the DLEU1-miRNA-mRNAs network.
Terms | Genes | P-value |
---|---|---|
GO | ||
Angiogenesis | CAV1, SRPX2, TNFRSF12A, HSPG2, ESM1, ELK3, ANGPT2, EPHB4 | 0.0003296 |
Positive regulation of cell proliferation | KIF14, NAMPT, SHMT2, TNFSF13B, RAC2, CLCF1, SOX4, ID4, ESM1, |
0.0003889 |
Positive regulation of fibroblast apoptotic process | BTG1, STK17B, STK17A | 0.0004981 |
Regulation of neutrophil migration | MYD88, RAC2 | 0.0116385 |
Canonical Wnt signaling pathway | 0.0126456 | |
Cellular response to heat | MKI67, C8ORF4, CXCL10 | 0.0196642 |
Single organismal cell-cell adhesion | SRPX2, |
0.0212821 |
Negative regulation of fat cell differentiation | 0.0249487 | |
Non-canonical Wnt signaling pathway via JNK cascade | 0.0288453 | |
Response to organic cyclic compound | NAMPT, MKI67, ANGPT2 | 0.0331943 |
Mammary gland involution | CAV1, IGFBP5 | 0.0345148 |
Signal transduction | NAMPT, PDPN, MRC2, GNG12, ELK3, CXCL10, MYD88, RAC2, TNFSF13B, CLEC5A, ANGPT2, TRAF4, IGFBP5 | 0.0375587 |
Negative regulation of protein export from nucleus | SOX4, BARD1 | 0.0401515 |
Kidney morphogenesis | SOX4, |
0.0457556 |
T cell mediated immunity | BTN3A3, BTN3A2 | 0.0457556 |
Cartilage development | 0.0465266 | |
Positive regulation of protein binding | 0.0493935 | |
Pathway | ||
Hippo signaling pathway | 0.010874 | |
Pathways in cancer | 0.024194 | |
Proteoglycans in cancer | 0.027611 | |
Basal cell carcinoma | 0.040012 | |
Wnt signaling pathway | 0.045011 | |
Signaling pathways regulating pluripotency of stem cells | 0.046643 |
Genes in bold indicate these genes were enriched in at least four significant GO or pathway terms. GO, Gene Ontology; miRNA, microRNA; DLEU1, deleted in lymphocytic leukemia 1.