Background
Glioma is the most prevalent malignant tumor in central nerve system in adults [
1]. The standards of care in glioma are the maximally safe surgical resection followed by radiotherapy and chemotherapy with temozolomide (TMZ)[
2,
3]. Regardless of multidisciplinary treatment, patients will experience tumor progression and recurrence with nearly universal mortality [
4,
5]. As a disease with such a poor prognosis, the advances on basic and clinical researches of glioma to improve survival and preserve the quality of life are urgently needed.
In recent years, the roles of lncRNAs in oncogenesis were widely studied, as well as their potential in development of new innovative therapies in future [
6,
7]. PVT1 encodes for a lncRNA, called lncPVT1 that has been shown to be associated with several tumors [
8,
9]. Since then, PVT1 has repeatedly emerged in many profiling studies as a prominently dysregulated lncRNA in tumors.
Previous studies suggested that PVT1 could promote the occurrence and development of cancers by affecting cell proliferation, migration, invasion and apoptosis [
10]. Using 97 patients’ data at Yiwu Central Hospital, Fang et al. found that PVT1 was highly expressed in tumor and was an unfavorable prognosis factor for glioma [
11]. However, the clinical and genetic features related PVT1 was not be clarified yet. Meanwhile, there have been few reports illustrating the role of PVT1 in TMZ chemoresistance. Thus, deeply investigating the molecular mechanism and functional diversity of PVT1 in glioma may help to get a potential therapeutic target in glioma. In this study, we gathered genomic and transcriptomic profiles from three independent cohorts (TCGA, CGGA and GSE16011) to comprehensively depict the role of PVT1 in gliomas. Interestingly, we found that PVT1 was not only played an important role in tumor progression, but was also tightly related to the sensitivity of TMZ chemotherapy via JAK/STAT signaling in glioma. This study was the first integrative study to characterize PVT1 expression in glioma in clinical and molecular aspects.
Methods
Patients and cohorts
In total, the 1210 eligible patients who were diagnosed with glioma according to 2016 World Health Organization classification for the central nervous system tumors were included in this study. The transcriptome data and corresponding clinical information were collected from the Chinese Glioma Genome Atlas (CGGA) cohort (n = 325) (
http://www.cgga.org.cn), The Cancer Genome Atlas (TCGA) cohort (n = 627) (
http://cancergenome.nih.gov/) and the GSE16011 (n = 258) (
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc = gse16011). The somatic mutation and copy-number alterations (CNAs) were obtained from TCGA cohort. Overall survival (OS) was calculated from the date of diagnosis until death or the end of follow-up. The clinicopathologic characteristics of the patients from three datasets were shown in Table
1.
Table 1
The clinical and molecular characteristics of glioma in three independent datasets
Age | | | |
≤40 years | 138 | 242 | 71 |
>40 years | 187 | 385 | 187 |
Gender | | | |
Male | 203 | 363 | 180 |
Female | 122 | 264 | 78 |
WHO grade | | | |
Grade II | 109 | 221 | 23 |
Grade III | 72 | 245 | 84 |
Grade IV | 144 | 161 | 151 |
IDH Status | | | |
Mutant | 167 | 390 | 77 |
Wild-type | 158 | 237 | 130 |
1p/19q Status | | | |
Codel | 64 | 154 | 48 |
Intact | 261 | 457 | 104 |
Molecular subtype | | | |
Proneural | 102 | 136 | 91 |
Neural | 81 | 71 | 26 |
Classical | 74 | 83 | 57 |
Mesenchymal | 68 | 92 | 84 |
Cell culture and transfection
Human glioma cell lines U87 and LN229 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All of these cell lines were cultured in DMEM culture medium with added FBS at 10% final concentration and added antibiotics penicillin and streptomycin at 1% final concentrations (Life Technologies, China). Cells were cultured in certified incubators at 37℃ under humidified conditions and 5% CO2. The small interference RNA (siRNA) and negative control (NC) of PVT1 were synthesized by Santa-cruz Biotechnology Co, Inc. In accordance with the manufacturer’s instructions, U87 and LN229 cell lines were transfected with siRNA or NC using the INTERFER in Transfection reagent (Polyplus-transfection Co., Ltd.) when the cell density reached 30–50%.
Western blot analysis
Total cellular proteins were lysed by RIPA lysis buffer (Beyotime, China). Total protein was extracted and quantified with the BCA Protein Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. Equal quantities of total protein (40 µg) from the cell lysates were subjected to 10% SDS-PAGE to separate the proteins and then transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After incubation with antibodies specific for JAK3 (1:1000, cat.no.ab45141, Abcam, USA), STAT3 (1:2000,cat.no.10253-2-AP, Proteintech, USA) or GAPDH (1:5000, cat.no.60004-1-Ig, Proteintech, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody. After washes, bands were detected using the Chemi-DocTM XRS + (Bio-Rad, USA). GAPDH was used as a loading control.
Cell scratch assays
U87 and LN229 cells were seeded in 6-well plates (1 × 105 cells per well) and incubated in a 37 °C, 5% CO2 incubator. At 48 h after transfected with PVT1 siRNA or a NC siRNA, the cell monolayer was scraped with a sterile 200-µL pipette tip. Then, fresh medium without serum was added to the plates and each well was photographed to mark the “zero point” of migration. After completion of 24 h incubation, the samples were washed twice very gently with PBS (Gibco; Thermo Fisher Scientific). Each well was photographed using computer-assisted microscopy. Phase-contrast images were taken at the beginning (0 h) and after 24 h under ×100 magnitude microscope.
Cell migration assays
Cell migration assay was performed using 24-well transwell chambers (8 μm pore size, Corning, Corning, NY, USA). After the transfection with PVT1 siRNA and NC siRNA, approximately 1 × 105 cells were seeded into upper chambers with serum-free medium in triplicate and medium with 20% FBS was added into the lower chamber. After incubation for 5 h, U87 and LN229 cells were fixed in 4% paraformaldehyde solution for 10 min, followed by staining with 5% crystal violet solution. Cell images were captured under an inverted microscope (IX51; Leica Microsystems GmbH, Wetzlar, Germany) in 6 randomly selected fields.
Real-time PCR (RT-PCR)
Moreover, 15 samples, including 5 WHO grade II gliomas, 5 WHO grade III gliomas and 5 WHO grade IV gliomas, were collected to assess differentially expression of PVT1 by real-time quantitative PCR. Total RNA was isolated from glioma samples using TRIzol reagent (Aidalb Biotechnologies Co., Ltd., Beijing, China) following the manufacturer’s instructions. RNA concentration was evaluated by NanoDrop (NanoDrop Technologies; Thermo Fisher Scientific, USA). The RNA quality was estimated by determining the optical density (OD)260/OD280 ratio; values between 1.8 and 2.1 were considered to meet the experimental requirements. Specifically, 1 ug of total RNA was reversely transcribed into cDNA using a Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The results of real-time PCR were normalized to the corresponding GAPDH mRNA levels and the analysis were performed in triplicate to remove the outliers. The PCR conditions were as follows: Pre-denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. Relative gene expression was normalized to the expression of β-actin and was calculated by applying the 2-dCt method. The primers were as follows: PVT1: forward: 5’- GGGGAATAACGCTGGTGGAA-3’, PVT1: reverse: 5’- CCCATGGACATCCAAGCTGT-3’, GAPDH: forward: 5’-AGGGCTGCTTTTAACTCTGGT-3’, GAPDH: reverse: 5’-CCCCACTTGATTTTGGAGGGA-3’.
Cell counting kit-8 (CCK-8) assay
U87 and LN229 cells successfully transfected with siRNA were cultured in medium containing different concentrations of temozolomide (TMZ) (0, 200, 400 and 800 µM) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 24 h. Then, cells were seeded in 96-well plates at a density of 2 × 103 cells/ well, and cell viability was assessed using the CCK-8 Assay Kit (Dojindo Molecular Technologies, Japan), as instructed by the manufacturer. Samples were measured at OD 490 nm using the Bio-Rad Microplate Reader Model 680 (Bio-Rad, China).
Gene ontology enrichment analysis
The functional enrichment analysis for PVT1 correlation genes was performed using DAVID software (
http://david.abcc.ncifcrf.gov/). Gene set enrichment analysis (GSEA) was performed to identify gene sets of statistical difference using GSEA R package.
Statistical analysis
The Student’s t-test, one-way ANOVA, or Chi-squared test was used to assess differences in variables between groups. The Kaplan-Meier analysis was performed to evaluated the overall survival of glioma patients using the “survival” and “survminer” packages. The genomic aberration was performed using the “maftools” package. Other statistical computations and figures drawing were performed with several packages (ggplot2, pheatmap and corrgram). All statistical analyses were conducted using R software. A two-sided p value of < 0.05 was considered statistically significant.
Discussion
Glioma is the most frequent malignant brain type of CNS in adults with poor prognosis [
4]. The intratumor heterogeneity and the sensitivity to radiotherapy and chemotherapy contributed to the poor survival for glioma patients [
21]. Therefore, understanding the molecular mechanisms underlying its progression behavior is urgently needed. Long non-coding RNAs are non-coding RNAs longer than 200 nucleotides. Numerous studies have shown that lncRNA play critical roles in translation and post-translational modification in glioma [
22]. PVT1, at the 8q24 chromosomal band, were associated with cancer susceptibility and tumorigenesis [
23]. However, there are few studies on the investigation the prognostic significance and clinical features of PVT1 in gliomas.
In this study, we collected the transcriptome and genomic data of 1210 glioma samples from CGGA, TCGA and GSE16011 cohorts to analyze the molecular and clinical characteristics of PVT1 comprehensively. Results showed that PVT1 expression was significantly increased with tumor grade in both three independent cohorts. Furthermore, PVT1 expression exhibited a tight relationship with classical and mesenchymal subtypes. These results were further validated by RT-PCR, which showed a high level of PVT1 expressed in the GBM tissue compared with the level in the LGG tissue. In addition, gliomas with chemotherapy tended to have a higher PVT1 expression. These findings showed that PVT1 contributed to the malignance of glioma.
To explore the mechanism of action of PVT1 in tumor progression in glioma, we investigated the distinct genomic alternations in order of increasing PVT1 expression.
We observed the events of somatic mutations and CNVs which were positively associated with PVT1 expression. Glioma with higher PVT1 expression was significantly associated with the somatic mutation of PTEN, EGFR and TTN. Meanwhile, the amplification of EGFR and CDK4, and the deletion of PTEN and CDKNA2A/B were observed in the gliomas with high PVT1 expression. The mutation rate of PTEN was 30 − 40% in GBM [
24], and these findings have had a significant impact on management of PTEN mutant subtypes of glioma [
25]. These findings suggested that PVT1 expression was associated with the malignant biological process. Exploring and revealing the mechanism of PVT1 in glioma may develop the therapeutic approaches to overcome this disease.
In addition, we found that patients with higher expression of PVT1 had a significantly shorter overall survival for glioma patients in each cohort. Our findings in line with the prognostic role of PVT1 in colon cancer [
26,
27], gastric cancer [
28] and melanoma cancer [
29]. The results verified that PVT1 played an important role in the malignancy of glioma and may serve as a potential therapeutic target for glioma patients.
Pathway enrichment analysis showed that PVT1 was significantly related N-glycan biosynthesis, TNF signaling pathway, response to drug, regulation of JAK-STAT cascade, Mismatch repair (MMR). The JAK/STAT pathway is the key pathway in tumor progression and chemoresistance. The results were consistent with existing findings on the major physiological function of PVT1 in tumor development [
10,
23,
30]. Then we depicted the relationship between PVT1, JAK/STAT pathway and chemoresistance. We found PVT1 expression was significantly positively related with genes in JAK/STAT pathway and DNA damage repair. Meanwhile, we found that PVT1 was highly expressed in recurrent GBMs, patients with chemotherapy, or patients with MGMT promoter unmethylated. As we know, the MGMT promotor was an important biomarker of tumor response to temozolomide (TMZ) chemotherapy [
31]. The western blot analysis found that protein levels of JAK3 and STAT3 were decreased after PVT1 knockdown. Thus, we implied that PVT1 was not only functions as an important factor of the tumor proliferation but also results in the resistance of TMZ chemotherapy through regulating JAK/STAT pathway. The further mechanism and intervention treatment required extensive studies to validate in vivo. In the future, the combined strategy of TMZ and anti-PVT1 may improve the prognosis of GBM.
Conclusion
Taken together, we elaborated the roles of PVT1 in glioma from transcriptomic and genomic levels. In addition, this study indicated that PVT1 promoted glioma TMZ chemoresistance through JAK/STAT signaling. The underlying mechanism provides a basis for PVT1 as a new molecular target for treatment of glioma.
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