Introduction
Glioma is one of the most malignant and prevalent tumor in adults [
1,
2]. Despite of the aggressive therapy including maximal surgical resection together with radio- and chemo- therapy, the survival time of glioblastoma patients is still less than 14 months [
3,
4]. Therefore, understanding the underlying mechanism of glioma progression is essential for improving patients’ prognosis.
Long non-coding RNAs (lncRNAs) are a class of RNAs that consists of more than 200 nucleotides and lacks the ability of coding proteins [
5,
6]. Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to be an important lncRNA participating in progression of a lot of tumors, including glioma [
7‐
10]. NEAT1 was shown closely associated with glioma patients’ prognosis [
11]. And, NEAT1 levels were characteristically overexpressed in glioma cell lines [
12]. However, little is known about the mechanism of NEAT1 regulating glioma development. By now, most researches of NEAT1 focused on its role of sponging miRNAs, like miR-107 [
13] and miR-139-5p [
14]. Qun Chen et al. [
10] identified that NEAT1 was critical for glioma progression by increasing β-catenin nuclear transport and H3K27 trimethylation. However, these results did not revealed the mechanism of NEAT1 on regulating glycolysis of glioma cells.
In this study, we found that NEAT1 was overexpressed in glioma. NEAT1 knockdown significantly suppressed proliferation and glycolysis of glioma cell. In addition, we identified a new NEAT1 interacted protein: PGK1. NEAT1 overexpression is essential for PGK1 stability in glioma cells, thus promoting glycolysis of glioma cells. Moreover, we identified the direct interaction between hairpin A of NEAT1 and M1 domain of PGK1. Lastly, NEAT1 expression was positively correlated with PGK1 in glioma samples, indicating NEAT1 and PGK1 are potential biomarkers for glioma.
Method and materials
Clinical samples
Five tumor adjacent tissues and twenty GBM samples were collected from The department of Neurosurgery, Lianyungang Clinical College of Nanjing Medical University. Samples were surgically resected and frozen immediately in liquid nitrogen for further study. The use of clinical samples was approved by Lianyungang Clinical College of Nanjing Medical University.
Cell culture
The human glioma cell lines (U251 and T98G) were purchased from ATCC. Cells were maintained in DMEM (Gibco, USA) supplemented with 10% FBS (Fetal Bovine Serum) (Hyclone, USA), 100 μg/ml penicillin, and 100 μg/ml streptomycin in 37 ℃. Cells were thawed fresh every 2 months.
Cell transfection
Polymerase chain reaction (PCR)-amplified full length or truncated human PGK1 was cloned into pcDNA3.1/hygro( +)-Flag. And the authenticity of all the constructs was confirmed by DNA sequencing. PGK1 CDS was amplified using primers described below:
Full length PGK1 | AATGCGGCCGCATGTCGCTTTCTAACAAG | GCCTCTAGACTAAATATTGCTGAGAGCATC |
PGK1 M1 | AATGCGGCCGCATGTCGCTTTCTAACAAG | GCCTCTAGATACACAGTCCTTCAAGAACAG |
PGK1 M2 | AATGCGGCCGCGGCCCAGAAGTGGAGAAA | GCCTCTAGAGGCCTTTGCAAAGTAGTTCAG |
PGK1 M3 | AATGCGGCCGCTTGGAGAGCCCAGAGCGA | GCCTCTAGATTGGCCAGTCTTGGCATTCTC |
PGK1 M4 | AATGCGGCCGCGCCACTGTGGCTTCTGGC | GCCTCTAGACTAAATATTGCTGAGAGCATC |
PGK1 ΔM4 | AATGCGGCCGCATGTCGCTTTCTAACAAG | GCCTCTAGATTGGCCAGTCTTGGCATTCTC |
LipofectamineTM 2000 reagent was used for plasmids transfection. Lentivirus carrying NEAT1 shRNA (the target sequence of NEAT1-shRNA is GCGCAAGTTAGCCACAAAT) was purchased from Ribobio (China) and transfected into GBM cells according to the manufacturer’s instruction.
RNA extraction and qRT-PCR analysis
Total RNA from U251 and T98G cells was extracted using TRIzol reagent (Invitrogen, CA) as previously described [
15]. qRT-PCR was performed using SYBR Green Premix Ex Taq (TaKaRa) according to the manufacturer’s recommendations. The sequence information of NEAT1 primers used in this study was shown below.
Forward: CCAGTTTTCCGAGAACCAAA.
Reversed: ATGCTGATCTGCTGCGTATG.
Protein extraction and western blot analysis
Protein samples preparation and western blot analysis were performed as previously described [
15]. Briefly, glioma cells were lysed using RIPA lysis buffer supplemented with protease inhibitors. Next, cell lysates were centrifugated at 12,000
g for 15 min at 4 ℃. The protein samples were separated by 10% SDS-PAGE and transferred to nitrocellulose filter membranes (Millipore). 5% nonfat milk was used to block the membranes for 2 h. After washing for three times using PBS/Tween-20, the membranes were incubated with the primary antibodies. Antibodies against CDK2 (#18048, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), CDK6 (#13331, Cell Signaling Technology), PGK1 (#68540, Cell Signaling Technology), Flag (F9291, Sigmaaldrich), HA (#3724, Cell Signaling Technology), and GAPDH (#5174, Cell Signaling Technology) were used in this study. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) was used to visualize subsequent. The western blotting results was quantified using Image J software.
CCK-8 assays
CCK-8 assays were performed to evaluate cell growth. Briefly, 2000 transfected U251 and T98G cells were seeded into a 96-well plates. CCK-8 reagent was added at indicated time and incubated for 1 h at 37 ℃. The absorbance was detected at 450 nm.
Colony formation assays were performed to evaluate cell growth. Briefly, 400 transfected U251 and T98G cells were seeded into a 6-well plate. And cells were maintained until colonies formed. Colonies were then fixed by paraformaldehyde for 30 min and stained using crystal violet. The colonies were then calculated for further analysis.
The XF Glycolysis Stress Test kit was used for the ECAR assay. Briefly, 1 × 104 transfected U251 and T98G cells were seeded into the Seahorse SF 96 cell culture microplates. 10 mM glucose, 1 um oligomycin, and 75 mM 2-DG were added to the cell medium successively at indicated time. Next, Seahorse SF-96 Wave software was used to analyze the results. ECAR was presented in mpH/min.
Measurement of intracellular Lactate levels
The accumulation of lactate in transfected glioma cells was measured using lactate assay kit II (Sigma Aldrich, MAK065). Briefly, glioma cells were homogenized and centrifuged at 13,000g for 10 min. The supernatant was collected and deproteinized using a 10 kDa MWCO spin filter to remove lactate dehydrogenase. Reaction Mixes were prepared according to manufacture. Add 50 μl mix to a 96-well plate and incubated the reaction for 30 min at room temperature. Measure the absorbance at 450 nm.
RNA pulldown assay
Biotin-labeled RNA NEAT1 was transcribed in vitro using the T7 High Yield RNA Transcription Kit (Vazyme) and Pierce™ RNA 3’ End Desthiobiotinylation Kit (Thermo Fisher). Next, Pierce™ Magnetic RNA–protein Pull-Down Kit (Thermo Fisher) was used to obtain the RNA–protein binding mixture. Mass spectrometry was performed to identify target proteins.
RNA immunoprecipitation (RIP) assay
Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore) was used for RIP assays. Briefly, whole cell lysis was extracted from 1 × 105 U251 and T98G cells. The protease inhibitor cocktail and RNase inhibitor were added into the cell lysis for 5 min. Meanwhile, magnetic beads were incubated with IgG or Flag antibodies for half an hour at room temperature. After centrifugating at 1000g for 10 min, the supernatant of cell lysis was incubated with coated beads and incubated at 4 ℃ overnight. At last, the purified RNA was quantified by qRT-PCR.
Animal study
The animal manipulations were approved by Animal Core Facility of Nanjing Medical University. 1 × 106 U251 cells, which were transfected with luciferase reporter, were suspended in 10 ul of DMEM, and intracranially injected into nude mice (female, 6-week-old). The tumor growth was monitored by bioluminescence imaging system at indicated time. At the end, mice were euthanatized by cervical dislocation under anesthesia, and the tumors from brains were fixed and embed using paraffin for further IHC analysis.
Immunohistochemistry analysis
Paraffin-embedded clinical human and mice samples were sliced into 4 mm slides followed by dewaxing and rehydration. Antibodies against ki-67 (#9027, Cell Signaling Technology) and PGK1 (ab233135, abcam) were incubated with tissues overnight at 4 ℃. The protein expression was detected using DAB (Gene Tech). The IHC score was judged by two independent pathologists. The following proportion scores were assigned: 0, 1, 2, 3, 4, and 5 if 0%, 0%–1%, 2%–10%, 11%–30%, 31%–70%, and 71%–100% of the tumor cells exhibited positive staining, respectively. Also, the staining intensity was rated on a scale of 0 to 3: 0, negative; 1, weak; 2, moderate; and 3, strong. A total score was obtained by adding the proportion score and intensity score.
Statistic analysis
Student t test or one-way ANOVA was performed to evaluate differences between two groups or between more than two groups, respectively. Unless stated otherwise, experiments were performed for three time and data represent the mean ± SD. Overall survival rates were calculated by the Kaplan–Meier method with the log-rank test applied for comparison. p < 0.05 was considered as statistically significant. All statistical analyses were performed using GraphPad Prism 8 software.
Discussion
Recent studies have demonstrated that lncRNAs dramatically participate in many biological processes of tumors [
17,
18]. Glioma is the most lethal tumor in central nervous system of adults. Despite of a great deal of researches on the treatment of glioma, the prognosis of glioma patients remains poor [
1,
19]. LncRNAs have been considered to be involved in metabolism reprogramming, proliferation, invasion, radio- and chemo-resistance of glioma [
20‐
24]. NEAT1 is a transcript of the multiple endocrine neoplasia type 1 (MEN1) gene located on human chromosome 11. It has been identified to be involved in various biological and pathological processes such as neurodegenerative diseases, viral infection [
25‐
29]. At the same time, NEAT1 is one of the most investigated lncRNAs in glioma. Studies revealed that NEAT1 promotes glioma progression via activating several important signaling pathways, including mTOR and Wnt signaling. In nuclear, NEAT1 could interact with EZH2 to promotes H3K27 trimethylation levels, resulting the activation of Wnt signaling pathway [
10]. In cytoplasm, NEAT1 functions as a ceRNA by sponging miR-185-5p to activate mTOR signaling [
30]. Additionally, NEAT1 dysregulation was found to be involved in regulating the expression of multiple significant genes by recruiting or sequestering RNA-/DNA-binding proteins to or from promoters or target gene transcripts to influence gene transcription, splicing, RNA stability, or translation [
31,
32]. However, the associated proteins of NEAT1 in cytoplasm have not been investigated. In this study, using RNA pulldown and mass spectrum analysis, we identified NEAT1 specifically interacted with PGK1 and blocked the degradation of PGK1. NEAT1 promotes glioma proliferation and glycolysis through regulating PGK1.
PGK1 is a pivotal ATP-generating enzyme in the glycolysis process, catalyzing the conversion of ADP and 1,3-BPG into ATP and 3-PG [
33,
34]. Recent studies have shown that PGK1 is overexpressed and functions as an oncogene in numerous tumors, including glioma [
35,
36]. The expression and kinase activity of PGK1 could be regulated by posttranslational modification. Ubiquitination is one of the most common and important modification of PGK1 [
37]. PGK1 Ubiquitination level was regulated by lots of factors, including lncRNAs. In lung cancer, MetaLnc9 promoted migration and invasion of NSCLC cells. Mechanistic research indicated that MetaLnc9 specifically interacted with PGK1, which blocked PGK1 ubiquitination to activate AKT/mTOR signaling pathway [
38]. In this study, we found for the first time that NEAT1 specifically interacted with PGK1 to block the ubiquitination of PGK1. Moreover, we precisely found that hairpin A of NEAT1 and M1 domain of PGK1 were the key connection points.
Clinically analysis using CGGA, TCGA datasets and our samples, we revealed that NEAT1 is overexpressed in GBM than low grade glioma and normal brain tissues. In addition, NEAT1 level was associated with poor prognosis of glioma patients. NEAT1 and PGK1 expression levels were positively correlated in glioma tissues, indicating NEAT1 and PGK1 are potential biomarkers for predicting glioma patients prognosis.
Collectively, our study found that NEAT1 enhances glioma cell proliferation and glycolysis in vitro and in vivo. Meanwhile, NEAT1 interacts with PGK1 increasing the stability of PGK1. Finally, we identified the role of NEAT1 as a potential biomarker and therapeutic target for glioma treatment.
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