Background
Gliomas are tumors that initiate from precursor or glial cells and include astrocytoma (including glioblastoma), oligodendroglioma, ependymoma, oligoastrocytoma (mixed glioma) and malignant glioma and account for about 25.5% of all primary brain and other central nervous system tumors and 80.8% of malignant tumors [
1]. The current clinical treatment of glioma includes surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy; however, the prognosis of glioma remains unfavorable with a high recurrence rate after initial treatment [
2]. Diffuse glioma comprises no more than 1% of all newly diagnosed cancers, though, it leads to high mortality and morbidity, especially the most lethal type, glioblastoma, which takes place with 70–75% of all glioma cases with a median overall survival of 14–17 months [
3]. Developing novel therapeutic options and identifying new molecular mechanisms are of great importance in glioma control.
Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are two large classes of non-protein-coding transcripts that participate in diverse essential cellular processes through multiple mechanisms [
4,
5]. LncRNAs are larger than 200 nucleotides, and their importance in gene expression, key cellular processes, metastasis and disease prognosis in cancer has been largely studied [
6,
7], including in glioma [
8,
9]. miRNAs are 20–25 nucleotides in length, and they down-regulate gene expression by binding with target mRNAs post-transcriptionally thus resulting in transcript degradation [
10]. miRNAs exert critical functions in fundamental cellular process such as cell proliferation, apoptosis, development and inflammation [
11]. Likewise, dysregulation of several miRNAs has been found to participate in human glioma progression [
12,
13]. Importantly, a competing endogenous RNA (ceRNA) theory proposed recently, which suggests that non-coding RNAs and protein-coding RNAs work as ceRNAs through competing for miRNAs via shared miRNA recognition elements [
14,
15], has aroused wide concerns. Several ceRNA networks have been validated to play key functions in metastasis, proliferation, and apoptosis in human glioma cells [
16,
17]. The possible ceRNA networks in glioma and the mechanisms involved remain largely unknown.
To this end, this study first figured out the representative differentially expressed lncRNAs through the glioma microarrays, and lncRNA NCK1-AS1, which has been noted to be aberrantly expressed in several human cancers [
18,
19] with its role in glioma remaining unknown, was selected as the subject of this study. We further determined tripartite motif-containing 24 (TRIM24) as an indirect target of NCK1-AS1 through the miR-138-2-3p sponge. TRIM24 is a member of the TRIM family with its oncogenic role being identified in prostate cancer [
20]. In addition, TRIM24 has been suggested to promote cancer progression by triggering the Wnt/β-catenin signaling pathway [
21,
22]. This pathway has been noted to be closely associated with cellular proliferation, migration, and angiogenesis and the following glioma malignancy [
23]. We assumed that NCK1-AS1 could affect glioma progression through the miR-138-2-3p/TRIM24 network and the following Wnt/β-catenin pathway, with gain- and loss-of functions of these molecules performed in both cell and animal experiments to validate this hypothesis.
Materials and methods
Clinical tissue sample collection
A total of 12 pairs of normal brain tissue samples and 32 pairs of glioma tissue samples were acquired from the Zhejiang Provincial Hospital of Traditional Chinese Medicine. Normal brain tissues collected from epileptic patients who subjected to surgery were used as control. The study gained the approval of the Clinical Ethical Committee of the Zhejiang Provincial Hospital of Traditional Chinese Medicine. All procedures were conducted as per the Declaration of Helsinki. All eligible participants signed the informed consent.
RNA-in situ hybridization (RNA-ISH)
RNA-ISH of lncRNA NCK1-AS1 was performed using an ISH assay kit (Boye Biotechnology Co., Ltd., Guangzhou, Guangdong, China). In brief, the tissue samples were fixed, embedded in paraffin, and warm-incubated in gradient alcohol and then in 3% H2O2 for 30 min. Then, the streptavidin-horseradish peroxidase (HRP) conjugate and biotin conjugate probes were introduced into the samples for hybridization. Then samples were then stained with hematoxylin and observed under an optical microscope (Leica, Solms, Germany).
Cell culture
Normal human glial cell line HEB from Yan-Yu Bio-technology Co., Ltd. (Shanghai, China) (
http://www.hdbsw.com/) and 4 glioma cell lines U251, SHG-441, U87 and T98 (ATCC, Manassas, USA) were incubated in DMEM (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL) (37 °C with 5% CO
2). The cells were passaged when they reached an 85% confluence.
Cell treatment and grouping
The glioma cells were trypsinized to 2 × 106 cells/mL. Then the cell suspension was sorted into 12-well plates at 1 mL per well and incubated in 5% CO2 at 37 °C. Next, the cells were transfected with different vectors using the Lipofectamine 2000 (Thermo Fisher, MA, USA) as per the protocols and allocated into the corresponding groups. Three batches of grouping were performed. As for the first batch, cells were allocated into over-expression (oe)-negative control (NC) group (oe-NC group, cells were transfected with NC of NCK1-AS1 oe vector), oe-NCK1-AS1 group (cells were transfected with NCK1-AS1 oe vector), short hairpin (sh)-NC group (cells were transfected with NC of shRNA of NCK1-AS1) and sh-NCK1–AS1 (cells were transfected with sh-NCK1-AS1).
As for the second batch, cells were allocated into mimic NC group (cells were transfected with mimic NC), miR-138-2-3p mimic group (cells were transfected with miR-138-2-3p mimic), inhibitor NC group (cells were transfected with inhibitor NC) and miR-138-2-3p inhibitor group (cells were transfected with miR-138-2-3p inhibitor).
In terms of the third batch, cells were allocated into mimic NC group, miR-138-2-3p mimic group, inhibitor NC group, miR-138-2-3p inhibitor group, miR-138-2-3p mimic + oe-NC group (cells were transfected with miR-138-2-3p mimic and NC of NCK1-AS1 oe vector) and miR-138-2-3p mimic + oe-TRIM24 group (cells were transfected with miR-138-2-3p mimic and TRIM24 oe vector).
After transfection, the cells were incubated in the transfection solution for 4 h and then in normal cell culture medium for following experiments. The sequences for the vectors or mimic are listed in Supplementary Table
1.
Dual luciferase reporter gene assay
The wide type (WT) sequence based on the binding site between NCK1-AS1 and miR-138-2-3p and the corresponding mutant type (MUT) sequence were inserted into the target sequence of the psiCheck2 vector to construct psiChech2-NCK1-AS1-WT vector and psiChech2-NCK1-AS1-MUT vectors. The psiChech2-TRIM24-WT psiChech2-TRIM24-MUT vectors were constructed in a similar manner. Next, well-constructed reporter vectors were co-transfected with miR-138-2-3p mimic or mimic NC into HEK293T cells, and then the luciferase activity was determined in accordance with the instructions of a dual luciferase reporter assay system (Promega, Madison, WI, USA). Three independent experiments were performed.
5-ethynyl-2′-deoxyuridine (EdU) labeling assay
Exponentially growing cells were sorted into 24-well plates, and to each group, 3 duplicated cells were set up. Next, EdU (RiboBio Co., Ltd., Guangdong, China) was filled into the culture medium and adjusted to 10 μmol/mL. After 2 h of incubation in 5% CO2 at 37 °C, the medium was absorbed, and the cells were fixed in 4% paraformaldehyde-supplemented phosphate buffer saline (PBS) for 15 min, washed twice in 3% bovine serum albumin (BSA)-supplemented PBS, and then incubated in PBS supplemented with 0.5% Triton-100 for 20 min. Following two 3%BSA-PBS washes, each well was filled with 100 μL Apollo® 567 (RiboBio) and incubated for 30 min without light exposure at room temperature. Then the cells were washed twice in 3% BSA-PBS again, and stained with 1× Hoechst 33342 for 30 min. Thereafter, the wells were washed 3 more times in PBS, sealed, and observed under a fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan) with 5 views randomly selected. The positive cells, which were stained in red under the scope, were counted and recorded. The procedures were repeated 3 times.
Scratch test
Forty-eight hours after transfection, each group of cells were sorted into 6-well plates at 5 × 105 cells/well. A 200 μL pipette tip (21–0200, Biologix, Shanghai, China) was used to produce a scratch through the midline of the wells when the confluence reached 85%. Then the floating cells were washed away with PBS, while the remaining cells were continually incubated in serum-free medium for 1 h of recovery. At 0 h and 24 h after recovery, the cells were photographed to measure the migration of cells on an Image-Pro Plus Anaysis software (Media Cybernetics, USA). The experiment was performed in triplicate.
Transwell assay
Extracellular matrix (ECM) gel was allowed to stand at 4 °C overnight. The next day (all the pipette tips and Transwells were pre-cooled on ice for half an hour), the gel was diluted in serum-free medium at 1:9 till the final concentration reaching 1 mg/mL. Each apical chamber of the 24-well Transwells was loaded with 40 μL ECM gel on the polycarbonate membrane and then placed in a 37 °C incubator with 5% CO2 for 5 h to polymerize the ECM gel. Next, the remaining liquid was removed, and each well was filled with 70 μL DMEM and incubated at 37 °C with 5% CO2 for 30 min to hydrate the gel again with the remaining culture medium discarded. After that, each group of cells were hungered in a serum-free condition for 24 h, detached, centrifuged, and diluted in FBS-free DMEM to 2.5 × 105 cells/mL. Then 0.2 mL suspension was loaded in each apical chamber where the substrate membraned was already hydrated, while each basolateral chamber was loaded with 700 μL pre-cooled 10% FBS-DMEM. The chambers were then incubated in 5% CO2 with saturated humidity at 37 °C for 24 h. Thereafter, the apical chambers were removed, and the cells in apical chambers and on the substrates were discarded using wet cotton swabs. The remaining cells were methanol-fixed, stained by 0.1% crystal violet, air dried, and photographed under a microscope with 5 random fields (200 ×) observed, and the average volume of invaded cells was calculated. The experiments were performed in triplicate.
Flow cytometry
Cells were washed 3 times in PBS 48 h after transfection and centrifuged at 3000 r/min for 20 min to discard the supernatant, and then diluted in PBS to adjust the concentration to 1 × 105 cells/mL. Then the suspension was successively treated with 1 mL − 20 °C pre-cooled 75% ethanol for 1 h of fixing, centrifuged for 5 min at 1500 r/min, washed in PBS, treated with 100 μL Rnase A (Thermo Fisher) without light exposure, bathed in 37 °C water for 30 min, and stained by 400 μL propidium iodide (PI) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) at 4 °C for 30 min. Next, the cell cycle was measured based on the red fluorescence at the 488 nm (excitation wavelength).
In terms of apoptosis detection, the cells were detached in EDTA-free trypsin (Thermo Fisher) 48 h after transfection and filled into the flow cytometer tubes. Following 30 min of centrifugation at 3000 r/min and 3 cold PBS washes, the cells were further centrifuged at 3000 r/min for 20 min to discard the supernatant. Then the Annexin-V-FITC/PI staining solution was compounded by HEPES buffer (Thermo Fisher), Annexin-V-FITC and PI (50:1:2). Then the cells were stained for 15 min at room temperature, and then treated with 1 mL HEPES buffer. After vibration, the cell apoptosis was measured on the flow cytometer at 488 nm.
Fluorescence in situ hybridization (FISH)
The sub-cellular localization of NCK1-AS1 in glioma cell line was assessed according to the protocols of a lncRNA FISH probe Mix (RiboBio). In brief, cover glasses were put into 6-well plates on which glioma cells were seeded. One day later when the cell confluence got to 80%, the cell slides were collected, washed in PBS, and treated with 1 mL 4% paraformaldehyde. Next, the cells were given Protease-K (2 μg/mL), glycine and acetylating agent, and then cultured with prehybridization agent (250 μL) at 42 °C for 1 h, and then with hybridization agent (250 μL) containing probes (300 ng/mL) at 42 °C overnight. After 3 PBST washes, the cells were treated with PBST-DAPI (1:800) for nucleus staining for 5 min. Next, the cell slides were washed in PBST (3 × 3 min), sealed with anti-fluorescence quencher, and then observed under the fluorescence microscope (× 400) with 5 random fields included.
RNA immunoprecipitation (RIP)
A RIP kit (Millipore Corp., Billerica, MA, USA) was applied to measure the binding relation between NCK1-AS1 and AGO2. Glioma cells were washed in PBS. Then the cells were lysed in an equal volume of RIPA cell lysis solution (Beyotime, Shanghai, China) on ice for 5 min, and then centrifuged at 14000 r/min at 4 °C to collect the supernatant. A part of cell lysis was used as Input, and another part was cultured with the antibodies for coprecipitation. In each coprecipitation system, 50 μL magnetic beads were resuspended in RIP wash buffer (100 μL) and then incubated with 5 μg antibodies according the grouping. The magnet bead-antibody compounds were resuspended in RIP wash buffer (900 μL), and further cultured with 100 μL cell lysis overnight at 4 °C. The samples were set on the magnet base to obtain the magnet bead-antibody compounds. The samples and Input were respectively detached with protease K with the RNA extracted for the following reverse transcription quantitative polymerase chain reaction (RT-qPCR). The antibodies are AGO2 (ab32381, 1:50) and immunoglobulin G (IgG, 1:100, A109489) (all provided by Abcam Inc., Cambridge, MA, USA).
RNA pull-down
Glioma cells were either transfected with MUT-biotinylated or WT-biotinylated miR-138-2-3p (50 nM for each). Cells were harvested 48 h after, washed in PBS, and whirled. Then the cells were cultured for 10 min in specific cell lysis solution (Ambion, Austin, Texas, USA). The cell lysates were co-cultured with RNase-free and yeast tRNA-precoated M-280 streptavidin beads (all from Sigma-Aldrich) for 3 h at 4 °C and then washed twice in cold lysis solution, 3 times in low salt buffer, and once in high salt buffer. The combined RNA was purified with Trizol, and the NCK1-AS1 expression was evaluated using RT-qPCR.
Western blot analysis
Glioma cells and tissues were lysed in protease inhibitor-supplemented enhanced RIPA lysis solution (Boster, Wuhan, China) and then to have the total protein collected, and a bicinchoninic acid kit (Boster) was applied to assess the protein concentration. The proteins were run on 10% SDS-PAGE and transferred onto PVDF membranes. Then the membranes were sealed in 5% BSA for 2 h to block non-specific binding, and then cultured with the primary antibodies (Table
1) at 4 °C overnight. Afterwards, the membranes were washed 3 times in PBST and incubated with horseradish peroxidase-labeled goat-anti-rabbit (ab205719, 1:2000, Abcam) for 1 h at room temperature. Then the membranes were further washed in PBST and measured using the enhanced chemiluminescence reagent (EMD Millipore, USA). The signal intensity of the protein bands was analyzed using Image J software with the value of GAPDH set as the internal reference. The procedures were conducted for 3 times.
Table 1
Antibodies used in western blot analysis
PCNA | ab18197 | 1:1000 |
N-cadherin | ab76011 | 1:5000 |
MMP-9 | ab38898 | 1:1000 |
Bcl-2 | ab196495 | 1:1000 |
Bax | ab53154 | 1:1000 |
Wnt1 | ab15251 | 1:5000 |
Wnt3a | ab28472 | 1:2000 |
β-catenin | ab6302 | 1:4000 |
GAPDH | ab37168 | 1:2000 |
RT-qPCR
The Trizol reagent (15,596,026, Invitrogen Inc., Carlsbad, CA, USA) was utilized to obtain the total RNA of cells and tissues. Reverse transcription was performed using a PrimeScript RT reagent kit (RR047A, Takara Holdings Inc., Tokyo, Japan) to produce cDNA. Quantification was performed as per the instructions of a RT-qPCR assay kit (Thermo Fisher). The relative expression of miR-148a was normalized by a U6 transcript. Three duplicate wells were set for each group. U6 was set as the internal reference for miR-138-2-3p while GAPDH for other genes. Relative gene expression was evaluated using the 2
-ΔΔCt method. The primers were synthetized by TransGen Biotech (Beijing, China) and the sequences are presented in Table
2.
Table 2
Primer sequences for RT-qPCR
NCK1-AS1 | F: 5′-GCCGCAGGAGAGACTTAACA-3′ |
R: 5′-CCTTCGGCTGGGATGACATT-3′ |
miR-138-2-3p | F: 5′-GCGACCCGATTGTCCATAAG-3′ |
R: 5′-AGCAAGCCTGTCTGATGTGA-3′ |
TRIM24 | F: 5′-CATATGCAGCAACAGCAACCG-3′ |
R: 5′-GAAAGCCATCTGTAGGGGGT-3′ |
β-catenin | F: 5′-CATATGCAGCAACAGCAACCG-3’ |
R: 5′-GAAAGCCATCTGTAGGGGGT-3’ |
U6 | F: 5′-CTCGCTTCGGCAGCACA-3’ |
R: 5′-AACGCTTCACGAATTTGCGT-3’ |
GAPDH | F: 5′-GAAGACGGGCGGAGAGAAAC-3’ |
R: 5′-CCATGGTGTCTGAGCGATGT-3’ |
Immunofluorescence staining
The expression of stemness-related proteins SOX2 (1:1000, ab97959, Abcam) and OCT4 (1:1000, ab18976, Abcam) in U251 and SHG441 cells was determined as guided by a previous study [
25].
U251 and SHG-441 cells were seeded into 6-well ultra-low attachment culture dishes (Corning Incorporated, Corning, NU, USA). Then the cells were cultured in serum-free DMEM/F12 supplemented with 1% B27, 20 ng/mL human epidermal growth factor (EGF) and 20 ng/mL human fibroblast growth factor (FGF) at 37 °C for 2 weeks to form tumor spheres. Two weeks later, the number of newly formed tumor spheres were counted under an optical microscope (Nikon Corporation, Tokyo, Japan) at a × 200 magnification.
Xenograft tumors in nude mice
Thirty female BALB/c nude mice (3–4 weeks old, 14 ± 2 g, provided by SJA Laboratory Animal Co., Ltd., Hunan, China) were fed at 25–27 °C with controlled humidity (45–50%) with free access to food and water. Then the mice were randomized into 6 groups: sh-NC group, sh-NCK1-AS1 group, mimic NC group, miR-138-2-3p mimic group, sh-NCK1-AS1 + oe-NC group and sh-NCK1-AS1 + oe-TRIM24 group, 5 mice in each. Cells with stable transfection were resuspended and adjusted to 1 × 107 cells/mL, and each nude mouse was implanted with 20 μL cell suspension. The tumor growth was observed and photographed every 7 d for a total of 35 d, and a tumor growth curve was drawn. The tumor volume was calculated as (a × b2)/2, in which “a” refers to the longest diameter while “b” refers to the shortest. The mice were euthanized on the 35th d via intraperitoneal injection of 150 mg/kg pentobarbital to collect and weigh the tumors. Thereafter, the tumor samples were preserved in liquid nitrogen for following RT-qPCR and western blot analysis. Animal studies were conducted as per the principles and procedures ratified by the Committee on the Ethics of Animal Experiments of the Zhejiang Provincial Hospital of Traditional Chinese Medicine. Great attempts were made to minimize the number and pain of animals.
Statistical analysis
SPSS 21.0 (IBM Corp. Armonk, NY, USA) was applied for data analysis. Data were in normal distribution. Measurement data were exhibited as mean ± standard derivation (mean ± SD). Differences between every two groups were analyzed using the t test, while those among multiple groups were analyzed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. Differences among multiple groups at different time points were analyzed using repeated measurement ANOVA and Bonferroni test. The Log-rank test was used for single-factor analysis. The p value was acquired from two-tailed tests, and p < 0.05 was considered to show significant difference.
Discussion
It is quite necessary to develop new potential target for glioma treatment since the overall 5-year survival rate of patients with glioma remains lower than 5% even following comprehensive chemotherapy, radiotherapy and surgery [
26]. LncRNAs can sponge miRNAs and block these miRNAs from regulating the target mRNAs, thus mediating target genes post transcriptionally [
27]. In light of this theory and several recently identified ceRNA networks in cancers, the study figured out a novel NCK1-AS1/miR-138-2-3p/TRIM24 ceNRA network in glioma.
The evidence concerning the correlation between abnormal lncRNA expression with cancers has been growing [
28,
29]. Initially, data on glioma microarrays suggested that NCK1-AS1 is aberrantly highly expressed in glioma, which was further identified by RT-qPCR. As for the other screened out lncRNAs, lncRNA CRNDE, for example, has already been noted to promote glioma cell growth [
30], while LINC00998, has hardly been investigated in human cancers. NCK1-AS1 is a relatively newly recognized lncRNA with its oncogenic role in many several human malignancies [
19,
31,
32] reported, thus, was selected as the current study subject. Then, we found that silencing of NCK1-AS1 led to decreased proliferation, invasion and migration abilities while increased cell cycle arrest and apoptosis of glioma cells. Moreover, silencing of NCK1-AS1 was also found to inhibit the expression of CSC biomarkers SOX2 and OCT4 in our study. As CSCs are a class of self-renewal cells with strong tumorigenic potency and are more resistant to conventional therapies than other cancer cells [
33]. This finding further evidenced the promoting role of NCK1-AS1 in the malignant behaviors of glioma cells. The same trends were reproduced in in vivo experiments, which showed that silencing of NCK1-AS1 inhibited tumor formation and growth in nude mice.
The findings above triggered us to identify the downstream mechanisms involved in the events. As above mentioned, lncRNAs might exert functions through the crosstalk with miRNAs and mRNAs [
14,
15]. Therefore, we analyzed the potential target miRNAs of NCK1-AS1 and the following genes via integrated online bioinformation system, glioma microarrays, dual luciferase reporter gene, RNA pull-down and RIP assays, after which we identified the interactions among NCK1-AS1, miR-138-2-3p and TRIM24. Importantly, we noticed that up-regulation of miR-138-2-3p decreased proliferation, invasion and migration while promoted apoptosis of SHG-441 cells with over-expressed NCK1-AS1, showing as decreased levels of PCNA, N-cadherin, MMP-9 and Bcl-2 while increased level of Bax. But artificial over-expression of TRIM24 led to opposite trends in U251 cells with silenced NCK1-AS1. PCNA is an important replication accessory factor that supports DNA replication, repair and recombination, and cell cycle regulation [
34,
35]. Abnormal expression of the N-cadherin is a crucial biomarker of epithelial-to-mesenchymal transition in many cancer types, promoting the aggressiveness of tumors [
36]. As one of the most studied MMPs, MMP-9 is well-known for the key roles in invasion of cancer cells and metastasis of tumors [
37]. Bcl-2 is an anti-apoptotic protein that mediates apoptosis by regulating the permeability of the mitochondrial membrane, while Bax can break the outer mitochondrial membrane thus promoting apoptosis [
38]. In addition, hsa-miR-487a-5p and hsa-miR-184 were also identified as NCK1-AS1 targets after integrated analysis. However, the role of miR-184 in glioma has been largely studied [
39,
40], while the role of miR-487a-5p in cancer development has been little concerned. miR-138-2-3p is a rarely mentioned miRNA, though, some same trends were found in a previous study [
41], which suggested that over-expression of miR-138-2-3p decreased proliferation and invasion but promoted apoptosis of human laryngeal CSCs following radiotherapy. Meanwhile, lowly-expressed miR-138-2-3p has been found in drug-resistant non-small cell lung cancer patients [
42]. As for TRIM24, it is a member of the tripartite motif (TRIM) family [
20], and the oncogenic role of TRIM24 in carcinogenesis has been demonstrated in several cancer types [
43‐
45]. Moreover, our study identified that inhibited miR-138-2-3p or promoted TRIM24 stimulated Wnt/β-catenin activation in glioma cells, which was in line with the previous studies [
21,
41]. Wnt/β-catenin controls diverse cellular processes and drives cancer progression [
46], with its activation revealed to promotes tumorigenesis of multiple cancers [
47,
48], including glioma [
23]. Taken the above discussion together, in can be inferred that NCK1-AS1 could promote the malignant behaviors of glioma cells and tumor metastasis through the crosstalk with miR-138-2-3p and TRIM24, and the following activation of the Wnt/β-catenin pathway.
By the way, GLIS2, the other intersected target gene of miR-138-2-3p, is a member of GLI-similar zinc finger protein family and is closely linked with acute myeloid leukaemia, has been also noted to correlated with tumor progression [
49,
50]. We would like to investigate if GLIS2 plays roles in glioma progression in our future experiments.
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