Glioma is the most common malignant brain tumour in adults, and the aetiology and mechanism of this tumour remain largely unknown. Previous studies have demonstrated that the long non-coding RNA X-inactive specific transcript (XIST) is upregulated in many cancers, and a high expression level of XIST is associated with poor clinical outcome. In the present study, the expression and function of XIST were investigated in the glioma cell line U251. XIST and microRNA (miR)-133a levels in glioma cell lines were detected by reverse transcription-quantitative polymerase chain reaction. Small hairpin RNA XIST (sh-XIST) and mimics/inhibitor of miR-133a were transfected in glioma cell lines and cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) were examined. Luciferase assays were used to verify the associations among XIST, miR-133a and SRY-box (SOX)4. When XIST was knocked down, the proliferation, metastasis and EMT of glioma cells decreased. Notably, downstream genes of SOX4 were also upregulated or downregulated upon sh-XIST treatment. Overexpression of miR-133a inhibited glioma proliferation, metastasis and EMT via reducing the expression of SOX4; in contrast, knockdown of miR-133a exhibited the opposite effect, which revealed that miR-133a negatively regulates glioma progression. Furthermore, using luciferase assays, it was demonstrated that XIST and SOX4 could bind miR-133a in the predicted binding site; XIST competed with SOX4 for miR-133a binding. In conclusion, a XIST/miR-133a/SOX4 axis and a mechanism of XIST glioma in promoting cell proliferation and metastasis were revealed. These findings revealed that XIST has an oncogenic role in the tumourigenesis of glioma and may serve as a potential therapeutic target for glioma.
Gliomas are the most pervasive and aggressive major type of primary tumour in the nervous system (
Long non-coding RNAs (lncRNAs), which are a class of transcripts >200 nucleotides in length, have been demonstrated to serve important roles in gene transcription and post-transcription regulation (
SOX4 is a major member of the SOX family that is located on 6p22.3 and encodes a protein of 474 amino acids with three distinguishable domains: A high mobility group box, a glycine-rich region, and a serine-rich region (
In the present study, it was demonstrated that XIST influenced glioma cell proliferation and migration by regulating the miR-133a/SOX4 axis. These findings suggested that XIST is potentially a therapeutic target for glioma.
U251 glioma cell lines, control cell line HEB and the 293 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured at 37°C in humidified 5% CO2. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All media contained 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). The 293 cells were used to investigate the regulatory mechanism between miR-133a and XIST or between miR-133a and SOX4.
On the day prior to transfection, U251 cells were digested by trypsin, counted and seeded in a 6-well plate at 1×105 cells/well. When the cell confluence reached 90%, the medium was changed with serum-free DMEM and incubated at 37°C overnight. An sh-XIST vector was used to achieve knockdown of XIST (GeneCopoeia, Inc.). miR-133a mimics (5′-UUUGGUCCCCUUCAACCAGCUG-3′ and 5′-GCUGGUUGAAGGGGACCAAAU-3′) or an miR-133a inhibitor (5′-CAGCUGGUUGAAGGGGACCAAA-3′; GeneCopoeia, Inc.) were used to achieve miR-133a overexpression and inhibition, respectively. Transient transfections were performed using Lipofectamine 2000 (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Sh-negative control (NC; sense, 5′-GATCCGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAACTTTTTG-3′; and antisense, 5′-AATTCAAAAAGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAACG-3′), mimics NC (5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′) and inhibitor NC (5′-CAGUACUUUUGUGUAGUACAA-3′; GeneCopoeia, Inc.) were used as respective controls. XIST shRNA (sense, 5′-CACCGCTCTTGAACAGTTAATTTGCTTCAAGAGAGCAAATTAACTGTTCAAGAGCTTTTTTG-3′ and antisense, 5′-GATCCAAAAAAGCTCTTGAACAGTTAATTTGCTCTCTTGAAGCAAATTAACTGTTCAAGAGC-3′) or miRNA (hsa-miR-133a mimics, 5′-UUUGGUCCCCUUCAACCAGCUG-3′ and 5′-GCUGGUUGAAGGGGACCAAAUU-3′; and hsa-miR-133a inhibitor, 5′-CAGUACUUUUGUGUAGUACAA-3′) was transfected into the indicated cell lines for 24 h at 37°C. Transfection efficiency was determined via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cells were transfected with mimics or inhibitors at a final concentration of 100 nM.
Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. cDNA was synthesized by reverse transcription of total RNA using the All-in-One™ First-Strand cDNA Synthesis kit (GeneCopoeia, Inc.) according to the manufacturers' instruction at room temperature. Then, using cDNA as the template, the gene expression levels were analysed by qPCR conducted using iTaq™ Universal One-Step SYBR RT-qPCR kits on a real-time PCR system (both Bio-Rad Laboratories, Inc.). The qPCR conditions were 95°C for 5 sec and 60°C for 15 sec, followed by 70°C for 15 sec, for 45 cycles. The relative gene expression level was calculated using the 2−ΔΔCq method (
Cells were washed with cold PBS twice and lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing the protease inhibitor for 30 min on ice. Following centrifugation at 12,000 × g and 4°C for 15 min, the protein concentration was determined using the enhanced bicinchoninic acid BCA protein assay kit (Beyotime Institute of Biotechnology). The supernatant was loaded on a 10% SDS-PAGE gel (10 µg total protein/lane) and proteins were separated. Proteins were then transferred to a polyvinylidene difluoride membrane, blocked with 5% bovine serum albumin (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature and incubated with primary antibody at 4°C overnight. The primary antibodies used in the present study were as follows: Anti-Sox4 antibody (1:1,000; sc-518016; Santa Cruz Biotechnology, Inc.), anti-E-cadherin antibody (1:1,000; sc-59778; Santa Cruz Biotechnology, Inc.), anti-vimentin antibody (1:1,000; sc-373717; Santa Cruz Biotechnology, Inc.), anti-N-cadherin antibody (1:1,000; sc-53488; Santa Cruz Biotechnology, Inc.), anti-α-catenin antibody (1:1,000; sc-9988; Santa Cruz Biotechnology, Inc.) and anti-β-actin antibody (1:5,000; #4970; Cell Signaling Technology, Inc.) Membranes were washed with TBS containing 0.1% Tween-20 five times and then incubated with donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody at room temperature (1:10,000; sc-2313; Santa Cruz Biotechnology, Inc.) for 1 h. Membranes were finally washed in TBS and developed with diaminobenzidine horseradish peroxidase color development kit (Beyotime Institute of Biotechnology) for chemiluminescence substrate. Densitometric analyses were performed using a chemiluminescence imaging system (model 5200; Tanon Science and Technology Co., Ltd.) and built-in Tanon MP software (version 2014).
U251 cells were suspended in DMEM culture medium containing 10% FBS and placed in a 96-well plate with approximately 2,000 cells/200 µl volume in each well. These cells were incubated under the condition of 5% CO2 and 37°C for 24 h. Then, 20 µl MTT (5 mg/ml) was added in every well, and these cells continued to be cultured for an additional 4 h. Culture medium was removed, and 150 µl DMSO was added for the dissolution of the crystals on the shaker for 10 min. Optical density values were measured at 490 nm.
Migration ability was determined using a wound-healing assay. U251 cells were plated into 12-well plates without antibiotics, and cells were transfected with sh-XIST or the control or with miR-133a mimics, miR-133a inhibitor or the control. Then, 24 h later, transfected cells were wounded with a sterile plastic 100 µl micropipette tip, the floating debris were washed with PBS, and the remaining cells were cultured in serum-free medium at 37°C. The width of the wound was measured at 0 and 24 h.
First, 80 µl diluted Matrigel was put into the upper chamber of a 24-well Transwell chamber (Corning Inc.) and incubated at 37°C for 30 min for gelling. U251 cells were harvested, and the cells were suspended in serum-free DMEM/F12. Then, all cells in various groups were placed in the upper chamber with 200 µl serum-free DMEM, and 600 µl DMEM culture medium containing 10% FBS was placed in the bottom chamber. Cells were incubated at 37°C for 20 h, and cells that migrated or invaded the lower surface of the membrane were removed from the 24-well plates. Cells adhering to the membrane were stained with crystal violet for 10 min at room temperature, and each insert was counted at ×100 magnification using a bright field microscope (Model DC 300F; Leica Microsystems GmbH).
Luciferase reporter gene assay was implemented using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's instructions. Cells were seeded into 24-well plates and transfected with a wild-type (wt)-XIST luciferase reporter gene vector, a mutant (mut)-XIST vector containing a 6-bp mutation on the predicted miR-133a binding site within XIST, a wt-SOX4 3′UTR vector, or a mut-SOX4 3′UTR vector (all from Shanghai GenePharma Co., Ltd.) containing a mutation in the predicted miR-133a binding site in the 3′UTR of SOX4, along with the miR-133a mimic or mimic NC using Lipofectamine 3000 (Life Technologies; Thermo Fisher Scientific, Inc.). Following 48 h, cells were lysed using passive lysis buffer (Promega Corporation) and the luciferase activity was detected. Luciferase activity was normalized against
Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS software 16.0 (SPSS, Inc.). Statistical significance was measured using Student's t-test or one-way analysis of variance in conjunction with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
The relative expression levels of XIST and miR-133a were detected via RT-qPCR in the glioma cell line U251. The data revealed that XIST was upregulated in U251 compared with that in the controlled HEB cells (
To evaluate the association of XIST expression with glioma cells' proliferation and metastasis. XIST knockdown was achieved by sh-XIST transfection, and the inhibitory efficiency was verified by RT-qPCR (
MTT assays revealed that knocking down XIST reduced cell proliferation of U251 cell lines (
Mimics or an inhibitor of miR-133a were used to assess the role of miR-133a in glioma cell behaviour. It was first demonstrated that elevated miR-133a expression could lower XIST expression in U251 glioma cells. In contrast, inhibitor of miR-133a significantly suppressed the level of miR-133a and elevated the level of XIST (
The above results collectively revealed that XIST and miR-133a served opposite roles in glioma cell proliferation, migration and invasion, as well as in the expression of SOX4 and EMT behaviour. A possible model was proposed in which XIST competed with SOX4 for miR-133a binding. To validate this hypothesis, four luciferase reporter gene vectors were constructed (wt-XIST luciferase reporter, mut-XIST vector containing a 6-bp mutation within the predicted miR-133a binding site within XIST, wt-SOX4 3′UTR, and mut-SOX4 3′UTR vector containing a 6-bp mutation within the predicted miR-133a binding site in the 3′UTR of SOX4;
A previous study revealed that ~18% of lncRNAs are associated with human tumours, and that only 9% of human protein-coding genes perform this function (
The lncRNA XIST is upregulated in many cancers (
The SOX4 transcription factor belongs to a large family of proteins that serves a fundamental role during embryogenesis and controls cell fate and differentiation (
Recently, an increasing number of studies (
lncRNAs are widely associated with regulating gene expression networks at epigenetic, transcriptional and post-transcriptional levels, which may affect the growth, proliferation, differentiation, metabolism, apoptosis and other important physiological processes of cells (
The present study still has some limitations at its current stage. For example,
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No funding was received.
All data generated or analysed during this study are included in this published article.
CL substantially contributed to the conception of this study and drafting the manuscript. CT, ZQ and BaZ were involved in the study conception and design. MZ performed the literature research and experimental studies. BoZ and ZQ performed data acquisition and edited the manuscript. SW and XL performed data and statistical analysis. CT was involved in revising it critically for important intellectual content. All authors read and approved the final version of the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
The expression of XIST and miR-133a in glioma cell lines. (A) XIST expression was higher in U251 compared with the control HEB cells. (B) miR-133a expression was lower in U251 compared with the control HEB cells. n=3. **P<0.01 vs. HEB. XIST, X-inactive specific transcript; miR, microRNA.
Effect of downregulation of XIST on glioma cell proliferation and metastasis. (A) Following transfection with sh-XIST, XIST was significantly downregulated. The transfection of sh-XIST further potentiated miR-133a in U251 cells. (B) Cell viability of U251 lines following sh-XIST transfection. (C) Cell migration (magnification, ×16) and (D) cell invasion assay of glioma cells (magnification, ×100). (E) Western blot results demonstrated the expression levels of E-cadherin N-cadherin, α-catenin and vimentin in glioma cells following sh-XIST transfection. (F) Expression levels of SOX4 transcription factor. n=3, *P<0.05, **P<0.01 and ***P<0.001 vs. sh-NC. XIST, X-inactive specific transcript; sh, small hairpin RNA; miR, microRNA; SOX, SRY-box; NC, negative control; OD, optical density.
Effects of miR-133a on glioma cell proliferation and metastasis. (A) Relative expression levels of miR-133a and XIST in glioma cells following transfection with miR-133a mimics or inhibitor. (B) Cell viability following transfection with miR-133a mimics or inhibitor assessed by MTT assay. (C) Migration (magnification, ×16) and (D) invasion (magnification, ×100) of cell lines detected by scratch-wound healing and Transwell migration assays, respectively. (E) Western blotting showing the expression of E-cadherin, N-cadherin, α-catenin and vimentin in glioma cells following transfection with miR-133a inhibitor or mimics. (F) Expression level of SOX4 transcription factor. n=3. *P<0.05, **P<0.01 and ***P<0.001. miR, microRNA; XIST, X-inactive specific transcript; SOX, SRY-box; NC, negative control; OD, optical density.
Binding affinity between SOX4, XIST and miR-133a. (A) Predicted complementary binding sites between SOX4 or XIST and miR-133a. (B) Binding affinity was detected via dual luciferase reporter assays using wild-type or mutant forms of XIST and miR-133a. (C) Dual luciferase activity demonstrating binding between miR-133a and SOX4 with wt or mut forms. n=3. ***P<0.001. SOX, SRY-box; XIST, X-inactive specific transcript; miR, microRNA; wt, wild-type; mut, mutant; NC, negative control.