What this is
- This research investigates the role of SNHG12 in glioblastoma (GBM) and its contribution to () resistance.
- The study identifies that SNHG12 is upregulated in -resistant GBM cells and tissues due to DNA demethylation and SP1 transcription factor binding.
- SNHG12 acts as a sponge for miR-129-5p, leading to increased expression of MAPK1 and E2F7, which promotes cell proliferation and inhibits apoptosis.
Essence
- SNHG12 promotes resistance in glioblastoma by regulating the MAPK/ERK pathway through miR-129-5p sponging. Its expression is linked to poor patient survival.
Key takeaways
- SNHG12 is significantly upregulated in -resistant GBM cells and tissues. Its overexpression correlates with poor overall survival in GBM patients undergoing treatment.
- Loss of DNA methylation in the SNHG12 promoter enhances its expression via increased binding of the SP1 transcription factor. This mechanism is crucial in regulating SNHG12 levels in -resistant cells.
- SNHG12 functions as a sponge for miR-129-5p, leading to elevated levels of MAPK1 and E2F7. This interaction contributes to cell cycle progression and reduced apoptosis in GBM.
Caveats
- The study's findings may not be universally applicable due to the heterogeneity of GBM tumors, which can affect treatment responses and resistance mechanisms.
- Further research is needed to fully elucidate the complex regulatory networks involving SNHG12 and its interactions with other molecular pathways in GBM.
Definitions
- lncRNA: Long non-coding RNA, a type of RNA longer than 200 nucleotides that regulates gene expression.
- temozolomide (TMZ): An oral chemotherapy drug used as a standard treatment for glioblastoma.
AI simplified
Background
Gliomas comprise the most common primary brain tumor and more than half of these are glioblastoma tumors, the most malignant of all the brain tumors. Even with the most aggressive treatment, the median survival of glioblastoma (GBM) patients is still less than 15 months [1, 2]. Temozolomide (TMZ) is a second-generation oral alkylating agent that readily passes through the blood-brain barrier, and it is the standard first-line chemotherapy for the clinical treatment of glioblastoma [3, 4]. However, improvements in the prognosis for these brain tumors are slow to fruition, owing to therapeutic resistance and postoperative tumor recurrence [5]. Thus, elucidating the underlying mechanisms of TMZ resistance and exploring reliable biomarkers to predict TMZ response in GBM patients are urgently needed.
Long non-coding RNA (lncRNA) is a class of heterogeneous RNA with a length of more than 200 nucleotides. LncRNAs play important roles in tumorigenesis, through effects on dose compensation, epigenetic regulation, cell cycle regulation, and drug resistance [6, 7]. Epigenetic changes have been identified as one of the hallmarks of tumorigenesis [8], and emerging evidence shows that epigenetic regulation is one of the main mechanisms regulating lncRNA expression and tissue specificity [9, 10]. However, the epigenetic regulation of lncRNAs and the subsequent effect on tumor progression, especially in terms of acquired chemoresistance, remain largely unknown. LncRNAs have been propounded to act as competitive endogenous RNA (ceRNA) that compete for microRNA (miRNA) binding, thereby playing a significant role in gene regulation [11, 12]. The ceRNA network participates in the mediation of postoperative treatment resistance in some cancers but its role in TMZ resistance is rarely reported.
In this study, we investigated the role of lncRNA SNHG12 (small nucleolar RNA host gene 12) in acquired TMZ resistance in GBM and the effect of epigenetic regulation on its abnormal expression. Our results showed that SNHG12 is epigenetically activated by DNA methylation at the CpG islands within its promoter region. SNHG12 regulates the MAPK/ERK signaling pathway and G1/S cell cycle transition by competitively binding to miR-129-5p, which, in turn, modulates TMZ resistance in GBM cells.
Methods
Patients and specimens
The 40 primary GBM specimens and 20 recurrent GBM specimens used in this study were obtained by surgical resection from patients undergoing TMZ chemotherapy (Department of Neurosurgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China). The experiment has passed ethical review by the medical ethics committee of the First Affiliated Hospital of Nanjing Medical University (Ethics number: 2019-SR-479). The diagnosis of glioma was confirmed by pathologists. Detailed patient information is presented in Additional file: Table S1. 1
Public data collection
Microarray datasets and their associated clinical information were downloaded from the Chinese Glioma Genome Atlas (CGGA; http://www.cgga.org.cn↗) and the Rembrandt microarray database (http://caintegrator.nci.nih.gov/rembrandt/↗), and raw microarray data from the Gene Expression Omnibus (GEO) databases were used to detect differential expression of SNHG12 (https://www.ncbi.nlm.nih.gov/geo/↗; including GSE4290↗, GSE7696↗, GSE15824↗, GSE50161↗, GSE59612↗, and GSE104267↗).
Cell lines and cell culture
The human embryonic kidney (HEK) 293 T cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). N3 patient-derived cells were obtained from the China National Clinical Research Center for Neurological Diseases, Beijing Tian Tan Hospital. Six drug-related cell lines (Pri GBM, Rec GBM, N3S, N3T3rd, U251, and U251T3rd) were as described in our previous report [13]. All cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2.
RNA extraction and quantitative real-time PCR assays
Total RNA was extracted from tissues and cell lines with the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The nuclear and cytoplasmic fractions were separated with the PARIS Kit (Invitrogen, CA, USA). cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara, Nanjing, China). Real-time quantitative PCR (qRT-PCR) analyses were performed with the SYBR Green Premix Ex Taq (Takara, Nanjing, China). The total RNA levels were normalized with GAPDH. U6 snRNA (small nuclear RNA) was used as the miRNA internal control. Relative RNA expression levels were measured with the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Primer sequences are shown in Additional file 2: Table S2. The relative quantitative value for each gene was determined as 2−∆∆CT.
Western blot assay
Western blot analysis was performed by following the manufacturer’s protocol as previously described [13]. Antibodies used are listed in Additional file 3: Table S3.
Plasmid construction and cell transfection
Lipofectamine 2000 (Invitrogen) was used to transfect the siRNAs, miRNA mimics, and plasmids into the GBM cells. All small interfering RNA (siRNA) and short hairpin RNA (shRNA) sequences designed for specific targets are listed in Additional file: Table S4. We synthesized full-length complementary cDNAs of human SNHG12, SP1, and MAPK1, and cloned these cDNAs into the expression vector pcDNA3.1 (Invitrogen). SNHG12 shRNAs and the negative control RNA (sh-Ctrl) were designed and synthesized by Genechem (Shanghai, China). Rec GBM and N3T3rd cells were used to establish stable cell lines and selected with puromycin at 48 h after injection. SP1 siRNA, MAPK1 siRNA, E2F7 siRNA, miR-129-5p mimics, and the miR-129-5p inhibitor were purchased from Genechem. 4
Methylation-specific PCR and bisulfite sequencing
Genomic DNA was extracted from GBM and normal tissues with a QIAamp DNA Mini Kit (Qiagen). The purified DNA was exposed to bisulfite with an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. Using the GeneAmp PCR System 2700 (Applied Biosystems, Grand Island, NY, USA), the methylation-specific PCR (MSP) of bisulfite-transformed DNA was carried out with a nested, two-stage PCR method. Amplified PCR products were separated by 3% agarose gel electrophoresis and visualized with GelRed (Vazyme, Nanjing, China). For bisulfite-sequencing PCR (BSP), bisulfite-converted genomic DNA was amplified using specific BSP primers and the sequencing library was prepared with the VAHTS Turbo DNA Library Prep Kit (Vazyme, Nanjing, China). The specific primers used for MSP and BSP are listed in Additional file: Table S2. 2
Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis
FISH analysis was performed on human tissues and GBM cells as previously described [14]. IHC was performed on mice xenogeneic tumor tissues as previously described [14].
Flow cytometry analysis of cell cycle transitions and apoptosis
For cell cycle analysis, the cells were harvested 24 h after serum starvation and fixed overnight in 70% ethanol at 4 °C. Cells were incubated with propidium iodide (PI) staining solution before flow cytometry detection. For apoptosis analysis, the cells were stained with PI and Annexin V-FITC according to the manufacturer’s instructions (Roche, Basel, Switzerland).
TUNEL assay
For the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assays, GBM cells were fixed in 4% paraformaldehyde for 15 min. Cells were then stained with the In Situ Cell Death Detection Kit, POD (Roche, Switzerland) according to the manufacturer’s protocol. Images were acquired with a Nikon ECLIPSE E800 fluorescence microscope.
CCK-8 and colony formation assays
GBM cells were seeded in 96-well plates and cell viability was evaluated with the Cell Counting Kit 8 (Dojindo, Shanghai, China). Absorbance was measured (OD value) at a wavelength of 450 nm.
For the colony formation assay, cells were seeded in six-well plates and cultured for 11 days with or without TMZ treatment. The resulting colonies were washed twice with PBS, fixed with 4% formaldehyde for 10 min, and stained with 0.1% crystal violet for 30 min.
Luciferase reporter assays
For the luciferase reporter assay at the SNHG12 promoter region, HEK293T cells were co-transfected with luciferase reporter packaging the sequence of SNHG12 promoter region and the empty vector, or the TFAP2A, TFAP4, SP1, STAT1 or IKZF1 plasmid (Genechem, Shanghai, China). The promoter region only contained the P1, P2 or P3 regions and these sequences were synthesized and cloned into the pGL3-basic luciferase reporter vector (Promega, Madison, USA). For miRNA target gene luciferase reporter assays, SNHG12, MAPK1, and E2F7 wild-type sequences with potential miR-129-5p-binding sites or mutants of each binding site were synthesized and co-transfected into N3T3rd and Rec GBM cells. All luciferase activities were measured with the Dual Luciferase Reporter Assay System (Promega) and normalized to Renilla luciferase activity.
Immunofluorescence
Cells were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.25% Triton X-100 (Beyotime, Shanghai, China) at room temperature. The cells were blocked with 1% bovine serum albumin for 20 min and then incubated with primary antibody at 4 °C overnight. After washing with PBS three times, the cells were incubated with goat anti-rabbit IgG secondary antibodies (FITC Green goat anti-rabbit; Molecular Probes, Shanghai, China) for 1 h at room temperature. The nucleic acids were stained with DAPI (Sigma-Aldrich, Shanghai, China). The images were captured with a Nikon ECLIPSE E800 fluorescence microscope.
RNA immunoprecipitation (RIP)
The RIP experiments were performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. GBM cell lysates were prepared and incubated with RIP buffer containing magnetic beads conjugated with human anti-argonaute-2 (anti-Ago2) antibody (Cat. ab32381; Abcam). Normal mouse IgG (Cat. 12–371; Millipore) functioned as the negative control. The RNA fraction precipitated by RIP was analyzed by qPCR.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed with an EZ-ChIP Kit (Millipore) according to the manufacturer’s instructions. Briefly, GBM cells were cross-linked with 1% formaldehyde for 10 min and then quenched with glycine. Cell lysates were then sonicated to generate chromatin fragments and then immunoprecipitated with H3K4me3 antibody (Cat. 39,915; Active Motif, Shanghai, China), H3K27me3 antibody (Cat. 39,155; Active Motif, Shanghai, China) or SP1 antibody (Cat. 9389; Cell Signaling Technology), and IgG antibody (Cat. 12–371; Millipore) was used as the negative control. The ChIP primer sequences are listed in Additional file: Table S2. 2
In vivo xenograft model
Four-week-old female BALB/c nude mice were purchased from the Experimental Animal Center of Nanjing Medical University. To establish the intracranial tumor model, 2.5 × 105 recurrent GBM cells were separately implanted stereotactically into the nude mice brain. After surgery, the mice were treated with or without TMZ by oral gavage at 1 week (66 mg/kg per day for 5 days). Bioluminescence imaging (IVIS Spectrum; PerkinElmer, USA) was used to confirm intracranial tumor formation and tumors were measured each week. The procedures used for animal treatments and experiments conformed with the Guide for the Care and Use of Laboratory Animals and this study was approved by the Nanjing Medical University Animal Experimental Ethics Committee.
Statistical analysis
All statistical analyses were performed with GraphPad software version 7.0 (GraphPad Software, San Diego, CA, USA) or IBM SPSS Statistics 23.0 software (SPSS, Chicago, IL, USA). The significance of the differences between groups was estimated with Student’s t test, chi-square test or one-way analysis of variance (ANOVA) as appropriate. The Kaplan–Meier method with the log-rank test was used to calculate the overall survival (OS) rate for comparison between different groups. The correlations between variables were analyzed with the Pearson correlation coefficient. GO and KEGG pathway analyses were performed on the DAVID website (https://david.ncifcrf.gov/↗). DIANA tools (http://diana.imis.athena-innovation.gr/↗) and miRcode (http://www.mircode.org/↗) were used to predict lncRNA-targeting miRNAs. All results are shown as the mean ± standard error of the mean (SEM) of three independent experiments. Statistical significance was considered to be represented by a value of p < 0.05. Additional file 5: Table S5-7.
Results
SNHG12 is highly expressed in TMZ-resistant GBM cell lines and tissues after TMZ treatment
Recent studies have shown that lncRNA-regulated TMZ-resistance mechanisms in GBM can be key nodes of therapeutic intervention. For example, lncRNA MALAT1 has the ability to promote TMZ resistance in GBM [15]. To determine whether lncRNAs play a crucial role in acquired TMZ-resistance, qRT-PCR of three paired TMZ-sensitive and TMZ-resistant GBM cell lines was used to detect the expression levels of three lncRNAs after TMZ treatment. SNHG12 expression was significantly up-regulated in all of the TMZ-resistant cells (Fig. 1c and Additional file 6: Figure S1a). These cells revealed poor response to TMZ as illustrated by an increased half-maximal inhibitory concentration (IC50) and enhanced proliferation ability when undergoing TMZ treatment (Additional file 6: Figure S1b–c). In addition, a high level of SNHG12 was found in two other GEO datasets (GSE59612↗ and GSE104267↗) and SNHG12 expression was found to be relatively high in the recurrent GBM tissues analyzed in this study compared with the primary GBM tissues in GSE7696↗ (this dataset describes samples of recurrent GBM tissues after receiving TMZ treatment) (Additional file 6: Figure S1d). Next, we measured SNHG12 levels in five normal brain tissues, 40 primary GBMs, and 20 recurrent GBMs (insensitive to TMZ treatment) by qRT-PCR. As shown in Fig. 1d, SNHG12 was significantly up-regulated in the GBMs compared with normal tissues. Furthermore, SNHG12 expression was markedly increased in recurrent GBM tissues compared with primary GBM tissues. Similar results were also observed with FISH (Fig. 1e).
Next, Kaplan–Meier analysis was used to determine whether SNHG12 expression levels in the GBM tissues were associated with clinical response to TMZ therapy. Survival analysis of the CGGA cohort revealed that a higher SNHG12 level was associated with poor overall survival (OS) in GBM patients (Additional file 6: Figure S1e). Moreover, Kaplan–Meier analysis of our cohort showed that patients with low SNHG12 expression exhibited a superior OS after TMZ therapy, while patients with high SNHG12 expression exhibited poor response to TMZ therapy (Fig. 1g). Taken together, these results suggest that lncRNA SNHG12 is upregulated in TMZ-resistant GBM cell lines and tissues, pointing to a possible relationship between SNHG12 and acquired TMZ resistance.
SNHG12 is up-regulated in TMZ-resistant GBM tissues and cell lines.Hierarchical clustering analysis of lncRNAs that were differentially expressed in glioma samples.Overlap of lncRNAs in the CGGA, Rembrandt, and GEO data setsThe expression level of SNHG12 in TMZ-resistant and TMZ-sensitive cells.Relative expression of SNHG12 in primary and recurrent GBM tissues. The SNHG12 expression was normalized to β-actin.FISH analysis of SNHG12 expression in normal brain tissues, primary GBM tissues and recurrent tissues. Scale bar = 50 μm.Kaplan-Meier survival analysis of OS in GBM patients with TMZ chemotherapy (= 40,= 0.0085). Data are presented as the mean ± SEM from three independent experiments. Significant results were presented as NS non-significant, *<0.05, **<0.01, ***<0.001, ****<0.0001 a b c d e f n P P P P P
Knockdown of SNHG12 restores TMZ sensitivity in TMZ-resistant GBM cells in vitro
Knockdown of SNHG12 restores TMZ sensitivity in TMZ-resistant cells in vitroqRT-PCR analysis of SNHG12 expression in sh-control, sh-SNHG12–1, sh-SNHG12–2, sh-SNHG12–3 treated TMZ-resistant cells.CCK-8 assay analysis revealed the effect of SNHG12 knockdown on the TMZ-resistant cells after TMZ treatment at the indicated concentrations for 48 h.Heatmaps of SNHG12-associated genes in 83 and 227 glioblastoma tissues sorted by the level of SNHG12 expression in CGGA and Rembrandt data sets.GO analyses were performed using the SNHG12 associated genes in CGGA and Rembrandt data sets.Western blot test of caspase-3 and PARP in TMZ-resistant cells treated with vehicle control or TMZ (200 μM) for 48 h. β-actin was used as the loading control.Flow cytometric analysis revealing the effect of SNHG12 knockdown on the apoptosis of TMZ- resistant cells with or without TMZ treatment (200 μM, 48 h).TUNEL analysis of SNHG12 knockdown cells or vehicle control with or without TMZ treatment (200 μM, 48 h). Scale bar = 50 μm.Colony formation assays of SNHG12 knockdown or vehicle control TMZ-resistant cells with or without TMZ treatment (200 μM, 48 h).Knockdown of SNHG12 decreased the levels of p-Rb, cyclin D1 but not CDK4 and CDK6 in TMZ-resistant cells. β-actin was used as the loading control.The cell cycle distribution was analyzed by flow cytometric analysis in TMZ-resistant cells transfected with sh-ctrl or sh-SNHG12–2. Data are presented as the mean ± SEM from three independent experiments. Significant results were presented as NS non-significant, **<0.01, ***<0.001 . P P a b c d e f g h i j
SNHG12 overexpression confers resistance to TMZ
SNHG12 overexpression confers TMZ resistance.qRT-PCR analysis of SNHG12 expression in Pri GBM and N3S cells transfected with empty vector or pcDNA-SNHG12.Western blot analysis of caspase-3 and PARP in TMZ-sensitive cells with or without TMZ treatment (200 μM, 48 h). β-actin was used as the loading control.Flow cytometric analysis revealing the effect of SNHG12 overexpression on the apoptosis of TMZ-sensitive cells with or without TMZ treatment (200 μM, 48 h).TUNEL analysis of SNHG12 overexpression cells or empty vector with or without TMZ treatment (200 μM, 48 h). Scale bar = 50 μm.Colony formation assays of SNHG12 overexpression or empty vector TMZ-sensitive cells with or without TMZ treatment (200 μM, 48 h).Overexpression of SNHG12 increased the levels of p-Rb, cyclin D1 but not CDK4 and CDK6 in TMZ-sensitive cells.The cell cycle distribution was analyzed by flow cytometric analysis in TMZ-sensitive cells transfected with empty vector or pcDNA-SNHG12. Data are presented as the mean ± SEM from three independent experiments. Significant results were presented as NS non-significant, *<0.05, **<0.01 a b c d e f g P P
DNA methylation and SP1 regulate the expression of SNHG12
Several studies have shown that transcription factors play an important role in regulating the expression of lncRNAs and DNA-methylated genes [18, 19]. We, therefore, used the JASPAR database (http://jaspar.genereg.net/↗) to perform bioinformatic analysis of the promoter region of SNHG12 to predict potential binding sites for transcription factors. Dual luciferase reporter assays were then used to detect the binding activity of the transcription factors among the top five predictions. HEK293T cells were transfected with a luciferase plasmid containing the promoter region of SNHG12 together with plasmids containing either the individual transcription factors or the control sequence. SP1 showed maximum luciferase activity (Fig. 4e) and depletion of SP1 decreased SNHG12 expression, while overexpression of SP1 increased SNHG12 levels (Fig. 4f and Additional file 8: Figure S3d). Furthermore, the expression levels of SP1 and SNHG12 were positively correlated in 20 recurrent GBM tissues, which was consistent with the analysis of CGGA and Rembrandt primary GBM tissue data (Fig. 4g and Additional file 8: Figure S3e). Next, bioinformatic analysis of the promoter region of SNHG12 predicted three potential binding sites for SP1 (Fig. 4h). A chromatin immunoprecipitation assay (CHIP) found enrichment at Site 3 (containing the BSP1 sequence) in TMZ-resistant cells (Fig. 4i). Furthermore, ChIP assays revealed that the enrichment of SP1 at Site 3 of SNHG12 was significantly increased in TMZ-resistant cells compared with TMZ-sensitive cells and NHAs (Fig. 4j). In order to further explore the mechanism of methylated changes in the promoter region of SNHG12, we detected the expression level of DNA methyltransferases (DNMTs) in TMZ-resistant cells. As is shown in Fig. 4k, DNMT1 expression was downregulated in TMZ-resistant cells compared with the parental TMZ-sensitive cells. Overexpression of DNMT1 led to increased methylation level in SNHG12 promoter region (Fig. 4l). Moreover, overexpression of DNMT1 significantly suppressed SNHG12 expression in TMZ-resistant cells,and knockdown of DNMT1 resulted in restoration of SNHG12 expression in TMZ-sensitive cells (Fig. 4m). ChIP assays revealed that the enrichment of SP1 at Site 3 of SNHG12 was decreased after DNMT1 overexpression, and knockdown of DNMT1 increased SP1 enrichment (Fig. 4n). These results suggest that DNMT1 was involved in epigenetic regulation process in SNHG12 promoter.
Several studies have shown that a high percentage of hypermethylated genes are pre-marked with H3K27me3 modifications, while hypomethylated genes show H3K4me3 modifications [20, 21]. Here, compared to TMZ-sensitive cells and NHAs, H3K27me3 was markedly decreased in TMZ-resistant cells, whereas H3K4me3 was significantly increased in TMZ-resistant cells (Additional file 8: Figure S3f). Thus, these data indicated that loss of DNA methylation makes the promoter region of SNHG12 more accessible to SP1, which leads to transcriptional activation of SNHG12.
DNA methylation and SP1 are involved in the activation of SNHG12.Schematic representation of the CpG islands and bisulfite sequencing; Magenta words, CG sites for bisulfite sequencing; Red region, input sequence; Blue region, CpG islands; BSP1 F1 and R1, BSP2 F1 and R1, bisulfite forward primer and reverse primer.Bisulfite genomic sequencing was performed to examine methylation status of CpG island 1 at the promoter region of SNHG12 in NHA, Pri GBM, Rec GBM, N3S, N3T3rd cells.MSP analysis was performed to examine methylation status of CpG island 1 at the promoter region of SNHG12 in normal brain tissues, primary GBM tissues and recurrent GBM tissues.qRT-PCR analysis detecting the SNHG12 levels in Pri GBM and N3S cells after the treatment with 5-azacytidine for 72 h and 144 h.The luciferase reporter plasmids carrying SNHG12 promoter region were co-transfected into HEK293T cells with five transcription factor plasmids, respectively. Relative luciferase activity in HEK293T cells were determined.The SNHG12 levels were detected in Rec GBM and Pri GBM cells either stably expressing SP1 or with SP1 depleted.The correlation between SP1 and SNHG12 in GBM tissues was analyzed.Predicted SP1-binding sites in the promoter region of SNHG12.ChIP-PCR assay of the enrichment of SP1 on the SNHG12 promoter region.ChIP analysis for the detection of SP1 binding to the promoter region of SNHG12.DNMT profiles using western blottingMSP analysis of CpG island 1 after DNMT1 overexpression.qRT-PCR analysis of SNHG12 expression after DNMT1 overexpression or knockdown.ChIP-PCR assay of the enrichment of SP1 on the SNHG12 promoter region after DNMT1 overexpression or knockdown. Data are presented as the mean ± SEM from three independent experiments. Significant results were presented as NS non-significant, *<0.05, **<0.01, ***<0.001 a b c d e f g h i j k . l m n P P P
SNHG12 serves as a sponge for miR-129-5p
To investigate whether miR-129-5p participates in the SNHG12-mediated mechanism involved in acquired TMZ resistance in GBM, we knocked down or overexpressed miR-129-5p in SNHG12-depleted TMZ-resistant GBM cells. Colony formation assays showed that miR-129-5p overexpression markedly suppressed TMZ resistance in SNHG12-depleted TMZ-resistant cells, while knockdown of miR-129-5p reversed SNHG12 knockdown-mediated suppression of tumor cell proliferation and chemoresistance (Fig. 5g). Similarly, miR-129-5p overexpression increased TMZ-induced apoptosis in SNHG12-depleted TMZ-resistant cells, while miR-129-5p knockdown reversed SNHG12 knockdown-mediated apoptosis in TMZ-resistant cells (Fig. 5h and Additional file 9: Figure S4d-e). Together, these data suggested that SNHG12 functions as a molecular sponge for miR-129-5p, and both of these molecules are involved in the molecular mechanisms underlying acquired TMZ resistance in GBM.
SNHG12 act as a sponge for miR-129-5p in the cytoplasm.FISH analysis indicated subcellular location of SNHG12 in Rec GBM and N3T3rd cells (green). Nuclei were stained by DAPI (blue).Relative SNHG12 expression levels in nuclear and cytosolic fractions of Rec GBM and N3T3rd cells. U6 was used as nuclear controls. β-actin was used as cytosolic controls.RIP experiments were performed using the Ago2 antibody, and specific primers were used to detect the enrichment of SNHG12 and miR-129-5p in Rec GBM and N3T3rd cells.Schematic drawing of the screening procedure of candidate miRNAs.The luciferase reporter plasmids carrying SNHG12 was co-transfected into HEK293T cells with 5 miRNA-coding plasmids.Up: Schematic representation of the miR-129-5p binding sites in SNHG12 and the site mutagenesis. Down: The luciferase reporter plasmid carrying wild type (WT) or mutant (MUT) SNHG12 was co-transfected into Rec GBM and N3T3rd cells with miR-129-5p in parallel with an empty vector. Relative luciferase activity in Rec GBM and N3T3rd cells were determined.Colony formation ability of Rec GBM and N3T3rd cells transfected with SNHG12 plasmid, miR-129-5p mimics + SNHG12 plasmid or miR-129-5p inhibitor + SNHG12 plasmid after 200 μM TMZ treatment for 48 h.Immunofluorescent staining of cleaved caspase-3 in Rec GBM cells transfected with SNHG12 plasmid, miR-129-5p mimics + SNHG12 plasmid or miR-129-5p inhibitor + SNHG12 plasmid after 200 μM TMZ treatment for 48 h. Scale bar = 50 μm. Data are presented as the mean ± SEM from three independent experiments. Significant results were presented as NS non-significant, *<0.05, **<0.01 a b c d e f g h P P
MAPK1 and E2F7 are miR-129-5p target genes and are responsible for SNHG12-mediated temozolomide resistance
MAPK1 and E2F7 are direct targets of miR-129-5p and is suppressed by SNHG12 detection.Schematic drawing of the screening procedure of candidate target genes.After transfected with miR-NC or miR-129-5p in Rec GBM and N3T3rd cells, the expression level of 8 potential targets for miR-129-5p was analyzed using real-time PCR.The luciferase reporter plasmid carrying wild type (WT) or mutant (MUT) MAPK1 or E2F7 was co-transfected into Rec GBM and N3T3rd cells with miR-129-5p in parallel with an empty vector. Relative luciferase activity in Rec GBM and N3T3rd cells were determined.RIP experiments were performed using the Ago2 antibody, and specific primers were used to detect the enrichment of MAPK1 and E2F7.RIP assay of the enrichment of Ago2 on SNHG12, MAPK1 and E2F7 transcripts relative to IgG in Pri GBM and N3S cells transfected with pcDNA-ctrl or pcDNA-SNHG12.RIP assay of the enrichment of Ago2 on SNHG12, MAPK1 and E2F7 transcripts relative to IgG in Rec GBM and N3T3rd cells transfected sh-ctrl or sh-SNHG12.Relative protein levels of E2F7 and MAPK1 in Rec GBM and N3T3rd cells transfected with control miRNA, miR-129-5p-inhibitor or miR-129-5p mimics.MAPK1 and E2F7 protein level in Rec GBM and N3T3rd cells following knockdown of SNHG12.andLuciferase activity of reporters which contained wild-type or mt MAPK1 or E2F7 3’UTR with indicated treatment in Rec GBM cells () and Pri GBM cells ()Relative protein levels of E2F7 and MAPK1 in Rec GBM and N3T3rd cells following knockdown of SNHG12 and/or inhibition of miR-129-5p.Western blot analysis of E2F7 and MAPK1 in Pri GBM cells and N3S cells transfected with pcDNA-Ctrl, pcDNA-SNHG12, or pcDNA-SNHG12mt along with miR-129-5p mimics. Data are presented as the mean ± SEM from three independent experiments. Significant results were presented as NS non-significant, **<0.01, ***<0.001 a b c d e f g h i j i j k l P P
MAPK1 and E2F7 exhibit different functions in SNHG12-mediated temozolomide resistance
The effects of MAPK1 and E2F7 on GBM cell proliferation and survival.CCK-8 assay analysis of the effect of MAPK1 or E2F7 overexpression on Rec GBM cells after knocking down SNHG12 upon TMZ treatment at the indicated concentrations for 48 h.Colony formation analysis of Rec GBM and N3T3rd cells after co-transfected with si-ctrl, si-MAPK1 or si-E2F7.Flow cytometric analysis of TMZ-induced (200 μM, 48 h) apoptosis in Rec GBM and N3T3rd cells transfected with si-ctrl or si-MAPK1.The cell cycle distribution was analyzed by a flow cytometer in Rec GBM and N3T3rd cells.Transfection of sh-SNHG12 or pcDNA-MAPK1 into Rec GBM and N3T3rd cells affected expression of MAPK signaling pathway-associated protein. Expression of the indicated proteins was detected by western blot.Rescue assays for immunoblotting analysis of MAPK signaling pathway-associated protein after transfection of pcDNA-SNHG12 or si-MAPK1 into Pri GBM and N3S cells.Western blot analysis of indicated proteins in Rec GBM cells and N3T3rd cells transfected with sh-SNHG12–2 and miR-129-5p inhibitor.Western blot analysis of indicated proteins in Pri GBM cells and N3S cells transfected with pcDNA-SNHG12 and miR-129-5p mimics. Data are presented as the mean ± SEM from three independent experiments. Significant results were presented as NS non-significant, **<0.01, ***<0.001 a b c d e f g h P P
Knockdown of SNHG12 restores TMZ sensitivity in vivo
Knockdown of SNHG12 restores TMZ sensitivity in TMZ-resistant GBM xenografts.Representative bioluminescence images of intracranial xenografts bearing SNHG12-depleted or control Rec GBM cells in the absence or presence of TMZ treatment on the days as indicated.Immunohistochemistry staining of Ki67, Cleaved caspase-3, MAPK1 and E2F7. Scale bar = 50 μm () Kaplan-Meier survival curve of nude mice is shown.The mechanistic scheme of DNA-methylation-mediated activating of lncRNA SNHG12 in regulating GBM cell proliferation and temozolomide resistance a b c d
Discussion
Surgical resection with postoperative radiotherapy and chemotherapy is currently the standard treatment for glioma patients. Clinical practice has proven that effective chemotherapy after successful surgery is one of the most effective methods for treating malignant glioma, and chemotherapy can significantly improve the survival rate and survival time of patients with malignant glioma. However, acquired TMZ resistance limits the range of therapeutic options available to GBM patients, particularly recurrent GBM patients. Elucidating the molecular mechanisms of TMZ resistance would greatly assist the rational design of combination therapies blocking TMZ chemotherapy resistance.
In recent years, the roles of lncRNAs in cancer have been widely studied. Many reports have shown that lncRNAs participate in several cancer-associated processes including chemoresistance [26, 27], and abnormal expression of lncRNAs have been shown to be involved in the malignant process of glioma [28–30]. However, lncRNA-mediated TMZ-resistant mechanisms have rarely been studied. Here, we identified lncRNA SNHG12 to be aberrantly expressed in TMZ-resistant cells and tissues, resulting in the TMZ-resistant phenotype. SNHG12 was shown to promote TMZ resistance by promoting cell growth and inhibiting cell apoptosis. Knockdown of SNHG12 increased the sensitivity of TMZ-resistant cells toward TMZ, which indicates that SNHG12 may be a key node of treatment intervention as part of GBM therapy.
Similar to protein-coding genes, lncRNA expression is affected by gene dosage and promoter utilization, through factors such as copy-number alterations and epigenetic regulation [31–34]. DNA methylation was one of the first modes of epigenetic regulation to be discovered. The DNA methylation landscape of progressive glioblastoma has been shown to exhibit extensive spatial and temporal heterogeneity, and the analysis of genome-wide DNA methylation patterns has proven helpful for glioma classification and diagnosis [35, 36]. However, aberrant changes in the DNA methylation patterns of lncRNAs are seldom reported. To identify the mechanisms underlying the abnormally high expression of SNHG12, we analyzed the promoter region of SNHG12. We observed abnormally low levels of methylation within this region, resulting in increased SNHG12 expression.
Studies have shown that transcription factors can also regulate the transcription of lncRNAs [37, 38]. We, therefore, investigated whether transcription factors play a role in the aberrant expression of SNHG12 and whether loss of DNA methylation would make the promoter region of SNHG12 more accessible to transcription factors. We found that SNHG12 is activated by the transcription factor, SP1. SP1 has been reported to play an important role in acquired TMZ resistance in GBM [39, 40], and our results revealed that SP1 activated the transcription of SNHG12. Moreover, loss of DNA methylation made the promoter of SNHG12 more accessible to this transcription factor. However, all the factors mediating the methylation of the SNHG12 promoter region have not yet been fully elucidated. Further study is needed to clarify these factors.
The molecular functions of lncRNAs are primarily determined by their subcellular localization [41]. Thus, to further unravel the downstream effects of SNHG12, we analyzed the localization of SNHG12 in TMZ-resistant cells. Our results showed that SNHG12 was expressed in both the nucleus and cytoplasm, with particularly high expression in the cytoplasm. LncRNAs have been shown to act as endogenous decoys for miRNAs, thereby forming complex regulatory networks [11, 42]. In this study, we determined that SNHG12 interacts with Ago2 in TMZ-resistant cells, which indicates that SNHG12 can act as a miRNA sponge. Further, by using bioinformatic analyses and luciferase reporter assays, we found miR-129-5p to be a potential target of SNHG12. Our previous study found that miR-129-5p overexpression could confer chemosensitivity to TMZ in GBM cells [43]. Taken together, these results indicate that the ceRNA network in which SNHG12 participates plays an important role in the mechanism of acquired TMZ resistance in glioma. Further, in the present study, we found that MAPK1 and E2F7 are potential targets of the SNHG12 ceRNA networks, which was validated by luciferase reporter assays and RIP analysis. MAPK1, as a member of the ERK signaling pathway, is associated with the mechanisms of chemoresistance in many cancers [44, 45]. The Ras/Raf/ERK (MAPK/ERK) pathway has been reported to be overactivated in many cancers, thereby promoting the malignant phenotype; it is, therefore, considered to be a potential drug target [46, 47]. E2F7, a member of the E2F family of transcription factors, is overexpressed in many cancers, which can induce cell proliferation in tumors such as glioma [25, 48]. In our study, we found that SNHG12 promotes TMZ resistance by competitively binding miR-129-5p, resulting in the dysregulation of MAPK1 and E2F7. We further validated that SNHG12-mediated G1/S cell cycle transitioning is mainly regulated by E2F7, while cell apoptosis is predominantly modulated by MAPK1. Furthermore, our results indicated that SNHG12 inhibit apoptosis by activating the MAPK/ERK pathway.
It is important to emphasize the potential limitations of this study. Firstly, our findings establish that SNHG12 axis is an important regulator of TMZ resistance. Thus, targeting this axis may present as a potential therapeutic strategy for treating GBM. However, Individual GBM are far from being uniform, and single tumors exhibit substantial cellular heterogeneity that includes small subpopulations termed glioma stem-like cells (GSCs). GSCs have been shown to contribute to tumor initiation, malignant phenotypes, recurrence and therapy-resistance. Drug resistance toward TMZ is multifactorial, involving not only the internal processes of cells but also factors within the GBM microenvironment [49, 50]. Secondly, analysis of recurrent glioma tissue demonstrated increased SNHG12 expression compared to primary, treatment naive tumors. Our previous study noted that serum ncRNAs were higher in GBM patients that responded poorly to TMZ. Given the ease of obtaining serum samples, analyzing serum SNHG12 level following TMZ treatment may be a potential strategy for predicting response to TMZ.
Conclusions
Collectively, our results revealed that lncRNA SNHG12, which is activated by abnormal DNA demethylation and increased binding of transcription factor SP1, induces TMZ resistance in GBM. SNHG12 was found to play an important role in promoting cell proliferation and inhibiting cell apoptosis by acting as a sponge of miR-129-5p, thereby increasing MAPK1 and E2F7 expression and activating the MAPK-ERK pathway. Moreover, we identified that SNHG12 is correlated with poor overall survival and drug sensitivity in the clinic. Therefore, SNHG12 is a promising prognostic biomarker and a potential therapeutic target for temozolomide resistance in GBM.
Supplementary information
Additional file 1: Table S1. Summary of clinical GBM patients.Additional file 2: Table S2. Primer sequence used in this study and SNHG12 promoter sequence.Additional file 3: Table S3. Information of antibodies.Additional file 4: Table S4. Sequences of siRNA and shRNA against specific targets.Additional file 5: Table S5. The differentially expressed lncRNAs in CGGA, Rembrandt and GEO data sets; Table S6. Predicted miRNAs targeting to SNHG12; Table S7. Predicted SNHG12 and miR-129-5p target genes.Additional file 6: Figure S1. SNHG12 is up-regulated in GBM, related to Fig. 1.Additional file 7: Figure S2. SNHG12 levels correlate with temozolomide resistance, related to Figs. 2-3.Additional file 8: Figure S3. DNA methylation and SP1 regulate SNHG12 expression level, related to Fig. 4.Additional file 9: Figure S4. SNHG12 act as a sponge for miR-129-5p in the cytoplasm, related to Fig. 5.Additional file 10: Figure S5. SNHG12 regulates MAPK1 and E2F7 expression by competitively binding miR-129-5p, related to Fig. 6Additional file 11: Figure S6. SNHG12 accelerates temozolomide resistance in GBM cells via MAPK1 and E2F7, related to Fig. 7.