Background
Glioblastoma (GBM) is the most common primary malignant brain tumor in adults [
1]. Multimodality treatment such as cytoreductive surgery followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ) chemotherapy has been widely accepted as the new standard of care for patients with newly diagnosed GBM. However, the prognosis of TMZ-treated patients remains dismal, with a median survival of 12.1–14.6 months [
2‐
4]. Intrinsic or acquired chemoresistance to TMZ is a major clinical obstacle for the treatment of GBM patients. Therefore, a better understanding of the molecular mechanisms underlying TMZ chemoresistance may lead to improved clinical outcomes in GBM patients.
Recently, several studies have shown that anticancer therapies can induce autophagy, which constitutes a novel mechanism of chemoresistance in cancer [
5‐
8]. Autophagy is a highly evolutionarily conserved process that occurs in virtually all eukaryotic cells and has been implicated in various physiological and pathological conditions [
9]. In some cases, autophagy induces apoptotic death in GBM cells upon TMZ treatment, and treatment with autophagy inducer rapamycin can further enhance chemotherapy-induced apoptosis [
10‐
14]. In other cases, TMZ-induced autophagy may delay cell death [
15‐
17]. Therefore, modulation of autophagy in response to TMZ treatment may hold great promise for circumventing chemotherapeutic resistance and improving anticancer efficacy in GBM patients. However, the roles of autophagy in regulating GBM cell death and survival remain controversial.
MicroRNAs (miRs) are small, non-coding RNA molecules (20–22 nucleotides in length) that negatively regulate gene expression by binding to the 3′-untranslated region (3′UTR) of target mRNAs [
18]. miRs have been shown to be key players in a wide range of biological processes, including proliferation, apoptosis, and migration [
19]. Recent evidence has indicated that miRs can regulate the chemosensitivity of glioma cells to TMZ by modulating autophagy signaling [
20]. Previously, we demonstrated that
miR-519a is closely related to improved prognosis of GBM patients [
21]. However, the molecular mechanisms underlying the role of
miR-519a in the chemoresistance of GBM remain unclear.
Signal transducer and activator of transcription 3 (STAT3) functions as a signal messenger and transcription factor, which regulates the transcription of downstream target genes during malignant transformation and tumor development. Several studies have demonstrated that STAT3 overexpression in glioma cells can promote tumor progression [
22‐
24]. A growing body of evidence has implicated STAT3 in the regulation of autophagy, from the assembly of autophagosomes to their maturation [
25]. In addition, differential localization of STAT3 may regulate autophagy in distinct ways [
25]. For instance, nuclear STAT3 may upregulate BCL2 expression and lead to autophagy inhibition [
26]. Therefore, a better understanding of the role of STAT3 signaling in regulating autophagy may provide new insights into the mechanisms of chemoresistance and the potential strategies to overcome TMZ chemoresistance in GBM.
In the present study, we evaluated whether miR-519a can affect the chemosensitivity of TMZ in GBM. Furthermore, the roles of miR-519a in the modulation of autophagy via STAT3/Bcl-2/Beclin-1 signaling pathway were investigated.
Methods
Cell lines and reagents
U87-MG cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO
2. The methods for culturing patient-derived GBM cell line G131212 were described previously [
21]. TMZ-resistant cell lines were generated by iterative pulse exposure of U87-MG and G131212 GBM cells to TMZ. The derived resistant cell lines were designated as U87-MG/TMZ and G131212/TMZ, respectively. Meanwhile, a stock solution of TMZ (100 mM; cat. no. T2577; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO; cat. no. D2650; Sigma-Aldrich) and stored at − 20 °C. 3-Methyladenine (3-MA; cat. no. M9281; Sigma-Aldrich) was prepared freshly in DMEM at 60 °C and then diluted to 5 mM before use.
Oligonucleotides and siRNA transfection
The miRNA mimic, miRNA inhibitor, STAT3 siRNA, and scrambled siRNA were synthesized by RiBoBio (China). The oligonucleotide sequences were listed in Table S1 (Additional file
1: Table S1). The miRNA overexpression vector pCMV-MIR519A (MI0003182) and the empty vector control were obtained from OriGene (Rockville, MD, USA). The
miR-519a sponge and empty vector control were purchased from GeneChem (China). Transfections were performed using Lipofectamine 2000 reagent (cat. no. 11668-019; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Cell viability assay
Cell viability was assessed by using MTT assays. First, cells were seeded in 96-well plates at a density of 8000 cells per well. After an overnight incubation, the cells were treated under the indicated conditions. At the end of the treatment, 0.5 mg/mL MTT was added to each well and incubated for 4 h. Then, the supernatants were aspirated carefully, and formazan crystals were dissolved in DMSO. Finally, the absorbance was measured at 550 nm using Thermo Varioskan Flash reader (Thermo Fisher Scientific, Waltham, MA, USA).
Cells (200 cells/well) were seeded onto 6-well culture plates and cultured in DMEM supplemented with 10% FBS. The cells were treated with the indicated agents and incubated for 10–14 days at 37 °C and 5% CO2. Colonies were then stained with 0.1% crystal violet (Sigma-Aldrich) and counted. In some experiments, cells were pre-treated for 1 h with 3-MA (Sigma-Aldrich) or rapamycin (Abcam, San Francisco, CA, USA), followed by an incubation period of 24 h. For each set of clones, three independent assays were carried out.
Cell apoptosis assay
GBM cells were transfected with miRNAs (or anti-miRNAs) and/or incubated with 400 μM TMZ for 36 h. Subsequently, cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) for apoptotic analysis. The percentage of apoptotic cells was calculated as the sum of early and late apoptotic cells located in the lower and upper right quadrants, respectively.
Green fluorescent protein-LC3 puncta assay
GBM green fluorescent protein (GFP)-LC3 stable cells were transfected with miRNAs or anti-miRNAs. Two days after the transfection, cells were fixed with 4% paraformaldehyde. GFP-LC3 dot formation was observed under a confocal laser scanning microscope (FLUOVIEW FV10i; Olympus, Japan). The average number of GFP-LC3 dots/cell was counted in at least 200 cells.
Transmission electron microscopy (TEM)
GBM cells were subjected to different treatments. The freshly harvested tumors from mice were fixed overnight with 2.5% glutaraldehyde at 4 °C and post-fixed in 1% osmic acid. The fixed samples were then dehydrated using a graded series of ethanol (70–100%) and embedded in EPON resin. Ultrathin sections were cut with an ultramicrotome and double-stained with uranyl acetate and lead citrate. The stained sections were then examined using a TEM (H-7650; Hitachi, Tokyo, Japan).
Construction of stable lentiviral clones
Lentiviral expressing GFP empty vector (NC-LV), GFP vector overexpressing
miR-519a (LV-
miR-519a), or GFP vector inhibiting
miR-519a expression (LV-anti-
miR-519a) was constructed by Systems Biosciences Inc. (Mountain View, CA, USA). Virus production and cell transduction in GBM cells were performed as previously described [
21], and cells were selected in puromycin (1 μg/mL). The selected cells were sorted by flow cytometry to maintain a GFP-positive rate for at least 95%.
Western blotting
Western blotting was performed as described previously [
27]. The antibodies including anti-LC3B (cat. no. 4445), anti-BECN1/Beclin (cat. no. 3495), anti-STAT3 (cat. no. 12640), anti-phospho-STAT3 (Tyr705; cat. no. 9145), and anti-CASP3/caspase-3 (cat. no. 9915) were purchased from Cell Signaling Technology, while anti-Bax (cat. no. sc-7480) and anti-Bcl-2 (cat. no. sc-509) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA.
RNA isolation and quantitative reverse transcription polymerase chain reaction
Total miRNA from cultured cells was extracted using TRIzol reagent (cat. no. 15596-026; Invitrogen), and the RNA purity was evaluated by A260/A280 ratio of 1.9–2.0. cDNA was synthesized from 1 μg of total RNA using PrimeScript RT reagent kit (cat. no. RR047A; Takara, Shiga, Japan). The primers used for PCR amplification were listed in Table S2 (Additional file
2: Table S2). The expression levels of target genes were quantified on a Stratagene Mx-3005p instrument (Agilent Technologies Inc., USA) by using Maxima SYBR Green/ROX qPCR Master Mix (cat. no. K0222; Thermo Scientific). Triplicate samples were examined.
Tumor xenograft assays in nude mice
BALB/c nude mice (4–5 weeks old) were provided by the Experimental Animal Center of Southern Medical University. U87-MG cells with stable expression of lentivirus miR-519a or U87-MG/TMZ cells with stable expression of miR-519a shRNA were injected into the left flank of the mice, while control cells were injected into the right flank of the mice. Mice were injected intraperitoneally (i.p.) with phosphate-buffered saline alone (control) or TMZ (Merck Co., NJ, USA; 20 mg/kg/mouse) once every other day for 3 weeks, starting on day 3. Tumor volume and animal weight were assessed every 4 days. Tumor volume was calculated using the following formula: volume (mm3) = 4/3 × 3.14 × radius (mm)3. Mice were humanely sacrificed on day 24. Tissue blocks were subjected to immunohistochemical staining and TEM analysis.
For survival analysis in the orthotopic xenograft model, nude mice were randomly divided into four groups: LV-anti-miR-519a group, LV-anti-NC group, LV-anti-NC+TMZ group, and LV-anti-miR-519a+TMZ group. In this model, 3 × 105 cells were stereotactically implanted into the right striatum of the mice. Twenty four days after injection, tumor burden of mice was assessed using magnetic resonance imaging (MRI) scanner (Bruker Medical Inc., Billerica, MA, USA). The number of surviving nude mice was recorded, and survival analysis was performed by Kaplan-Meier survival curves.
Patient samples
In this study, 24 patients with recurrent GBM treated with TMZ before second surgery and 24 patients with primary GBM without TMZ treatment were recruited from Nanfang Hospital (Guangzhou, China). Tissue samples were retrieved from the Department of Pathology and subjected to sectioning process. Subsequently, the tissue sections were fixed and immunohistochemically stained with anti-LC3B anti-STAT3, and anti-CASP3/caspase-3 antibodies (cat. no. 4445, 12649, and 9915, respectively; Cell Signaling Technology, Danvers, MA, USA).
Statistical analysis
All experiments were performed in triplicate and repeated at least once. All data were expressed as means ± standard deviation. If the homogeneity of variance assumption was met, one-way analysis of variance (ANOVA) was performed to determine the differences between groups, while least significant difference (LSD) test was used to compare the means of two groups. If the variance was heterogeneous, Welch test was applied to compare the differences between groups, while Dunnett’s T3 test was used for pairwise comparisons. p values of less than 0.05 were considered statistically significant.
Discussion
GBM is the most common type of malignant brain tumor [
30]. Even after multimodality treatment including radical surgery, radiation, and chemotherapy, the median survival time of GBM is approximately 1 year from diagnosis. De novo and acquired resistance to TMZ in GBM cells have emerged as a challenging problem in clinical practice [
31]. Therefore, identifying the mechanisms underlying TMZ chemoresistance shed light on a novel combination therapy strategy to circumvent acquired resistance in GBM patients. Numerous studies have reported that miRNA dysfunction may be involved in tumor progression and therapeutic resistance [
20,
32,
33]. Moreover, the therapeutic potential of miRNAs in cancer, either alone or in combination with conventional drugs, has been demonstrated in several published studies (reviewed in [
34]). Previously, we reported that
miR-519a is downregulated in GBM cells, and overexpression of
miR-519a may suppress GBM cell proliferation [
21]. However, the pivotal role of
miR-519a in the modulation of TMZ sensitivity is still not fully understood. In this study, we demonstrated that the expression of
miR-519a was reduced in chemoresistant GBM tissues and TMZ-resistant cells, thus suggesting that low levels of
miR-519a were associated with TMZ resistance. In addition, our results showed that
miR-519a enhanced chemosensitivity in GBM cells, mainly through TMZ-induced autophagy and apoptosis.
Both prosurvival and prodeath roles of autophagy have been proposed in GBM cells in response to metabolic and therapeutic stress and are dependent on the cellular context and duration or degree of stress stimuli. Moreover, accumulating evidence has revealed a correlative relationship between chemoresistance and reduced autophagic activity in GBM cells [
35‐
37]. Consistent with previous studies, we found that the basal level of autophagy was lower in TMZ-resistant U87-MG/TMZ cells than in parental GBM cells, suggesting an inverse correlation between reduced autophagy and chemoresistance (Additional file
11). Therefore, restoration of autophagic activity in resistant GBM cells may be a promising strategy to overcome chemoresistance and improve the effectiveness of chemotherapy. In this study, we demonstrated that the enhanced autophagy by forced
miR-519a expression can sensitize GBM cells to TMZ. Additionally, these effects were attenuated by co-treatment with autophagic blockers (3-MA), suggesting the involvement of autophagic pathway. These results are consistent with previous research demonstrating that autophagy induction can lead to the suppression of GBM cell growth [
10‐
14]. Furthermore, the combination of
miR-519a and TMZ induced prodeath autophagy, suggesting that the autophagic response of GBM cells to TMZ can be modified when administered in combination with other antitumor agents.
Because the autophagy-related signal pathway is complex, additional studies are needed to elucidate the mechanisms of cell autophagy regulation. Beclin-1 may bind to and be inhibited by Bcl-2 protein to prevent cell autophagy [
38,
39]. Furthermore, STAT3/Bcl-2/Beclin-1 signaling is associated with the induction of autophagy. Previous reports have showed that STAT3 has the ability to transcriptionally activate the apoptosis-inhibitory protein BCL2, which also inhibits the induction of autophagy by dissociating the Bcl-2/Beclin-1 complex. Upon activation, STAT3 upregulates BCL2 expression and consequently leads to autophagy inhibition [
33,
40,
41]. In our study, transfection with STAT3 siRNA also inhibited both basal autophagy and TMZ-induced autophagy, suggesting that STAT3 was involved in the induction of autophagy in GBM. Moreover, dysfunction of miRNAs can modulate autophagy through a variety of mechanisms in GBM [
20,
32,
33]. We previously demonstrated that
miR-519a functions as a tumor suppressor in glioma by targeting STAT3 [
21]; therefore, we hypothesized that
miR-519a-mediated prodeath autophagy may occur via targeting of STAT3 to sensitize U87-MG/TMZ cells to TMZ. Indeed, in this study, we found that
miR-519a promoted the autophagy of GBM cells by enhancing dissociation of the Bcl-2/Beclin-1 complex and enhanced therapeutic efficacy in vivo and in vitro. We further improved our understanding of the molecular basis of
miR-519a in GBM. Advances in molecular biology have promoted our understanding of the molecular basis of GBM and provide tools with which to improve therapy. There are many tools, including decoy oligonucleotides/antisense oligonucleotide/RNA interference and guanine-rich oligonucleotides, that have been very promising in modulating STAT3 pathway and facilitating the development of new drugs for clinical applications [
42]. miR-519a, as a small molecule, may be a favorable candidate for analysis in clinical trials.
More recently, several studies have suggested that autophagy plays a prodeath role in GBM cells treated with chemotherapeutic agents, by enhancing autophagy-mediated apoptosis instead of autophagic cell death [
41‐
43]. Mu et al. [
43] observed an induced autophagy and apoptosis in GBM cells treated with a combination treatment of β-elemene and gefitinib. Bak et al. [
44] reported that enhanced autophagy contributes to the synergistic effects of vitamin D in TMZ-based GBM chemotherapy. Peng-Hsu et al. [
45] found that
miR-128 promotes apoptotic death in glioma cells through non-protective autophagy formation. Our results, in agreement with previous findings [
41‐
43], showed that autophagy inhibition by 3-MA may reduce apoptosis during combined treatment of
miR-519a and TMZ, whereas rapamycin-induced autophagy can enhance apoptosis following combined treatment of anti-
miR-519a and TMZ. Nevertheless, emerging evidence has suggested a crosstalk between autophagic and apoptotic pathways [
46].
Since the autophagy-related pathway network can be complex, additional studies are required to elucidate the molecular mechanisms underlying cell autophagy. STAT3/Bcl-2/Beclin-1 signaling has been proposed to be associated with autophagy induction. Notably, Beclin-1 may be bound to or inhibited by Bcl-2 protein in order to prevent cell autophagy [
38,
39]. STAT3 is able to induce BCL2 transcriptional activation, which inhibits the induction of autophagy by dissociating the Bcl-2/Beclin-1 complex [
33,
40,
41]. In this study, transfection with STAT3 siRNA inhibited both basal autophagy and TMZ-induced autophagy, suggesting that STAT3 signaling pathway is involved in TMZ-induced autophagy in GBM cells. Additionally, dysfunction of miRNAs can modulate autophagy in GBM cells through various mechanisms [
20,
32,
33]. We previously demonstrated that
miR-519a functions as a tumor suppressor in glioma by targeting STAT3 [
21]. In the present study, we confirmed that
miR-519a sensitized U87-MG/TMZ cells to TMZ and triggered autophagy-mediated apoptosis via STAT3 pathway. Indeed, we found that
miR-519a promoted the autophagy of GBM cells via dissociation of Bcl-2/Beclin-1 complex. These results significantly improved our understanding of the molecular basis of
miR-519a in GBM cells with TMZ resistance.