Temozolomide (TMZ) is one of the most commonly used drugs for the clinical treatment of glioblastomas. However, it has been reported that treatment with TMZ can induce autophagy, which leads to tumor resistance and increases the survival of tumor cells. MicroRNA-30a (miR-30a) has been found to have inhibitory effects on autophagy by directly targeting beclin 1. However, the exact role of miR-30a in TMZ-treated glioblastoma cells has not been studied previously. The present study aimed to investigate whether miR-30a increased the cytotoxicity of TMZ to glioblastoma U251 cells, as well as the underlying mechanism. MTT and flow cytometry assay results showed that treatment with TMZ inhibited the proliferation of U251 cells while inducing cell apoptosis in a dose-dependent manner. Western blotting data showed that the expression levels of LC3-II and beclin 1 as well as the ratio of LC3-II to LC3-I were markedly increased in TMZ-treated U251 cells compared with the untreated control cells, indicating that treatment with TMZ induced autophagy. Moreover, reverse transcription-quantitative polymerase chain reaction data showed that treatment with TMZ led to a significant reduction in miR-30a levels in a dose-dependent manner in U251 cells. Elevation of the miR-30a level significantly inhibited TMZ-induced autophagy, demonstrated by the decreased LC3-II and beclin 1 levels and ratio of LC3-II to LC3-I, accompanied by the reduced proliferation and increased apoptosis in TMZ-treated U251 cells. Furthermore, luciferase reporter assay data indicated that beclin 1 was a direct target of miR-30a in U251 cells. In summary, this study demonstrated that miR-30a increases the chemosensitivity of glioblastoma U251 cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy. Therefore, autophagy may be a promising target for the treatment of TMZ-resistant tumors.
Gliomas are the most common brain tumors, accounting for ~30% of central nervous system tumors and 80% of all malignant brain tumors (
Autophagy, an evolutionarily conserved function, is a cellular self-catabolic degradation process, responsible for the lysosomal degradation of long-lived proteins as well as aged or damaged organelles (
MicroRNAs (miRs), a class of non-coding RNAs, 18–25 nucleotides in length, are able to induce mRNA degradation or suppress protein translation via binding to the 3′-untranslated regions (3′-UTRs) of mRNA of specific genes (
In the present study, the aim was to investigate whether miR-30a has an effect on TMZ-induced autophagy in glioblastomas. In addition, the involvement of beclin 1 in the underlying molecular mechanism was explored.
Human glioblastoma U251 cells were obtained from the China Cell Culture Center (Shanghai, China). The U251 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (all from Thermo Fisher Scientific, Inc.). To mimic chemotherapy, U251 cells were treated with TMZ (1, 5, 10 or 30 µg/ml) for 6 h, and then examined by a series of assays.
TRIzol Reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from U251 cells, in accordance with the manufacturer's instructions. A RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA) was used to reverse transcribe total RNA into cDNA, according to the manufacturer's protocol. The miRNA expression was determined using a PrimeScript® miRNA RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China), in accordance with the manufacturer's instructions. The PCR conditions were 95°C for 10 min, and 40 cycles of denaturation at 95°C for 30 sec and annealing/elongation at 60°C for 30 sec. The primer sequences for miR-30a were: Forward, 5′-GGGGTGTAAACATCCTCGACTG-3′ and reverse, 5′-ATTGCGTGTCGTGGAGTCG-3′. The primer sequences for U6 were: Forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. They were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). All miRNA data are expressed relative to a U6 small nuclear RNA from the same sample. Independent experiments were repeated three times. The relative expression levels of mRNA were analyzed by use of the 2−∆∆Cq method (
Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) was used to perform transfection according to the manufacturer's protocol. Briefly, U251 cells were cultured to 70% confluence, and resuspended in serum-free DMEM. Serum-free DMEM was used to dilute Lipofectamine 2000, miR-30a mimic, or scrambled miR mimic, respectively. The diluted Lipofectamine 2000 was then added to the diluted miR-30a mimic or diluted scrambled miR mimic. After incubation for 20 min at room temperature, the mixture was added to the cell suspension. After incubation at 37°C with 5% CO2 for 6 h, the medium was replaced by DMEM supplemented with 10% FBS. Following transfection for 48 h, the following assays were performed.
An MTT assay was performed to evaluate the cell proliferation. In brief, 1×104 U251 cells from each group were plated in a 96-well plate, and incubated for 6, 12, 24 and 48 h at 37°C with 5% CO2. MTT (5 mg/ml; Thermo Fisher Scientific, Inc.) was then added to each well, and the plate was incubated for 4 h at 37°C with 5% CO2. The supernatant was removed, and 100 µl dimethylsulfoxide (Thermo Fisher Scientific, Inc.) was added to dissolve the precipitate. The absorbance was detected at 492 nm using the BioTek™ ELX800™ Absorbance Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
Cell apoptosis was determined using an Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, San Diego, CA, USA), according to the manufacturer's instruction. In brief, U251 cells were harvested and washed with cold PBS twice. After that, U251 cells (1×106) were resuspended in 200 µl binding buffer with 10 µl Annexin-V-FITC and 5 µl PI-PE, and incubated in the dark for 30 min. Following incubation, 300 µl binding buffer was added and the cells were analyzed by flow cytometry (C6 cytometer; Beckman Coulter, Inc. (Brea, CA, USA).
U251 cells were solubilized in cold radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) to extract protein, which was separated by 10% SDS-PAGE (Pierce; Thermo Fisher Scientific, Inc.), and transferred onto a polyvinylidene difluoride (PVDF) membrane (Pierce). The PVDF membrane was incubated with rabbit anti-LC3-II polyclonal antibody (1:50; ab48394; Abcam, Cambridge, MA, USA), rabbit anti-LC3-I polyclonal primary antibody (1:50; ab128025; Abcam), rabbit anti-beclin 1 monoclonal antibody (1:100; ab55878; Abcam) and rabbit anti-GAPDH polyclonal primary antibody (1:100; ab9485; Abcam), respectively, at 4°C overnight. After washing with PBST three times, the PVDF membrane was then incubated with mouse anti-rabbit secondary antibody (1:5,000; ab99697; Abcam) at room temperature for 40 min. Chemiluminescent detection was conducted using an ECL kit (Pierce). The protein expression was analyzed using Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and the expression levels were represented as the density ratio vs. GAPDH.
Targetscan software (version 3.1;
All data are represented as the mean of at least triplicate samples ± standard deviation. Statistical analysis of differences was performed by one-way analysis of variance using SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
U251 cells were treated with TMZ (1–30 µg/ml). Following treatment for 6–48 h, an MTT assay was performed to examine the cell proliferation. As shown in
As miR-30a has been demonstrated to play a suppressive role in autophagy, the expression level of miR-30a in U251 cells with or without treatment with TMZ (1–30 µg/ml) was determined. As shown in
To further determine the role of miR-30a in the beclin 1-mediated autophagy of TMZ-treated glioblastoma cells, U251 cells were transfected with miR-30a mimic or scrambled miR mimic as a negative control prior to TMZ treatment. As shown in
Whether miR-30a upregulation could promote the TMZ-induced inhibition of proliferation and/or TMZ-induced apoptosis of U251 cells was investigated. As shown in
Finally, the relationship between miR-30a and beclin 1 in U251 cells was examined. Bioinformatic prediction data indicated that beclin 1 is a direct target gene of miR-30a (
Autophagy has been demonstrated to be important not only in the recirculation of degraded components to sustain metabolic homoeostasis, but also in the prevention of the toxic accumulation of damaged components (
TMZ has been widely used for the treatment of many types of human cancers, including glioblastoma. It has been reported that the antitumor activity of TMZ is achieved through DNA damage via induction of loop structures as well as DNA condensation (
miR-30a has been found to play a key role in multiple types of human cancers, mainly acting as a tumor suppressor. For instance, Fu
miR-30a has been found to sensitize tumor cells to several different chemotherapy drugs. Zou
In conclusion, the present study demonstrated that treatment with TMZ induced an activation of autophagy as well as a downregulation of miR-30a, while overexpression of miR-30a inhibited the expression of beclin 1, and thus suppressed TMZ-induced autophagy in U251 cells. Inhibition of autophagy by the elevation of miR-30a expression enhanced the cytotoxicity of TMZ to U251 cells. Based on these findings, it is suggested that autophagy may be a promising target for the treatment of TMZ-resistant tumors.
This study was supported by the National Natural Science Foundation of China (grant no. 81201740) and as a project of the Hunan Province Science and Technology Department (grant no. 2012FJ6075).
Treatment with TMZ inhibits proliferation, and induces apoptosis and autophagy in U251 cells. (A) MTT assay was performed to examine the proliferation of U251 cells treated with TMZ (1–30 µg/ml). *P<0.05 vs. control. &P<0.05 vs. TMZ (1 µg/ml). (B) Flow cytometry was performed to examine the apoptosis of U251 cells treated with TMZ (1–30 µg/ml). *P<0.05 vs. control. #P<0.05 vs. TMZ (5 µg/ml). &P<0.05 vs. TMZ (10 µg/ml). (C) Western blot analysis was conducted to determine the levels of autophagy-related proteins in U251 cells treated with TMZ (30 µg/ml). *P<0.05 vs. control. (D) The ratio of LC3-II to LC3-I was calculated in U251 cells treated with TMZ (30 µg/ml). *P<0.05 vs. control. Non-treated U251 cells were used as the control in each assay. TMZ, temozolomide.
Treatment with TMZ decreases the expression of miR-30a in U251 cells. Reverse transcription-quantitative polymerase chain reaction was conducted to examine the relative miR-30a levels (relative to U6) in U251 cells treated with TMZ (1–30 µg/ml). Non-treated U251 cells were used as a control. *P<0.05 vs. control. #P<0.05 vs. TMZ (1 µg/ml). &P<0.05 vs. TMZ (5 µg/ml). $P<0.05 vs. TMZ (10 µg/ml). TMZ, temozolomide.
Restoration of miR-30a level suppresses TMZ-induced autophagy in U251 cells via inhibition of beclin 1. (A) Reverse transcription-quantitative polymerase chain reaction was conducted to examine the relative miR-30a levels in U251 cells transfected with miR-30a mimic or miR-NC, respectively. (B) Western blot analysis was conducted to determine the levels of autophagy-related proteins in the transfected U251 cells treated with TMZ (30 µg/ml). (C) The ratio of LC3-II to LC3-I was calculated. Non-transfected U251 cells treated with TMZ were used as the control. *P<0.05 vs. TMZ. TMZ, temozolomide; miR-NC, scrambled miR.
Overexpression of miR-30a increases the cytotoxicity of TMZ to U251 cells. (A) MTT assay was performed to examine the proliferation of TMZ (30 µg/ml)-treated U251 cells transfected with miR-30a mimic or miR-NC, respectively. (B) Flow cytometry was conducted to examine cell apoptosis. Non-transfected U251 cells treated with TMZ (30 µg/ml) were used as control. *P<0.05 vs. TMZ. TMZ, temozolomide; miR-NC, scrambled miR.
Beclin 1 is a direct target of miR-30a in U251 cells. (A) Bioinformatic prediction data indicated that beclin 1 was a direct target gene of miR-30a. (B and C) WT or MUT BECN1 3′-UTR was cloned downstream of the firefly luciferase coding region of the pmirGLO™ vector, to form pMIR-WT BECN1 and pMIR-MUT BECN1, respectively. (D) U251 cells were co-transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and miR-30a mimic or miR-NC, and pRL-TK plasmid for internal normalization, respectively. Luciferase reporter assay data showed that co-transfection with pMIR-WT BECN1 and miR-30a mimic significantly decreased the luciferase activity; however, co-transfection with pMIR-MUT BECN1 and miR-30a mimic caused no change in luciferase activity. Control cells were transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and the pRL-TK plasmid. *P<0.05 vs. control. WT, wild type; MUT, mutant; BECN1, beclin 1; UTR, untranslated region; miR-NC, scrambled miR mimic.