miR-130a can predict response to temozolomide in patients with glioblastoma multiforme, independently of O6-methylguanine-DNA methyltransferase

The Cancer Genome Atlas (TCGA) database [12] included miRNA expression profiling of 184 adult patients who had GBM and underwent radiation therapy and TMZ chemotherapy. Data on these patients were used as a discovery set. We downloaded the mRNA expression profiling and clinical information on these patients, as well as the miRNA expression profiling of another 128 glioblastoma patients in TCGA who only received radiation therapy. Additionally, we obtained miRNA expression profiling of 28 adult patients who had glioblastoma and underwent TMZ chemotherapy from the Chinese Glioma Genome Atlas (CGGA) database [13]. Data on these 28 patients were used as a validation set.

Human glioma cell lines U87, LN229, SHG44, and U251 were cultured in DMEM/F-12 culture medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco). Cells were maintained in a humidified incubator with 5% CO₂ at 37°C. The All-in-One miRNA qRT-PCR Detection Kit (FulenGen, Guangzhou, China) was used to evaluate the miR-130a levels in cells.

Regarding miR-130a mimics and control miRNA, inhibitors for miRNA-130a and controls were purchased from FulenGen in China. Transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. Transfection efficiency was also evaluated using the All-in-One miRNA qRT-PCR Detection Kit.

After transfection, cells were seeded at a density of 6000 cells per well on 96 well plates in triplicates. TMZ (Sigma Aldrich, St. Louis, MO, USA) was added at various concentrations ranging from 0 to 1000 μmol/L for 48 hours. The Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) was used to determine the number of viable cells in the TMZ cytotoxicity assay. To test the cleavage of nuclear poly (ADP-ribose) polymerase (PARP), proteins of SHG44 and U251 were extracted after transfection and 96 hours 500 μM TMZ treatment.

At 48 hours after transfection, cells were washed twice with PBS, then incubated in complete medium supplemented with 100 μM hydrogen peroxide to induce oxidative stress. The incubation continued for 24 hours before determination of Ape1 expression using western blotting.

Proteins were extracted using RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitor. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane. After blocking with 5% skim milk in TBST, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then washed and incubated with secondary antibodies for 1 hour. Antibody binding was assessed by enhanced chemiluminescence substrate (BioRad). Antibodies to PARP and Ape1 were purchased from Proteintech in China.

For each miRNA, the association between the expression level and OS in the discovery set was calculated using a univariate Cox proportional hazards regression analysis. The 4 miRNAs that were found to be significantly associated with OS in the discovery set were then evaluated in the validation set using the Kaplan-Meier method and 2-sided log-rank tests. Subsequently, the miRNA that was significantly associated with OS in both sets was further analyzed using a Cox multivariate analysis that included O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, age, and gender as covariates. The association between the expression of this miRNA and OS was also investigated in the group of patients from TCGA who had received radiation therapy alone. As previously, the Kaplan-Meier method and 2-sided log-rank tests were also used in this analysis. The cutoff value for this miRNA in TCGA was defined as the median expression level.

The miRNA-associated gene set was identified using a Spearman correlation analysis. The negatively correlated genes were included in a gene ontology (GO) analysis that was performed using DAVID [14]; (National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA). Additionally, we performed a Gene Set Enrichment Analysis (GSEA) with GSEA software downloaded from the Broad Institute [15,16] (Cambridge, MA, USA).

Paired-sample t-tests were employed to assess the significance of differences between TMZ cytotoxicity assay results.

Statistical analyses of TCGA and CGGA datasets were performed using BRB-Array Tools 4.3.0 Beta_2, which was developed by Dr. Richard Simon and the BRB-Array Tools Development Team (National Cancer Institute, Bethesda, MD, USA). Statistical analyses for in vitro experimental results were performed using SPSS 16.0 (IBM SPSS, Inc., Chicago, IL, USA). Two-sided p-values less than 0.05 were regarded as statistically significant.

Further, the Kaplan-Meier method and 2-sided log-rank tests showed that miR-130a was not significantly associated with OS in the patients who only received radiation therapy (Figure 1F).

In addition, GSEA showed enrichment of genes related to response to oxidative stress (ROS) among patients with low miR-130a expression (normalized enrichment score [NES] =1.51, p = 0.037).

The miR-130a levels were relatively high in U251 cells (Figure 3A). This cell line was then transfected with inhibitors (5′-AUGCCCUUUUAACAUUGCACUG-3′) for miR-130a and the scrambled control (Figure 3D). Western blotting showed that U251 transfected with the scrambled control (U251-SC) expressed lower Ape1 than cells transfected with miR-130a inhibitors (U251-I) did under oxidative stress (Figure 2E).

The TMZ cytotoxicity assay showed that SHG44-M were more sensitive to TMZ than SHG44-SC (p = 0.027, Figure 3C). Western blotting showed that overexpression of miR-130a increased PARP cleavage in SHG44 induced by TMZ (Figure 3C).

U251-I were found to be more resistant to TMZ than U251-SC (p = 0.039, Figure 3E). Western blotting showed knockdown of miR-130a decreased PARP cleavage in U251 induced by TMZ (Figure 3E).