Oncosuppressive Role of RUNX3 in Human Astrocytomas

Abstract

Background: Gliomas are the most common and aggressive among primary malignant brain tumours with significant inter- and intratumour heterogeneity in histology, molecular profile, and patient outcome. However, molecular targets that could provide reliable diagnostic and prognostic information on this type of cancer are currently unknown. Recent studies show that certain phenotypes of gliomas such as malignancy, resistance to therapy, and relapses are associated with the epigenetic alterations of tumour-specific genes. Runt-related transcription factor 3 (RUNX3) is feasible tumour suppressor gene since its inactivation was shown to be related to carcinogenesis.

Aim: The aim of the study was to elucidate RUNX3 changes in different regulation levels of molecular biology starting from epigenetics to function in particular cases of astrocytic origin tumours of different grade evaluating significance of molecular changes of RUNX3 for patient clinical characteristics as well as evaluate RUNX3 reexpression effect to GBM cells.

Methods: The methylation status and protein expression levels of RUNX3 were measured by methylation-specific PCR and Western blot in 136 and 72 different malignancy grade glioma tissues, respectively. Lipotransfection and MTT were applied for proliferation assessment in U87-MG cells.

Results: We found that RUNX3 was highly methylated and downregulated in GBM. RUNX3 promoter methylation was detected in 69.4% of GBM (n=49) as compared to 0 to 17.2% in I-III grade astrocytomas (n=87). Weighty lower RUNX3 protein level was observed in GMB specimens compared to grade II-III astrocytomas. Correlation test revealed a weak but significant link among Runx3 methylation and protein level. Kaplan-Meier analysis showed that increased RUNX3 methylation and low protein level were both associated with shorter patient survival (p<0.05). Reexpression of RUNX3 in U87-MG cells significantly reduced glioma cell viability compared to control transfection.

Conclusions: The results demonstrate that RUNX3 gene methylation and protein expression downregulation are glioma malignancy dependent and contribute to tumour progression.

Figure 1

(a) Representative results for methylation-specific PCR of RUNX3 gene in different malignancy grade gliomas. M: PCR with primers for methylated RUNX3. U: PCR with primers for unmethylated RUNX3. Positive control: bisulfite converted universal methylated human DNA standard (Zymo Research, USA). Negative control: bisulfite converted normal lymphocyte DNA. Normal: “normal human brain DNA” (Zymo Research, cat. no. D5018). Arrows indicate methylated allele. (b) RUNX3 promoter methylation frequency in different astrocytoma grade. GBM revealed significant increase in RUNX3 gene promoter methylation frequency compared to I-III WHO grade astrocytomas (p<0.001, chi-square test). (c) Kaplan-Meier curves for survival (months) of all glioma patients (n=136) stratified by RUNX3 gene methylation status revealed significantly shorter survival of patients with RUNX3 methylated tumours (Log-rank test, χ²=44.68, df=1, p<0.001).

Figure 2

(a) Representative Western blot result of RUNX3 protein expression in astrocytomas. AI-AIII: astrocytoma malignancy grades I-III, respectively; GBM: glioblastoma. Two isoforms of RUNX3 were identified in all the specimens that is consistent with what has been described in the literature [6]. (b) RUNX3 protein expression levels in different astrocytoma grade. Protein expression was significantly downregulated in glioblastomas (GBM) as compared to grade II astrocytomas (p<0.005, Kruskal-Wallis test) and a tendency as compared to grade III astrocytomas (p=0.124, Kruskal-Wallis test). (c) Relative RUNX3 protein expression stratified by promoter methylation groups. Significant association between RUNX3 gene methylation and protein expression was found (p=0.026, Kruskal-Wallis test). (d) Kaplan-Meier curves for survival of all astrocytoma patients (n=72) stratified in two groups (low; high) according to protein expression revealed significant better survival rates for patient with high RUNX3 protein level (Log-rank test, χ2=6.11, df=1, p=0.013). (e) Kaplan-Meier survival curves of high malignancy grade (III-IV) astrocytoma patients only (n=39) (Log-rank test, χ2=13.74, df=1, p<0.001).

Figure 3

(a) WB of RUNX3 protein expression after U87-MG cells transfection with pcDNA3-RUNX3 construct. NTC: nontransfected cells lysate; 1: cells transfected with 100 ng of pcDNA3-RUNX3 vector; 2: cells transfected with 50 ng of pcDNA3-RUNX3 vector. Equal amount of total protein extract (60 μg) was loaded per each gel lane. The same amount of cell (b) RUNX3 promoter methylation status in U87-MG cells. (c) Results from glioblastoma U87-MG cells viability assay applying MTT test after 24 h of 100 ng of DNA transfection. RUNX3: RUNX3 gene in pcDNA3 expression vector (pcDNA3-RUNX3); GFP: green fluorescing protein in pcDNA4TO expression vector (pcDNA4TO-GFP); pcDNA3: empty (control) vector; NTC: nontransfected cell control. Overexpression of RUNX3 significantly decreased U87-MG cell viability starting from 50 ng of vector used as compared to the cells transfected with GFP vector. Moreover even bigger effect of decreased cell viability was obtained when 100 ng of RUNX3 was transfected.