Up-regulation of microRNA-15b correlates with unfavorable prognosis and malignant progression of human glioma

Abstract

Recent studies have demonstrated that microRNA-15b (miR-15b) regulates cell cycle progression, proliferationnd apoptosis in glioma cells by targeting Cyclins. However, the clinical significance of miR-15b in human glioma remains unclear. Therefore, the aim of this study was to investigate the significance of miR-15b expression in diagnosis, prognosis and malignant progression of glioma. Quantitative real-time reverse transcriptive-PCR (qRT-PCR) was performed to examine miR-15b expression levels in 76 glioma tissues (13 grade II, 13 grade III and 50 grade IV gliomas) and seven glioma cell lines, as well as 10 non-neoplastic brain tissues and human astrocyte as control. MiR-15b showed significant increased expression in high-grade gliomas (P≤0.001) and glioma cells (fold change 2.8-7.6) relative to non-neoplastic brains and astrocyte, respectively. Additionally, high miR-15b expression was significantly associated with advanced WHO grade (P≤0.001), advanced patient age (P≤0.001) and low Karnofsky performance score (KPS, P≤0.001). Furthermore, Kaplan-Meier survival analysis and Cox regression analysis showed that patients with high miR-15b expression had significantly poor overall survival rate (P≤0.001) and miR-15b expression was an independent prognosis-predicting factor for glioma patients (P≤0.001; risk ratio=5.6), respectively. Moreover, miR-15b expression was examined in seven independent patients with primary grade II or III gliomas that spontaneously progressed to grade III or IV gliomas. Statistically significant higher expression (P=0.01) in the recurrent tumor compared with the corresponding primary tumor was observed in all of the seven patients. Our results suggest that miR-15b may be a prognostic predictor and be involved in malignant progression of glioma.

Keywords: glioma; malignant progression; miRNA-15b; microRNA; prognosis; up-regulation.

Figure 1

miR-15b expression in 76 glioma tissues (50 primary GBMs, 13 AAs and 13 DAs), 10 non-neoplastic brain tissues, 7 glioma cell lines (U87, U251, U373, T98G, LN18, LN229 and SF295) and normal human astrocyte detected by quantitative reverse transcriptive real-time polymerase chain reaction (qRT-PCR) analysis. A. Dots indicate log2 of the relative quantification (RQ) values of miR-15b expression levels, normalized by RNU6B. Bars indicate log2 of the average RQ values for each group. The expression level of miR-15b was found to be remarkably increased in glioma tissues compared to non-neoplastic tissues (P ≤ 0.001). Both primary GBMs and AAs showed significantly higher expression of miR-15b than non-neoplastic brains tissues (P ≤ 0.001, respectively). Expression of miR-15b showed a distinctly upward tendency along with the increasing malignancy degree of gliomas. B. Columns indicate RQ values of miR-15b expression levels in human astrocyte and glioma cell lines. Glioma cell lines showed about 3- to 7.5-fold higher expression of miR-15b in comparison with normal human astrocyte.

Figure 2

Kaplan-Meier survival curves for glioma patients with high and low expression levels of miR-15b. A. Among 76 glioma patients (50 primary GBMs, 13 AAs and 13 DAs), those with high miR-15b expression (left, dotted line, n = 33) had significantly shorter survival periods than did patients with low miR-15b expression (right, solid line, n = 43; P ≤ 0.001). B. Among 63 high-grade glioma patients (50 primary GBMs and 13 AAs), those with high miR-15b expression (left, dotted line, n = 33) had significantly shorter survival periods than did patients with low miR-15b expression (right, solid line, n = 30; P ≤ 0.001).

Figure 3

Expression levels of miR-15b in seven patients with primary lower-grade glioma that recurred as higher-grade glioma. MiR-15b showed an about 1.3- to 41.6-fold higher expression in the recurrent glioma as compared to the corresponding primary tumor. Patient numbers 1-7 encode the individual patient; Gray and black columns indicate expression levels of miR-15b in primary and recurrent tumors, respectively. P, primary tumor; R, recurrent tumor.

Figure 4

Validation experiment for miR-15b expression in an independent series of 9 DAs, 8 AAs and 8 secondary-GBMs. miR-15b showed a significant progression-associated up-regulation in gliomas with different pathological-grade (P = 0.015, 0.016 and ≤ 0.001 for grade III vs. II, grade IV vs. III and grade IV vs. II, respectively).