MiR-16 modulate temozolomide resistance by regulating BCL-2 in human glioma cells

Abstract

Temozolomide (TMZ) with radiotherapy is the current standard of care for newly diagnosed glioma. However, glioma patients who are treated with the drug often develop resistance to it and some other drugs. Recently studies have shown that microRNAs (miRNAs) play an important role in drug resistance. In present study, we first examined the sensitivity to temozolomide in six glioma cell lines, and established a resistant variant, U251MG/TR cells from TMZ-sensitive glioma cell line, U251MG. We then performed a comprehensive analysis of miRNA expressions in U251MG/TR and parental cells using cancer microRNA PCR Array. Among the downregulated microRNAs was miR-16, members of miR-15/16 family, whose expression was further validated by qRT-PCR in U251MG/TR and U251MG cells. The selective microRNA, miR-16 mimics or inhibitor was respectively transfected into U251MG/TR cells and AM38 cell. We found that treatment with the mimics of miR-16 greatly decreased the sensitivity of U251MG/TR cells to temozolomide, while sensitivity to these drugs was increased by treatment with the miR-16 inhibitor. In addition, the downregulation of miR-16 in temozolomide-sensitive AM38 cells was concurrent with the upregulation of Bcl-2 protein. Conversely, overexpression of miR-16 in temozolomide-resistant cells inhibited Bcl-2 expression and decreased temozolomide resistance. In conclusion, MiR-16 mediated temozolomide-resistance in glioma cells by modulation of apoptosis via targeting Bcl-2, which suggesting that miR-16 and Bcl-2 would be potential therapeutic targets for glioma therapy.

Keywords: BCL-2; Human glioma cells; miR-16; temozolomide resistance.

Figure 1

Screen and identify the resistant glioma cell lines to temozolomide. A: The IC50 values of temozolomide in different glioma cell lines. B: Cell viability was assessed by CCK-8 assay. C: Cell apoptosis was evaluated by flow cytometry. All experiments were performed in triplicate.

Figure 2

Eighty-eight cancer-related microRNAs were profiled using cancer microRNA PCR Array in U251 MG/TR and U251 MG cells. A: A graphic representation of the 88 microRNA readouts for U251 MG/TR and U251 MG cells. Each vertical line represents a single microRNA value of relative expression in U251 MG/TR and U251 MG cells. B: miR-150, let-7a, miR-9, miR-16, miR-98 and miR-125b were determined to be differentially expressed by cancer microRNA PCR Array in U251 MG/TR and U251 MG cells, and this result was validated by quantitative polymerase chain reaction result (qPCR). miR-150, let-7a and miR-125b, 3 specifically up-regulated microRNAs in U251MG/TR cells, were confirmed to be highly expressed in U251MG/TR cells, while miR-16 was confirmed to be down-regulated in U251MG/TR cells.

Figure 3

Correlation between miR-16 expression and temozolomide resistance in glioma cells. A: The expression of miR-16 in different glioma cell lines by qRT-PCR. B: The correlation between the relative miR-16 expression and the IC50 values in glioma cells was quantified by Spearman’s rank correlation. C: Temozolomide-sensitive AM38 cells were transfected with specific inhibitor to miR-16. D: Temozolomide-resistant U251MG/TR cells were transfected with miR-16 mimics. After incubation with temozolomide for 48 hr, cell viability was assessed using CCK-8 assay and IC50 value to temozolomide was calculated. All experiments were performed in triplicate.

Figure 4

MiR-16 inhibits temozolomide induced apoptosis via upregulation of Bcl-2. A and B: AM38 cells transfected with miR-16 inhibitor and U251MG/TR cells were transfected with miR-16 mimics were labeled with FITC-Annexin V/PI, and cell apoptosis was evaluated by flow cytometry. Western blot analysis showed miR-16 upregulated Bcl-2 expression in AM38 cells and downregulated Bcl-2 expression in U251MG/TR cells, *P<0.05, **P<0.01 compared to NC, ##P<0.01. β-actin was used as an internal control. All data represent the means ± SEM of three replications.