The Use of Pro-Angiogenic and/or Pro-Hypoxic miRNAs as Tools to Monitor Patients with Diffuse Gliomas

Abstract

IDH (isocitrate dehydrogenase) mutation, hypoxia, and neo-angiogenesis, three hallmarks of diffuse gliomas, modulate the expression of small non-coding RNAs (miRNA). In this paper, we tested whether pro-angiogenic and/or pro-hypoxic miRNAs could be used to monitor patients with glioma. The miRNAs were extracted from tumoral surgical specimens embedded in the paraffin of 97 patients with diffuse gliomas and, for 7 patients, from a blood sample too. The expression of 10 pro-angiogenic and/or pro-hypoxic miRNAs was assayed by qRT-PCR and normalized to the miRNA expression of non-tumoral brain tissues. We confirmed in vitro that IDH in hypoxia (1% O₂, 24 h) alters pro-angiogenic and/or pro-hypoxic miRNA expression in HBT-14 (U-87 MG) cells. Then, we reported that the expression of these miRNAs is (i) strongly affected in patients with glioma compared to that in a non-tumoral brain; (ii) correlated with the histology/grade of glioma according to the 2016 WHO classification; and (iii) predicts the overall and/or progression-free survival of patients with glioma in univariate but not in a multivariate analysis after adjusting for sex, age at diagnosis, and WHO classification. Finally, the expression of miRNAs was found to be the same between the plasma and glial tumor of the same patient. This study highlights a panel of seven pro-angiogenic and/or pro-hypoxic miRNAs as a potential tool for monitoring patients with glioma.

Keywords: angiogenesis; glioma; hypoxia; miRNA.

Figure 1

Expression of pro-angiogenic or pro-hypoxic miRNAs according to the presence (HBT-14 (U-87 MG) IDH1^R132H cells) or absence (HBT-14 (U-87 MG) IDH1^WT cells) of the IDH1 R132H mutation. The miRNAs were extracted from cell lines using miRNAeasy (Qiagen™), then retrotranscribed (RT) and amplified (PCR) using the TaqMan MiRNA Reverse transcription kit (Applied Biosystem). The RT-PCR data were normalized to the small nucleolar house-keeping RNA, RNA RNU48 (SNORD48) (assay ID 001006). Each miRNA was expressed in base 100 (100 being attributed to the delta-CT of the miRNA measured in HBT-14 (U-87 MG) IDH1^WT cells) (n = 3, ANOVA followed by a post hoc Dunnett’s test, *: p < 0.05, **: p < 0.01). The mir-126-5p was undetectable in these lines.

Figure 2

Morphological appearance of HBT-14 (U-87 MG) IDH1^WT and HBT-14 (U-87 MG) IDH1^R132H grown in normoxia or hypoxia (1% O₂, 24 h). HBT-14 (U-87 MG) IDH1^WT and HBT-14 (U-87 MG) IDH1^R132H cells, as validated by immunohistochemical staining carried out according to standard procedures against IDH1^R132H (left panel), reaching 60% confluence, were cultivated for an additional 24 h in physoxia or hypoxia (0.1% O₂). The appearance of these cells was imaged under a phase contrast microscope: the right panel presents representative photos of these cells according to the culture condition (normoxia/hypoxia).

Figure 3

Expression of pro-angiogenic or pro-hypoxic miRNAs in HBT-14 (U-87 MG) IDH1^R132H or in HBT-14 (U-87 MG) IDH1^WT cells grown in hypoxia (1% O₂, 24 h). HBT-14 (U-87 MG) IDH1^WT and HBT-14 (U-87 MG) IDH1^R132H cells at 60% confluence were cultivated for an additional 24 h in normoxia or hypoxia (0.1% O₂). The miRNAs from HBT-14 (U-87 MG) IDH1^WT and HBT-14 (U-87 MG) IDH1^R132H cells were extracted using miRNAeasy (Qiagen™), then retrotranscribed (RT) and amplified (PCR) using the TaqMan MiRNA Reverse transcription kit (Applied Biosystem). The RT-PCR data were normalized to the small nucleolar house-keeping RNA, RNAS RNU48 (SNORD48) (assay ID 001006). Each miRNA ((a): mir-100-5p; (b): mir-128-3p; (c): mir-200b-3p; (d): mir-200c-3p; (e): mir-210-3p; (f): mir-221-3p; (g): mir-424-5p; (h): mir-451-5p) was thus finally expressed in base 100 (100 being attributed to the delta-CT of the miRNA measured in HBT-14 (U-87 MG) IDH1^WT or HBT-14 (U-87 MG) IDH1^R132H cells grown in normoxia) (n = 3, ANOVA followed by a post hoc Dunnett’s test, *: p < 0.05; **: p < 0.01; ***: p < 0.001, NS: non-significant).

Figure 4

The expression of pro-angiogenic or pro-hypoxic miRNAs varied markedly in patients with glioma. miRNA from the 97 FFPE surgically resected tumor specimens or the 8 healthy brain tissues were extracted using miRNAeasy-FFPE kit (Qiagen™), then retrotranscribed (RT) and amplified (PCR) using the TaqMan MiRNA reverse transcription kit (Applied Biosystem). The RT-PCR data were normalized to the small nucleolar house-keeping RNA, RNAS RNU48 (SNORD48) (assay ID 001006). Results are expressed as the fold change in glioma samples ± ESM compared to healthy brain tissue.

Figure 5

The expression levels of pro-angiogenic or pro-hypoxic miRNAs were correlated between the tumor sample and plasma from patients with glioma. For 7 patients with glioma, the miRNAs from the FFPE surgically resected tumor specimens or the plasma samples were extracted using, miRNAeasy-FFPE kit (Qiagen™) and NucleoSpin miRNA Plasma (Macherey-Nagel™), respectively, then retrotranscribed (RT) and amplified (PCR) using the TaqMan MiRNA Reverse transcription kit (Applied Biosystem). Results illustrated here for (a) mir-100-5p, (b) mir-132-3p, (c) mir-200b-3p, and (d) mir-221-3p are expressed as cycle thresholds (CTs) assayed for each miRNA normalized to the small nucleolar house-keeping RNA, RNAS RNU48 (SNORD48) (assay ID 001006).