The U2AF2 /circRNA ARF1/miR-342\u20133p/ISL2 feedback loop regulates angiogenesis in glioma stem cells

Yang Jiang,Jiangfeng Hu,Zhitao Jing,Junshuang Zhao,Lian Chen,Hao Li,Haiying Zhang,Jinpeng Zhou,Long Li,Wei Zheng

doi:10.1186/s13046-020-01691-y

Abstract

BackgroundGlioma is the most common and lethal primary brain tumor in adults, and angiogenesis is one of the key factors contributing to its proliferation, aggressiveness, and malignant transformation. However, the discovery of novel oncogenes and the study of its molecular regulating mechanism based on circular RNAs (circRNAs) may provide a promising treatment target in glioma.MethodsBioinformatics analysis, qPCR, western blotting, and immunohistochemistry were used to detect the expression levels of ISL2, miR-342–3p, circRNA ARF1 (cARF1), U2AF2, and VEGFA. Patient-derived glioma stem cells (GSCs) were established for the molecular experiments. Lentiviral-based infection was used to regulate the expression of these molecules in GSCs. The MTS, EDU, Transwell, and tube formation assays were used to detect the proliferation, invasion, and angiogenesis of human brain microvessel endothelial cells (hBMECs). RNA-binding protein immunoprecipitation, RNA pull-down, dual-luciferase reporter, and chromatin immunoprecipitation assays were used to detect the direct regulation mechanisms among these molecules.ResultsWe first identified a novel transcription factor related to neural development. ISL2 was overexpressed in glioma and correlated with poor patient survival. ISL2 transcriptionally regulated VEGFA expression in GSCs and promoted the proliferation, invasion, and angiogenesis of hBMECs via VEGFA-mediated ERK signaling. Regarding its mechanism of action, cARF1 upregulated ISL2 expression in GSCs via miR-342–3p sponging. Furthermore, U2AF2 bound to and promoted the stability and expression of cARF1, while ISL2 induced the expression of U2AF2, which formed a feedback loop in GSCs. We also showed that both U2AF2 and cARF1 had an oncogenic effect, were overexpressed in glioma, and correlated with poor patient survival.ConclusionsOur study identified a novel feedback loop among U2AF2, cARF1, miR-342–3p, and ISL2 in GSCs. This feedback loop promoted glioma angiogenesis, and could provide an effective biomarker for glioma diagnosis and prognostic evaluation, as well as possibly being used for targeted therapy.

Highlights

Glioma is the most common and lethal primary brain tumor in adults, and angiogenesis is one of the key factors contributing to its proliferation, aggressiveness, and malignant transformation
Opposite results were obtained after additional miR-342–3p mimic treatment, when compared with circRNA ARF1 (cARF1)-overexpressed GSC205-glioma conditioned medium (GCM) treatment alone (Fig. 4k-p). These results suggested that higher cARF1 expression in glioma stem cells (GSCs) promoted the proliferation, invasion, and angiogenesis of human brain microvessel endothelial cells (hBMECs), while miR-342–3p reversed these functions of cARF1 in GSCs
Compared to U2AF2-silenced GSC406-GCM treatment alone, the results showed that the proliferation, invasion, and angiogenesis of hBMECs were increased after cARF1 overexpression when combined with U2AF2-silenced GSC406-GCM treatment (Fig. 6j-k)

Summary

Introduction

Glioma is the most common and lethal primary brain tumor in adults, and angiogenesis is one of the key factors contributing to its proliferation, aggressiveness, and malignant transformation. The discovery of novel oncogenes and the study of its molecular regulating mechanism based on circular RNAs (circRNAs) may provide a promising treatment target in glioma. Glioma is the most common and lethal primary brain tumor in adults. There is a great need to identify the molecular mechanisms responsible for GSCs proliferation and progression, as well as to identify novel molecular targets for treatment of glioma. ISL2 is a LIM/homeodomain-type transcription factor of the Islet-1 family, and is mainly expressed in the primary sensory and motor neurons [5]. As a transcription factor involved in development of the nervous system, it is doubtful whether ISL2 affects the development and progress of glioma

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