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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: The U2AF2 /circRNA ARF1/miR-342–3p/ISL2 feedback loop regulates angiogenesis in glioma stem cells

Fig. 4

The cARF1-mediated GCM promoted proliferation, invasion, and angiogenesis of hBMECs by serving as a miRNA sponge of miR-342–3p. a Graphical illustration showing the predicted position of the cARF1 target on the miR-342–3p sequence. b A schematic representation showing that cARF1 was generated from ARF1 gene, located at chr1. c The qPCR measured the relative expression of cARF1 and linear ARF1 mRNA in GSC406 with the presence or absence of RNase R. d The luciferase reporter assays showed that miR-342–3p mimic or inhibitor affected the luciferase promoter activities of cARF1. e The anti-AgO2 RNA immunoprecipitation (RIP) assay was performed in GSC406 after the miR-342–3p mimic or negative control was transfected, followed by qPCR to detect the enrichment of cARF1 and miR-342–3p. f The qPCR showed the expression of cARF1 in GSCs after miR-342–3p mimic or inhibitor treatment. g The expression of miR-342–3p in GSCs after cARF1 overexpression or knockdown were detected by qPCR. h The cARF1 was expressed at higher levels in different grade glioma tissues, compared with NBT as measured by qPCR. (grade II, n = 20; grade III, n = 25; grade IV, n = 25; NBT n = 10). i The mRNA expression correlation between ISL2 and cARF1 in 70 cases of glioma patients was measured by qPCR. j The prognostic significance of the total 70 glioma patients with high versus low cARF1 expressions as detected by qPCR. k MTS assays showed that cARF1 knockdown or overexpression of GCM affected hBMEC cell viability and was reversed by the miR-342–3p mimic or inhibitor treatment, respectively. l The EDU assay showed that cARF1 knockdown or overexpression of GCM affected the proliferation of hBMECs and was reversed by miR-342–3p inhibitor or mimic treatment, respectively. Scale bar = 50 μm. m A representative Transwell assay showed that cARF1 knockdown or overexpression of GCM affected the invasion of hBMECs and was reversed by the miR-342–3p inhibitor or mimic treatment, respectively. Scale bar = 100 μm. n A representative tube formation assay showed that cARF1 knockdown or overexpression of GCM affected the tubulogenesis of hBMECs and was reversed by the miR-342–3p inhibitor or mimic treatment, respectively. Scale bar = 100 μm. o, p The qPCR (o) and ELISA assay (p) indicated that cARF1 knockdown or overexpression of GCM regulated the mRNA expression and secretion of VEGFA in GSCs and was reversed by miR-342–3p inhibitor or mimic treatment, respectively. EV: empty vector, OE: overexpression, NC: negative control, KD: knockdown. All data are expressed as the mean ± SD (three independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001

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