Fig 2: ICAM1 is more highly expressed in GBM cells expressing greater levels of MSI1-compared to GBM cells expressing lower levels of MSI1. (A) Gene ontology classification. (B) Schema for identifying genes under conditions of >3-fold differences between U87MG/MSI1 and U87MG/Ctrl cells, <0.75-fold differences between 05MG/sh-MSI1 and 05MG/sh-Scr cells, and migration-associated factors. (C) qPCR analysis of ICAM1 in U87MG /Ctrl, U87MG /MSI1, 05MG/sh-Scr, and 05MG /sh-MSI1 cells. *P < .01 by Student's t-test. Data shown are the mean ± SD of three independent experiments.

Fig 3: (A) IHC staining detected MSI1 in LGG and GBM patient samples. Scale bars, 100 μm. All data are presented as the mean ± SD, n = 10, *P < .01 by Student's t-test. (B) IHC staining detected ICAM1 in LGG and GBM patient samples. Scale bars, 100 μm. All data are presented as the mean ± SD, n = 10, *P < .01 by Student's t-test. (C) MSI1 and ICAM1 protein co-localization in tissues by IHC staining. Scale bars,100 μm. All data are presented as the mean ± SD, n = 10, *P < .01 by Student's t-test. (D) Different Kaplan–Meier plots of GBM patients with high MSI1 expression, high ICAM1expression, and high MSI1/ICAM1 expression. (E) Schematic illustration depicting that MSI1-mediated mRNA interaction promotes ICAM1 translation and tumor invasion.

Fig 4: MSI1-mediated mRNA interaction promotes ICAM1 translation. Representative immunoblotting analysis of ICAM1, Flag, and LIN28 in (A) U87MG cells. ß-Actin was used as the loading control (upper panel). RNA-immunoprecipitation of Flag-control or Flag-MSI1-overexpressing cell lines was performed using an antibody against Flag (IP-Flag). RT-PCR analysis for detecting mRNA expression of ICAM1, p21, and GAPDH (lower panel). To confirm RNA-mediated inhibition of MSI1, RNase A was used to eliminate total RNA in vitro, demonstrating that MSI1 promotes ICAM1 translation in an RNA-dependent manner. Input was 5% of total mRNA, and IgG was used as a negative control. (B) Identification of the effect of MSI1 on stabilizing ICAM1 mRNA in the U87MG cell line. Control or MSI1-overexpressing U87MG cells were treated with the transcriptional inhibitor actinomycin D (Act D, 6.5 µg/ml), and total RNA was harvested at 0, 1, 2 3 and 4 hr. posttreatment. ICAM1 mRNA levels were determined relative to GAPDH by RT-PCR and quantitated using ImageJ software.