CircSMARCA5 Regulates VEGFA mRNA Splicing and Angiogenesis in Glioblastoma Multiforme Through the Binding of SRSF1

Abstract

Circular RNAs are a large group of RNAs whose cellular functions are still being investigated. We recently proposed that circSMARCA5 acts as sponge for the splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1) in glioblastoma multiforme (GBM). After demonstrating by RNA immunoprecipitation a physical interaction between SRFS1 and circSMARCA5, we assayed by real-time PCR in a cohort of 31 GBM biopsies and 20 unaffected brain parenchyma controls (UC) the expression of total, pro-angiogenic (Iso8a) and anti-angiogenic (Iso8b) mRNA isoforms of Vascular Endothelial Growth Factor A (VEGFA), a known splicing target of SRSF1. The Iso8a to Iso8b ratio: (i) increased in GBM biopsies with respect to UC (p-value < 0.00001); (ii) negatively correlated with the expression of circSMARCA5 (r-value = -0.46, p-value = 0.006); (iii) decreased in U87-MG overexpressing circSMARCA5 with respect to negative control (p-value = 0.0055). Blood vascular microvessel density, estimated within the same biopsies, negatively correlated with the expression of circSMARCA5 (r-value = -0.59, p-value = 0.00001), while positively correlated with that of SRSF1 (r-value = 0.38, p-value = 0.00663) and the Iso8a to Iso8b ratio (r-value = 0.41, p-value = 0.0259). Kaplan-Meier survival analysis showed that GBM patients with low circSMARCA5 expression had lower overall and progression free survival rates than those with higher circSMARCA5 expression (p-values = 0.033, 0.012, respectively). Our data convincingly suggest that circSMARCA5 is an upstream regulator of pro- to anti-angiogenic VEGFA isoforms ratio within GBM cells and a highly promising GBM prognostic and prospective anti-angiogenic molecule.

Keywords: RNA binding proteins; VEGFA; alternative splicing; angiogenesis; circular RNA; glioblastoma multiforme; hsa_circ_0001445.

Figure 1

CircSMARCA5 and SRSF1 physically interact within U87-MG cells. (A) SRSF1 binding sites predicted by RBPMap database (http://rbpmap.technion.ac.il/) are highlighted with different colors within circSMARCA5 sequence. * indicates sequence conservation among twelve primates, including Homo sapiens (see [19]). (B) Fold enrichment (FC) of circSMARCA5, SRSF3 and GAPDH are shown as IPed samples/input. See [22] and Section 4 Materials and Methods for further details (* p-value < 0.05; ** p-value < 0.01, N = 4, two-samples t-test). (C) Representative western blot of U87-MG Input, SRSF1 and normal IgG RIPed samples. Antibodies against SRSF1 and Actin Beta (ACTB) were used as described in Section 4 Materials and Methods.

Figure 1

CircSMARCA5 and SRSF1 physically interact within U87-MG cells. (A) SRSF1 binding sites predicted by RBPMap database (http://rbpmap.technion.ac.il/) are highlighted with different colors within circSMARCA5 sequence. * indicates sequence conservation among twelve primates, including Homo sapiens (see [19]). (B) Fold enrichment (FC) of circSMARCA5, SRSF3 and GAPDH are shown as IPed samples/input. See [22] and Section 4 Materials and Methods for further details (* p-value < 0.05; ** p-value < 0.01, N = 4, two-samples t-test). (C) Representative western blot of U87-MG Input, SRSF1 and normal IgG RIPed samples. Antibodies against SRSF1 and Actin Beta (ACTB) were used as described in Section 4 Materials and Methods.

Figure 2

SRSF1, circSMARCA5 and VEGFA expression in GBM biopsies and unaffected controls (UC). (A) Box-and-whisker plots, representing the expression of SRSF1, circSMARCA5 and total VEGFA RNA in the studied cohort. Data are represented as log₂ fold change (FC) values versus UC. (*** p-value < 0.001, N_(GBM) = 31, N_(UC) = 20, Mann–Whitney test). (B) Western blot of SRSF1 in a selection of UC (N) and GBM (T) samples. ACTB was used as a loading control. (C) Bar graph representing densitometric quantification of SRSF1. Data, shown as mean ± standard deviation, represent fold change versus UC (* p-value < 0.05, N_(GBM) = 14, N_(UC) = 8, two-sample t-test).

Figure 3

Iso8a-to-Iso8b ratio in GBM biopsies vs. UC. (A) Box-and-whisker plots, representing the ratios between fold changes of Iso8a and total VEGFA (VEGFA_tot) and Iso8b and VEGFA_tot in GBM compared to UC (*** p-value < 0.00001, N = 27, Mann–Whitney test) (see Section 4 Materials and Methods for details on data analysis). (B) Correlation matrix among the expression of circSMARCA5 and SRSF1 and Iso8a-to-Iso8b ratio. Positive and negative correlations are displayed in blue and red colors, respectively. The color scale bar indicates r values. Color intensity and the size of the circle are proportional to the correlation coefficients into the correlogram. (C) Box-and-whisker plots, representing the ratios between fold changes of Iso8a and VEGFA_tot and Iso8b and VEGFA_tot in U87-MG overexpressing circSMARCA5 with respect to U87-MG transfected with the empty vector (NC) (** p-value < 0.001, N = 3, two-sample t-test) (see Section 4 Materials and Methods for details on data analysis).

Figure 4

MVD in GBM and UC samples. (A) Representative immunohistochemical staining for CD31 showing areas of high MVD with multiple branching vessels in glioblastoma tissue (1 and 2) and a lower MVD in unaffected brain tissue (3 and 4). Immunoperoxidase staining; 400× magnification (B) Bar graph representing the mean MVD in GBM and UC samples. Data are represented as mean ± standard deviation (*** p-value < 0.00001, N_(GBM) = 31, N_(UC) = 18, two sample t-test). (C) Correlation matrix among MVD, circSMARCA5 and SRSF1 expression and Iso8a to Iso8b ratio. Positive and negative correlations are displayed in blue and red color, respectively. Color scale bar indicates r values. Color intensity and the size of the circle are proportional to the correlation coefficients into the correlogram.

Figure 5

Kaplan–Meier overall survival (OS) and progression-free survival (PFS) curves of GBM patients, based on the expression of circSMARCA5. Patients having a lower expression of circSMARCA5 (DCt ≥ Median DCt) survive less (A) show a shorter PFS (B) than patients with a higher expression of circSMARCA5 (DCt < Median DCt).