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

Davide Barbagallo,Giuseppe Broggi,Thomas Hansen,Francesco Certo,Rosario Caltabiano,Michele Purrello,Cinzia Di Pietro,Duilia Brex,Giovanni Lauretta,Antonio Morrone,Angela Caponnetto,Giuseppe Barbagallo,Federica Mirabella,Cristina Barbagallo,Vittoria Spina-Purrello,Marco Ragusa

doi:10.3390/cancers11020194

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.

Highlights

Circular RNAs are a recently discovered large group of RNAs, of which biogenesis and functions within cells remain mostly to be investigated [1,2,3,4]
Serine and Arginine Rich Splicing Factor 1 (SRSF1) RNA immunoprecipitation (RIP) allowed us to validate this interaction, showing a significant enrichment of circSMARCA5 in addition to SRSF3 mRNA, a known splicing target of SRSF1, in immunoprecipitated U87-MG cells lysate with respect to a negative control (Figure 1B,C)
We demonstrated that total Vascular Endothelial Growth Factor A (VEGFA) mRNA was upregulated in the same cohort (p-value < 0.00001, Mann–Whitney test), while circSMARCA5 was downregulated (p-value < 0.00001, Mann–Whitney test), confirming our data previously obtained in an independent glioblastoma multiforme (GBM) cohort (Figure 2A)

Summary

Introduction

Circular RNAs (circRNAs) are a recently discovered large group of RNAs, of which biogenesis and functions within cells remain mostly to be investigated [1,2,3,4]. Notwithstanding the published data on the involvement of circRNAs and splicing in GBM, many questions remain without an answer [15,16,17,18]. We hypothesized that circSMARCA5 may carry out its function by tethering the splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1) and that through this mechanism it may regulate GBM cells migration [19]. SRSF1 is involved in a plethora of other cellular functions as: (i) regulation of RNA metabolism; (ii) mRNA translation; (iii) miRNA processing;

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Conclusion