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.

Circular RNAs (circRNAs) have recently emerged as a new class of RNAs, highly enriched in the brain and very stable within cells, exosomes and body fluids. To analyze their involvement in glioblastoma multiforme (GBM) pathogenesis, we assayed the expression of twelve circRNAs, physiologically enriched in several regions of the brain, through real-time PCR in a cohort of fifty-six GBM patient biopsies and seven normal brain parenchymas. We focused on hsa_circ_0001445 (circSMARCA5): it was significantly downregulated in GBM biopsies as compared to normal brain tissues (p-value < 0.00001, student’s t-test), contrary to its linear isoform counterpart that did not show any differential expression (p-value = 0.694, student’s t-test). Analysis of a public dataset revealed a negative correlation between the expression of circSMARCA5 and glioma’s histological grade, suggesting its potential negative role in the progression to malignancy. Overexpressing circSMARCA5 in U87MG cells significantly decreased their migration, but not their proliferation rate. In silico scanning of circSMARCA5 sequence revealed an enrichment in binding motifs for several RNA binding proteins (RBPs), specifically involved in splicing. Among them, serine and arginine rich splicing factor 1 (SRSF1), a splicing factor known to be a positive controller of cell migration and known to be overexpressed in GBM, was predicted to bind circSMARCA5 by three different prediction tools. Direct interaction between circSMARCA5 and SRSF1 is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP) data for SRSF1 in K562 cells from Encyclopedia of DNA Elements (ENCODE). Consistently, U87MG overexpressing circSMARCA5 showed an increased expression of serine and arginine rich splicing factor 3 (SRSF3) RNA isoform containing exon 4, normally skipped in a SRSF1-dependent manner, resulting in a non-productive non-sense mediated decay (NMD) substrate. Interestingly, SRSF3 is known to interplay with two other splicing factors, polypyrimidine tract binding protein 1 (PTBP1) and polypyrimidine tract binding protein 2 (PTBP2), that positively regulate glioma cells migration. Collectively, our data show circSMARCA5 as a promising druggable tumor suppressor in GBM and suggest that it may exert its function by tethering the RBP SRSF1.

Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) contribute to the onset of many neoplasias through RNA-RNA competitive interactions; in addition, they could be secreted by cancer cells into biological fluids, suggesting their potential diagnostic application. By analyzing the expression of 17 lncRNAs and 31 circRNAs in biopsies and serum exosomes from colorectal cancer (CRC) patients through qRT-PCR, we detected CCAT1, CCAT2, HOTAIR, and UCA1 upregulation and CDR1AS, MALAT1, and TUG1 downregulation in biopsies. In serum exosomes, UCA1 was downregulated, while circHIPK3 and TUG1 were upregulated. Combined receiver operating characteristic (ROC) curves of TUG1:UCA1 and circHIPK3:UCA1 showed high values of sensitivity and specificity. Through in vitro (i.e., RNA silencing and mitogen-activated protein kinase [MAPK] inhibition) and in silico analyses (i.e., expression correlation and RNA-RNA-binding prediction), we found that UCA1 could (1) be controlled by MAPKs through CEBPB; (2) sequester miR-135a, miR-143, miR-214, and miR-1271, protecting ANLN, BIRC5, IPO7, KIF2A, and KIF23 from microRNA (miRNA)-induced degradation; and (3) interact with mRNA 3′-UTRs, preventing miRNA binding. UCA1 and its co-regulated antisense LINC01764 could interact and reciprocally mask their own miRNA-binding sites. Functional enrichment analysis of the RNA-RNA network controlled by UCA1 suggested its potential involvement in cellular migration. The UCA1 regulatory axis would represent a promising target to develop innovative RNA-based therapeutics against CRC.

Glioblastoma multiforme (GBM) is the most frequent and deadly human brain cancer. Early diagnosis through non-invasive biomarkers may render GBM more easily treatable, improving the prognosis of this currently incurable disease. We suggest the use of serum extracellular vesicle (sEV)-derived circular RNAs (circRNAs) as highly stable minimally invasive diagnostic biomarkers for GBM diagnosis. EVs were isolated by size exclusion chromatography from sera of 23 GBM and 5 grade 3 glioma (GIII) patients, and 10 unaffected controls (UC). The expression of two candidate circRNAs (circSMARCA5 and circHIPK3) was assayed by droplet digital PCR. CircSMARCA5 and circHIPK3 were significantly less abundant in sEVs from GBM patients with respect to UC (fold-change (FC) of −2.15 and −1.92, respectively) and GIII (FC of −1.75 and −1.4, respectively). Receiver operating characteristic curve (ROC) analysis, based on the expression of sEV-derived circSMARCA5 and circHIPK3, allowed us to distinguish GBM from UC (area under the curve (AUC) 0.823 (0.667–0.979) and 0.855 (0.704 to 1.000), with a 95% confidence interval (CI), respectively). Multivariable ROC analysis, performed by combining the expression of sEV-derived circSMARCA5 and circHIPK3 with preoperative neutrophil to lymphocyte (NLR), platelet to lymphocyte (PLR) and lymphocyte to monocyte (LMR) ratios, three known diagnostic and prognostic GBM markers, allowed an improvement in the GBM diagnostic accuracy (AUC 0.901 (0.7912 to 1.000), 95% CI). Our data suggest sEV-derived circSMARCA5 and circHIPK3 as good diagnostic biomarkers for GBM, especially when associated with preoperative NLR, PLR and LMR.

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.