The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential.

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.

LINC00483 Has a Potential Tumor-Suppressor Role in Colorectal Cancer Through Multiple Molecular Axes.

Long non-coding RNAs (lncRNAs) are the most heterogeneous class of non-protein-coding RNAs involved in a broad spectrum of molecular mechanisms controlling genome function, including the generation of complex networks of RNA-RNA competitive interactions. Accordingly, their dysregulation contributes to the onset of many tumors, including colorectal cancer (CRC). Through a combination of in silico approaches (statistical screening of expression datasets) and in vitro analyses (enforced expression, artificial inhibition, or activation of pathways), we identified LINC00483 as a potential tumor suppressor lncRNA in CRC. LINC00483 was downregulated in CRC biopsies and metastases and its decreased levels were associated with severe clinical features. Inhibition of the MAPK pathway and cell cycle arrest by starvation induced an upregulation of LINC00483, while the epithelial to mesenchymal transition activation by TGFß-1 and IL-6 caused its down-modulation. Moreover, enforced expression of LINC00483 provoked a slowing down of cell migration rate without affecting cell proliferation. Since LINC00483 was predominantly cytoplasmic, we hypothesized a "miRNA sponge" role for it. Accordingly, we computationally reconstructed the LINC00483/miRNA/mRNA axes and evaluated the expression of mRNAs in different experimental conditions inducing LINC00483 alteration. By this approach, we identified a set of mRNAs sharing the miRNA response elements with LINC00483 and modulated in accordance with it. Moreover, we found that LINC00483 is potentially under negative control of transcription factor HNF4α. In conclusion, we propose that LINC00483 is a tumor suppressor in CRC that, through an RNA-RNA network, may control cell migration and participate in proliferation signaling.

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

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.

LncRNA UCA1, Upregulated in CRC Biopsies and Downregulated in Serum Exosomes, Controls mRNA Expression by RNA-RNA Interactions.

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.

CircSMARCA5 Inhibits Migration of Glioblastoma Multiforme Cells by Regulating a Molecular Axis Involving Splicing Factors SRSF1/SRSF3/PTB.

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.

Molecular Crosstalking among Noncoding RNAs: A New Network Layer of Genome Regulation in Cancer.

Over the past few years, noncoding RNAs (ncRNAs) have been extensively studied because of the significant biological roles that they play in regulation of cellular mechanisms. ncRNAs are associated to higher eukaryotes complexity; accordingly, their dysfunction results in pathological phenotypes, including cancer. To date, most research efforts have been mainly focused on how ncRNAs could modulate the expression of protein-coding genes in pathological phenotypes. However, recent evidence has shown the existence of an unexpected interplay among ncRNAs that strongly influences cancer development and progression. ncRNAs can interact with and regulate each other through various molecular mechanisms generating a complex network including different species of RNAs (e.g., mRNAs, miRNAs, lncRNAs, and circRNAs). Such a hidden network of RNA-RNA competitive interactions pervades and modulates the physiological functioning of canonical protein-coding pathways involved in proliferation, differentiation, and metastasis in cancer. Moreover, the pivotal role of ncRNAs as keystones of network structural integrity makes them very attractive and promising targets for innovative RNA-based therapeutics. In this review we will discuss: (1) the current knowledge on complex crosstalk among ncRNAs, with a special focus on cancer; and (2) the main issues and criticisms concerning ncRNAs targeting in therapeutics.

Asymmetric RNA Distribution among Cells and Their Secreted Exosomes: Biomedical Meaning and Considerations on Diagnostic Applications.

Over the past few years, exosomes and their RNA cargo have been extensively studied because of the fascinating biological roles they play in cell-to-cell communication, including the signal exchange among cancer, stromal, and immune cells, leading to modifications of tumor microenvironment. RNAs, especially miRNAs, stored within exosomes, seem to be among the main determinants of such signaling: their sorting into exosomes appears to be cell-specific and related to cellular physiopathology. Accordingly, the identification of exosomal miRNAs in body fluids from pathological patients has become one of the most promising activity in the field of biomarker discovery. Several analyses on the qualitative and quantitative distribution of RNAs between cells and their secreted exosomes have given rise to questions on whether and how accurately exosomal RNAs would represent the transcriptomic snapshot of the physiological and pathological status of secreting cells. Although the exact molecular mechanisms of sorting remain quite elusive, many papers have reported an evident asymmetric quantitative distribution of RNAs between source cells and their exosomes. This phenomenon could depend both on passive and active sorting mechanisms related to: (a) RNA turnover; (b) maintaining the cytoplasmic miRNA:target equilibrium; (c) removal of RNAs not critical or even detrimental for normal or diseased cells. These observations represent very critical issues in the exploitation of exosomal miRNAs as cancer biomarkers. In this review, we will discuss how much the exosomal and corresponding donor cell transcriptomes match each other, to better understand the actual reliability of exosomal RNA molecules as pathological biomarkers reflecting a diseased status of the cells.