PARP-14 Promotes Survival of Mammalian α but Not β Pancreatic Cells Following Cytokine Treatment

PARP-14 (poly-ADP Ribose Polymerase-14), a member of the PARP family, belongs to the group of Bal proteins (B Aggressive Lymphoma). PARP-14 has recently appeared to be involved in the transduction pathway mediated by JNKs (c Jun N terminal Kinases), among which JNK2 promotes cancer cell survival. Several pharmacological PARP inhibitors are currently used as antitumor agents, even though they have also proved to be effective in many inflammatory diseases. Cytokine release from immune system cells characterizes many autoimmune inflammatory disorders, including type I diabetes, in which the inflammatory state causes β cell loss. Nevertheless, growing evidence supports a concomitant implication of glucagon secreting α cells in type I diabetes progression. Here, we provide evidence on the activation of a survival pathway, mediated by PARP-14, in pancreatic α cells, following treatment of αTC1.6 glucagonoma and βTC1 insulinoma cell lines with a cytokine cocktail: interleukin 1 beta (IL-1β), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). Through qPCR, western blot and confocal analysis, we demonstrated higher expression levels of PARP-14 in αTC1.6 cells with respect to βTC1 cells under inflammatory stimuli. By cytofluorimetric and caspase-3 assays, we showed the higher resistance of α cells compared to β cells to apoptosis induced by cytokines. Furthermore, the ability of PJ-34 to modulate the expression of the proteins involved in the survival pathway suggests a protective role of PARP-14. These data shed light on a poorly characterized function of PARP-14 in αTC1.6 cells in inflammatory contexts, widening the potential pharmacological applications of PARP inhibitors

Profiling of circulating microRNAs in body fluids from Autism Spectrum Disorder patients

Autism Spectrum Disorder is the name for a heterogeneous group of neurodevelopmental conditions, clinically defined by: defects in social interaction and communication; fixed interests and repetitive behaviors. Molecular basis of ASD is heterogeneous and only partially known. ASD-associated variants have been characterized in hundreds of genes and separate transcriptome studies have identified points of convergence among these loci, proving that common biological processes play a role in this disorder. However, no common ASD-associated variants with large effect size, that would be appropriate for its molecular diagnosis, have been identified to date, and therefore, diagnosis just relies on clinical assessment and confirmation. Many factors, including disorders comorbid with ASD, like Tourette Syndrome, complicate ASD behavior-based diagnosis and make it vulnerable to bias. Extracellular microRNAs have attracted researchers for their potential as non-invasive tools for diagnosis, prognosis, and treatment evaluation of human diseases and disorders. Circulating miRNAs can be detected in all mammalian body fluids, from serum to saliva. Stability and general consistency of levels among individuals, along with the existence of specific expression signatures in association with both physiological and pathological conditions, make circulating miRNAs appropriate biomarkers. To investigate ASD etiology and to identify potential biomarkers to support its diagnosis, we used TLDA technology to profile serum miRNAs from ASD, TS, and TS+ASD patients and NCs (unaffected controls). Through validation assays, we demonstrated that miR-140-3p is upregulated in ASD vs: NC, TS, and TS+ASD. We found that delta Ct values for miR-140-3p and YGTSS scores are positively correlated. Our network functional analysis showed that nodes controlled by miR-140-3p, especially CD38 and NRIP1, are involved in processes convergingly dysregulated in ASD, such as synaptic plasticity, immune response, and chromatin binding. Through biomarker analysis, we proved that serum miR-140-3p can discriminate among ASD and NC, ASD and TS, and ASD and TS+ASD, showing that it could be useful to strengthen the behavior-based diagnosis of either ASD or TS+ASD, which can be challenging in some clinical cases. Among all body fluids, saliva represents the most accessible and complete source of different types of molecules that could reflect genetic, epigenetic, environmental, metabolic, emotional, and behavioral alterations in ASD. Therefore, we also used NanoString nCounter technology to profile supernatant saliva circulating miRNAs from ASD patients and NCs. Through validation assays, we demonstrated that both miR-29a-3p and miR-141-3p are upregulated in ASD saliva compared to NC one. We observed that delta Ct values for both miRNAs are correlated with neuropsychiatric scores evaluating ASD defects in social interaction and verbal communication. Target genes of these miRNAs represent main components and regulators of pathways and processes known to be dysregulated in ASD. Through biomarker performance evaluation, we proved that saliva miR-29a-3p and miR-141-3p when used in combination could be useful and non-invasive tools for discriminating ASD patients. In particular, these miRNAs could be used as supportive means for the recognition of ASD verbal and social defects. Overall, our findings suggest that profiling of circulating miRNAs in body fluids can represent an easy and innovative approach to address important biomedical issues, such as the need for biomarkers and the necessity to investigate neurodevelopmental disorders through more accessible patient biopsies. In fact, through the characterization of miRNAs in ASD serum and saliva, we identified three miRNAs that could facilitate ASD clinical assessment and that are worth being further investigated for their potential role in neurodevelopment

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

Expression and Regulatory Network Analysis of miR-140-3p, a New Potential Serum Biomarker for Autism Spectrum Disorder

Given its prevalence and social impact, Autism Spectrum Disorder (ASD) is drawing much interest. Molecular basis of ASD is heterogeneous and only partially known. Many factors, including disorders comorbid with ASD, like TS (Tourette Syndrome), complicate ASD behavior-based diagnosis and make it vulnerable to bias. To further investigate ASD etiology and to identify potential biomarkers to support its precise diagnosis, we used TaqMan Low Density Array technology to profile serum miRNAs from ASD, TS, and TS+ASD patients, and unaffected controls (NCs). Through validation assays in 30 ASD, 24 TS, and 25 TS+ASD patients and 25 NCs, we demonstrated that miR-140-3p is upregulated in ASD vs.: NC, TS, and TS+ASD (Tukey's test, p-values = 0.03, = 0.01, < 0.0001, respectively). ΔCt values for miR-140-3p and YGTSS (Yale Global Tic Severity Scale) scores are positively correlated (Spearman r = 0.33; Benjamini-Hochberg p = 0.008) and show a linear relationship (p = 0.002). Network functional analysis showed that nodes controlled by miR-140-3p, especially CD38 and NRIP1 which are its validated targets, are involved in processes convergingly dysregulated in ASD, such as synaptic plasticity, immune response, and chromatin binding. Biomarker analysis proved that serum miR-140-3p can discriminate among: (1) ASD and NC (Area under the ROC curve, AUC: 0.70; sensitivity: 63.33%; specificity: 68%); (2) ASD and TS (AUC: 0.72; sensitivity: 66.66%; specificity: 70.83%); (3) ASD and TS+ASD (AUC: 0.78; sensitivity: 73.33%; specificity: 76%). Characterization of miR-140-3p network would contribute to further clarify ASD etiology. Serum miR-140-3p could represent a potential non-invasive biomarker for ASD, easy to test through liquid biopsy

The contributions of rare inherited and polygenic risk to ASD in multiplex families.

Autism spectrum disorder (ASD) has a complex genetic architecture involving contributions from both de novo and inherited variation. Few studies have been designed to address the role of rare inherited variation or its interaction with common polygenic risk in ASD. Here, we performed whole-genome sequencing of the largest cohort of multiplex families to date, consisting of 4,551 individuals in 1,004 families having two or more autistic children. Using this study design, we identify seven previously unrecognized ASD risk genes supported by a majority of rare inherited variants, finding support for a total of 74 genes in our cohort and a total of 152 genes after combined analysis with other studies. Autistic children from multiplex families demonstrate an increased burden of rare inherited protein-truncating variants in known ASD risk genes. We also find that ASD polygenic score (PGS) is overtransmitted from nonautistic parents to autistic children who also harbor rare inherited variants, consistent with combinatorial effects in the offspring, which may explain the reduced penetrance of these rare variants in parents. We also observe that in addition to social dysfunction, language delay is associated with ASD PGS overtransmission. These results are consistent with an additive complex genetic risk architecture of ASD involving rare and common variation and further suggest that language delay is a core biological feature of ASD

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

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. Keywords: colorectal cancer, ceRNA network, circRNAs, lncRNAs, miRNAs, LINC01764, TUG1, circHIPK3, CEBP