Role of non-coding RNA alterations in melanoma

Cutaneous melanoma (CM) is a potentially lethal form of skin cancer and its most important histopathologic factor for staging is Breslow thickness (BT). Its correct determination is fundamental for pathologists. A deeper understanding of the molecular processes guiding CM pathogenesis could improve diagnosis, treatment and prognosis. MicroRNAs (miRNAs) play a key role in CM biology. The firs aim was to investigate miRNA expression in reference to BT assessment. We found that the combined miRNA expression of miR-21-5p and miR-146a-5p above or below 1.5 was significantly associated with overall survival and successfully identified all superficially spreading melanoma (SSM) patients with relapsing suggesting that the combined assessment of these miRNAs expression could aid in SSM staging. Secondly, we focus on multiple primary melanoma (MPM) patients, which develop multiple primary melanomas in their lifetime, and represent a model of high-risk CM occurrence. We explored the miRNome of single CM and MPM: CM and MPM present several dysregulated miRNAs, including key miRNAs involved in epithelial-mesenchymal transition. A different miRNA profile was observed between 1st and 2nd melanoma from the same patient. MiRNA target analysis revealed a more differentiated and less invasive status of MPMs compared to CMs. This characterization of the miRNA regulatory network of MPMs highlights molecular features differentiating this subtype from CM. Recently, NGS experiments revealed the existence of miRNA variants (isomiRs) with different length and sequence. We identified a shorter 3’isoform as tenfold over-represented compared to the canonical form of miR-125a-5p. Target analysis revealed that miRNA shortening could change the pattern of target gene regulation. Finally, we study miRNA and isomiR dysregulation in benign nevi (BN) and CM and in CM and melanoma metastasis. The reported non-random dysregulation of specific isomiRs contributes to the understanding of the complex melanoma pathogenesis and serves as the basis for further functional studies.

Characterisation of RNase Y in H. pylori: a non-essential enzyme involved in the processing of cag-PAI non coding RNA 1 (CncR1) sRNA

The importance of Helicobacter pylori as a human pathogen is underlined by the plethora of diseases it is responsible for. The capacity of H. pylori to adapt to the restricted host-associated environment andto evade the host immune response largely depends on a streamlined signalling network. The peculiar H. pylori small genome size combined with its paucity of transcriptional regulators highlights the relevance of post-transcriptional regulatory mechanisms as small non-coding RNAs (sRNAs). However, among the 8 RNases represented in H. pylori genome, a regulator guiding sRNAs metabolism is still not well studied. We investigated for the first time the physiological role in H. pylori G27 strain of the RNase Y enzyme. In the first line of research we provide a comprehensive characterization of the RNase Y activity by analysing its genomic organization and the factors that orchestrate its expression. Then, based on bioinformatic prediction models, we depict the most relevant determinants of RNase Y function, demonstrating a correlation of both structure and domain organization with orthologues represented in Gram-positive bacteria. To unveil the post-transcriptional regulatory effect exerted by the RNase Y, we compared the transcriptome of an RNase Y knock-out mutant to the parental wild type strain by RNA-seq approach. In the second line of research we characterized the activity of this single strand specific endoribonuclease on cag-PAI non coding RNA 1 (CncR1) sRNA. We found that deletion or inactivation of RNase Y led to the accumulation of a 3’-extended CncR1 (CncR1-L) transcript over time. Moreover, beneath its increased half-life, CncR1-L resembled a CncR1 inactive phenotype. Finally, we focused on the characterization of the in vivo interactome of CncR1. We set up a preliminary MS2-affinity purification coupled with RNA-sequencing (MAPS) approach and we evaluated the enrichment of specific targets, demonstrating the suitability of the technique in the H. pylori G27 strain.

Role of Non-coding RNAs in chemotherapeutic treatments

The transcribed ultraconserved regions (T-UCRs) are a group of long non-coding RNAs involved in human carcinogenesis. The factors regulating the expression of T-UCRs and their mechanism of action in human cancers are unknown. In this work it was shown that high expression of uc.339 associates with lower survival in 204 non-small cell lung cancer (NSCLC) patients. Moreover, it was shown that uc.339 found up-regulated in archival NSCLC samples, acts as a decoy RNA for miR-339-3p, -663-3p and -95-5p. So, Cyclin E2, a direct target of three microRNAs is up-regulated, inducing cancer growth and migration. Evidence of this mechanism was provided from cell lines and primary samples confirming that TP53 directly regulates uc.339. These results support a key role for uc.339 in lung cancer.

Identification and characterization of the sRNA network in Neisseria meningitidis

Bacterial small regulatory RNAs (sRNAs) are posttranscriptional regulators involved in stress responses. These short non-coding transcripts are synthesised in response to a signal, and control gene expression of their regulons by modulating the translation or stability of the target mRNAs, often in concert with the RNA chaperone Hfq. Characterization of a Hfq knock out mutant in Neisseria meningitidis revealed that it has a pleiotropic phenotype, suggesting a major role for Hfq in adaptation to stresses and virulence and the presence of Hfq-dependent sRNA activity. Global gene expression analysis of regulated transcripts in the Hfq mutant revealed the presence of a regulated sRNA, incorrectly annotated as an open reading frame, which we renamed AniS. The synthesis of this novel sRNA is anaerobically induced through activation of its promoter by the FNR global regulator and through global gene expression analyses we identified at least two predicted mRNA targets of AniS. We also performed a detailed molecular analysis of the action of the sRNA NrrF,. We demonstrated that NrrF regulates succinate dehydrogenase by forming a duplex with a region of complementarity within the sdhDA region of the succinate dehydrogenase transcript, and Hfq enhances the binding of this sRNA to the identified target in the sdhCDAB mRNA; this is likely to result in rapid turnover of the transcript in vivo. In addition, in order to globally investigate other possible sRNAs of N. meningitdis we Deep-sequenced the transcriptome of this bacterium under both standard in vitro and iron-depleted conditions. This analysis revealed genes that were actively transcribed under the two conditions. We focused our attention on the transcribed non-coding regions of the genome and, along with 5’ and 3’ untranslated regions, 19 novel candidate sRNAs were identified. Further studies will be focused on the identification of the regulatory networks of these sRNAs, and their targets.