Profiling of Small Nucleolar RNAs by Next Generation Sequencing: Potential New Players for Breast Cancer Prognosis

<div><p>One of the most abundant, yet least explored, classes of RNA is the small nucleolar RNAs (snoRNAs), which are well known for their involvement in post-transcriptional modifications of other RNAs. Although snoRNAs were only considered to perform housekeeping functions for a long time, recent studies have highlighted their importance as regulators of gene expression and as diagnostic/prognostic markers. However, the prognostic potential of these RNAs has not been interrogated for breast cancer (BC). The objective of the current study was to identify snoRNAs as prognostic markers for BC. Small RNA sequencing (Illumina Genome Analyzer IIx) was performed for 104 BC cases and 11 normal breast tissues. Partek Genomics Suite was used for analyzing the sequencing files. Two independent and proven approaches were used to identify prognostic markers: case-control (CC) and case-only (CO). For both approaches, snoRNAs significant in the permutation test, following univariate Cox proportional hazards regression model were used for constructing risk scores. Risk scores were subsequently adjusted for potential confounders in a multivariate Cox model. For both approaches, thirteen snoRNAs were associated with overall survival and/or recurrence free survival. Patients belonging to the high-risk group were associated with poor outcomes, and the risk score was significant after adjusting for confounders. Validation of representative snoRNAs (SNORD46 and SNORD89) using qRT-PCR confirmed the observations from sequencing experiments. We also observed 64 snoRNAs harboring piwi-interacting RNAs and/or microRNAs that were predicted to target genes (mRNAs) involved in tumorigenesis. Our results demonstrate the potential of snoRNAs to serve (i) as novel prognostic markers for BC and (ii) as indirect regulators of gene expression.</p></div

qRT-PCR confirmation of snoRNA expression.

<p>SNORD46 and SNORD89 were confirmed to be down–regulated in tumor, relative to normal samples using qRT-PCR platform. The Ct values obtained for snoRNAs were normalized to Ct values obtained for RNU6. * indicates statistical significance p<0.05.</p

Univariate and multivariate results.

<p>Univariate and multivariate results.</p

snoRNA-piRNA pairs with same direction of expression, fold change > 2.0 and FDR ≤ 0.05.

<p>snoRNA-piRNA pairs with same direction of expression, fold change > 2.0 and FDR ≤ 0.05.</p

Hierarchical clustering of differentially expressed snoRNAs.

<p>The 40 differentially expressed snoRNAs were subjected to unsupervised hierarchical clustering with average linkage and Euclidean as distance measure. The tumor samples (orange horizontal bars) were clearly separated from the normal samples (red horizontal bars).</p

Kaplan–Meier plots for case–control approach.

<p>Kaplan-Meier plots for risk scores were constructed to determine survival differences between low–risk and high–risk groups. Significant survival differences existed between the two risk groups, as indicated by the log–rank p–values. (A) OS for CC approach. (B) RFS for CC approach. (C) OS for CO approach and (D) RFS for CO approach. In all these approaches, patients belonging to high–risk group showed poor OS and RFS.</p

Additional file 2: Table S1. of Next generation sequencing profiling identifies miR-574-3p and miR-660-5p as potential novel prognostic markers for breast cancer

Differentially expressed miRNAs. Description: miRNAs were profiled from apparently healthy normal (n = 11) reduction mammoplasty breast tissues and breast tumor tissues (n = 104). RNAs were filtered for low read counts (minimum 10 read counts in at least 90 % of the samples). Following batch effects correction, sample outlier removal and RPKM normalization, differentially expressed RNAs with fold change > 2.0 and a false discovery rate (FDR) < 0.05 were identified. (PDF 63 kb

Discovery of ABT-267, a Pan-Genotypic Inhibitor of HCV NS5A

We describe here <i>N</i>-phenylpyrrolidine-based inhibitors of HCV NS5A with excellent potency, metabolic stability, and pharmacokinetics. Compounds with 2<i>S</i>,5<i>S</i> stereochemistry at the pyrrolidine ring provided improved genotype 1 (GT1) potency compared to the 2<i>R</i>,5<i>R</i> analogues. Furthermore, the attachment of substituents at the 4-position of the central <i>N</i>-phenyl group resulted in compounds with improved potency. Substitution with <i>tert</i>-butyl, as in compound <b>38</b> (ABT-267), provided compounds with low-picomolar EC₅₀ values and superior pharmacokinetics. It was discovered that compound <b>38</b> was a pan-genotypic HCV inhibitor, with an EC₅₀ range of 1.7–19.3 pM against GT1a, -1b, -2a, -2b, -3a, -4a, and -5a and 366 pM against GT6a. Compound <b>38</b> decreased HCV RNA up to 3.10 log₁₀ IU/mL during 3-day monotherapy in treatment-naive HCV GT1-infected subjects and is currently in phase 3 clinical trials in combination with an NS3 protease inhibitor with ritonavir (r) (ABT-450/r) and an NS5B non-nucleoside polymerase inhibitor (ABT-333), with and without ribavirin

Highlights of the Structure–Activity Relationships of Benzimidazole Linked Pyrrolidines Leading to the Discovery of the Hepatitis C Virus NS5A Inhibitor Pibrentasvir (ABT-530)

Curative interferon and ribavirin sparing treatments for hepatitis C virus (HCV)-infected patients require a combination of mechanistically orthogonal direct acting antivirals. A shared component of these treatments is usually an HCV NS5A inhibitor. First generation FDA approved treatments, including the component NS5A inhibitors, do not exhibit equivalent efficacy against HCV virus genotypes 1–6. In particular, these first generation NS5A inhibitors tend to select for viral drug resistance. Ombitasvir is a first generation HCV NS5A inhibitor included as a key component of Viekira Pak for the treatment of patients with HCV genotype 1 infection. Since the launch of next generation HCV treatments, functional cure for genotype 1–6 HCV infections has been achieved, as well as shortened treatment duration across a wider spectrum of genotypes. In this paper, we show how we have modified the anchor, linker, and end-cap architecture of our NS5A inhibitor design template to discover a next generation NS5A inhibitor pibrentasvir (ABT-530), which exhibits potent inhibition of the replication of wild-type genotype 1–6 HCV replicons, as well as improved activity against replicon variants demonstrating resistance against first generation NS5A inhibitors