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

ABSTRACT: 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.

SUBMITTER: Krishnan P

PROVIDER: S-EPMC5025248 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Krishnan Preethi P Ghosh Sunita S Wang Bo B Heyns Mieke M Graham Kathryn K Mackey John R JR Kovalchuk Olga O Damaraju Sambasivarao S

PloS one 20160915 9

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 ...[more]

PMID: 27631501

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Project description:BackgroundSmall ncRNAs (sncRNAs) offer great hope as biomarkers of disease and response to treatment. This has been highlighted in the context of several medical conditions such as cancer, liver disease, cardiovascular disease, and central nervous system disorders, among many others. Here we assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing of small RNA sequencing data.MethodsA total of 45 biological samples were included in the present study. All libraries were prepared using the Illumina TruSeq Small RNA protocol and sequenced using the HiSeq2500 or MiSeq Illumina sequencers. Small RNA sequencing data was validated using qRT-PCR. At each stage, we evaluated the pros and cons of different techniques that may be suitable for different experimental designs. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.ResultsOur results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS. In addition, we describe the strengths and limitations of three commercially available library preparation methods: (1) Novex TBE PAGE gel; (2) Pippin Prep automated gel system; and (3) AMPure XP beads. We describe our bioinformatics pipeline, provide recommendations for sequencing coverage, and describe in detail the expression and distribution of all sncRNAs in four human tissues: whole-blood, brain, heart and liver.ConclusionsUltimately this study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate and effective high-throughput sequencing quantification method for various study designs, and overall generating valuable information that can contribute to our understanding of small ncRNAs as potential biomarkers and mediators of biological functions and disease.

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

Publications

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

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