Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood

Candida Vaz,Pratibha Sharma,Rashi Gupta,Hafiz M Ahmad,Ritu Kulshreshtha,Lalit Kumar,Alok Bhattacharya

doi:10.1186/1471-2164-11-288

Candida Vaz, Pratibha Sharma + Show 5 more

Open Access

https://doi.org/10.1186/1471-2164-11-288

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Abstract

BackgroundMicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post - transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack the ability to identify novel miRNAs and accurately determine expression at a range of concentrations. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts.ResultsThe results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 and HL60 are presented. In general K562 cells displayed overall low level of miRNA population and also low levels of DICER. Some of the highly expressed miRNAs in the leukocytes include several members of the let-7 family, miR-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 or HL60 cells revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Relative expression levels of individual miRNAs belonging to a cluster were found to be highly variable. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by Real-time RT-PCR and or RNase protection assay. Organization of some of the novel miRNAs in human genome suggests that these may also be part of existing clusters or form new clusters.ConclusionsWe conclude that about 904 miRNAs are expressed in human leukocytes. Out of these 370 are novel miRNAs. We have identified miRNAs that are differentially regulated in normal PBMC with respect to cancer cells, K562 and HL60. Our results suggest that post - transcriptional processes may play a significant role in regulating levels of miRNAs in tumor cells. The study also provides a customized automated computation pipeline for miRNA profiling and identification of novel miRNAs; even those that are missed out by other existing pipelines. The Computational Pipeline is available at the website: http://mirna.jnu.ac.in/deep_sequencing/deep_sequencing.html

Highlights

MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post transcriptional level and thereby many fundamental biological processes
Our major findings are a list of expressed miRNAs (534 known and 370 novel) in leukocytes and the discovery that the expression of miRNAs may be controlled by regulating post - transcriptional events, such as manipulating the level of DICER, an enzyme involved in biosynthesis of miRNAs in tumor cells (K562)
Our analysis shows that some of the novel miRNAs are likely to be clustered in the genome similar to many known clusters of miRNAs

Summary

Introduction

MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post transcriptional level and thereby many fundamental biological processes. Small non-coding RNAs participate in a variety of processes from cell development and differentiation, stress responses to carcinogenesis by regulating gene expression [1,2,3,4]. The profiles generated revealed altered levels of miRNAs in different systems, such as oncogenesis and development [15,16] These studies showed strong correlation between specific miRNA expression and phenotype suggesting it can be used as a potential biomarker for diagnosis and prognosis in human cancer [17]. Many of the fundamental processes are regulated by miRNAs, such as cell differentiation (miR-223, miR-145) [28,29], apoptosis (miR-34, miR-16) [21,30], body patterning (miR-9, miR-196) [31,32], nervous system and muscle development (miR-134, miR-1 and miR-133) [33,34]

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