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

BMC Genomics, May 2010

Background MicroRNAs 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. Results The 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. Conclusions We 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

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

Candida Vaz 0 Hafiz M Ahmad 2 Pratibha Sharma 1 Rashi Gupta 0 Lalit Kumar 1 Ritu Kulshreshtha 2 Alok Bhattacharya 0 2 0 School of Information Technology, Jawaharlal Nehru University , New Delhi , India 1 Department of Medical Oncology, Institute Rotary Cancer Hospital, All India Institute of Medical Science , New Delhi , India 2 School of Life Sciences, Jawaharlal Nehru University , New Delhi , India Background: MicroRNAs 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. Results: The 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. Conclusions: We 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 - Background Small non-coding RNAs participate in a variety of processes from cell development and differentiation, stress responses to carcinogenesis by regulating gene expression [1-4]. Regulatory non-coding RNAs have been reported from almost all organisms from bacteria to mammals [5]. Among the various classes of non-coding small RNAs (sRNAs), the most conserved and prominent ones are the microRNAs or miRNAs [for a recent review see, [6]]. Mature miRNA sequences are single stranded, typically 18-24 nucleotides long and encoded as a precursor molecule of about 60-120 nucleotides (in humans) [7]. These precursors are derived from processing primiRNA (usually in kilobases) by a ribonuclease, such as DROSHA [8]. Pre-miRNAs are also further cleaved to generate active mature miRNAs with the help of DICER [9]. So far more than 800 miRNAs have been described in human [10]. miRNAs interact with 3'UTR of mRNAs through base pairing and bring about their degradation, destabilization, or repression of translation through RISC, a complex of multiple proteins and miRNA-mRNA adduct [11,12]. Occasionally it can also up regulate gene expression [13]. The expression profiles of miRNAs have been determined in order to understand the role of miRNAs in a specific biological process [14]. 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]. Several groups of miRNAs have been identified that regulate the expression of tumorassociated genes while others seem to hold prognostic value in predicting patient survival. For example, miR-21 is frequently over expressed in various cancers [18]. It is now considered as an oncomiR that acts by down regulating PTEN, a tumor suppressor gene [19]. Similarly let-7 family of miRNAs is frequently down regulated in lung cancers [20]. miR-15a and 16-1 are often deleted in chronic lymphocytic leukemia [21]. These miRNAs target RAS, HMGA2 and BCL2 oncogenes, respectively, thereby regulating tumorigenesis [ (...truncated)


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Candida Vaz, Hafiz M Ahmad, Pratibha Sharma, Rashi Gupta, Lalit Kumar, Ritu Kulshreshtha, Alok Bhattacharya. Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood, BMC Genomics, 2010, pp. 288, 11, DOI: 10.1186/1471-2164-11-288