Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood.
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.
Vaz
Candida
C
School of Information Technology, Jawaharlal Nehru University, New Delhi, India.
Ahmad
Hafiz M
HM
Sharma
Pratibha
P
Gupta
Rashi
R
Kumar
Lalit
L
Kulshreshtha
Ritu
R
Bhattacharya
Alok
A
D020869:Gene Expression Profiling / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D017422:Sequence Analysis, DNA
D035683:MicroRNAs
Cell. 2009 May 1;137(3):586-586.e1
19410551
Nat Rev Mol Cell Biol. 2007 Jan;8(1):23-36
17183358
Mol Cell. 2008 May 23;30(4):460-71
18498749
Biochem Soc Trans. 2008 Dec;36(Pt 6):1197-200
19021524
Brief Bioinform. 2009 Sep;10(5):490-7
19332473
Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13944-9
16166262
BMC Bioinformatics. 2010 Jan 18;11 Suppl 1:S14
20122185
Genome Biol. 2004;5(3):R13
15003116
Clin Cancer Res. 2008 Mar 1;14(5):1340-8
18316553
BMC Genomics. 2007 Jun 12;8:166
17565689
Mol Cancer Res. 2008 Dec;6(12):1830-40
19074828
J Cell Mol Med. 2009 Jan;13(1):12-23
19175697
Genome Res. 2009 Nov;19(11):2009-20
19767416
Cell Death Differ. 2010 Feb;17(2):193-9
19461653
Leuk Res. 2006 May;30(5):643-7
16226311
Nature. 2005 Dec 1;438(7068):671-4
16319892
Science. 2005 Sep 2;309(5740):1567-9
16141074
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W339-44
17553836
Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39
19165215
BMC Bioinformatics. 2010 Jan 18;11 Suppl 1:S29
20122201
Fertil Steril. 2006 Oct;86(4 Suppl):1193-209
16949591
Nat Cell Biol. 2006 Mar;8(3):278-84
16489342
Cell. 2009 May 15;137(4):606-8
19450510
Nature. 2009 Jul 23;460(7254):529-33
19626115
J Exp Med. 2006 Oct 30;203(11):2519-27
17060477
Blood. 2007 May 15;109(10):4399-405
17284533
Genome Res. 2008 Jun;18(6):957-64
18469162
Cancer Res. 2009 Feb 15;69(4):1279-83
19190326
J Cell Mol Med. 2009 Nov-Dec;13(11-12):4551-9
19017354
Nature. 2007 Jul 5;448(7149):83-6
17589500
Nat Methods. 2005 Apr;2(4):269-76
15782219
Cancer Cell. 2006 Mar;9(3):189-98
16530703
Development. 2005 Nov;132(21):4653-62
16224045
Blood. 2008 Jan 15;111(2):588-95
17906079
Semin Immunol. 2005 Apr;17(2):155-65
15737576
Int J Cancer. 2008 Mar 1;122(5):969-77
18098138
Nat Biotechnol. 2008 Apr;26(4):407-15
18392026
Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10141-6
19497860
Cell. 2005 Mar 11;120(5):635-47
15766527
Genes Chromosomes Cancer. 2008 Jan;47(1):8-20
17910043
Genome Res. 2008 Apr;18(4):610-21
18285502
Biochem Biophys Res Commun. 2008 Aug 8;372(4):831-4
18522801
Nat Genet. 2006 Jun;38 Suppl:S8-13
16736023
Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2257-61
16461460
Nat Genet. 2005 Jul;37(7):766-70
15965474
PLoS One. 2007 Oct 10;2(10):e1020
17925868
Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15535-40
18832181
Gynecol Oncol. 2009 Sep;114(3):457-64
19501389
Dev Biol. 2003 Jul 15;259(2):364-79
12871707
Cancer Res. 2004 Jun 1;64(11):3753-6
15172979
Nucleic Acids Res. 2005 May 12;33(8):2697-706
15891114
Nucleic Acids Res. 2008 Jan;36(Database issue):D154-8
17991681
Nat Rev Genet. 2009 Feb;10(2):94-108
19148191
Platelets. 2007 Dec;18(8):566-78
18041647
RNA. 2006 Jul;12(7):1161-7
16738409
Biochem Biophys Res Commun. 2006 Oct 13;349(1):59-68
16934749
J Cell Biochem. 2006 Feb 15;97(3):485-93
16294322
Cold Spring Harb Symp Quant Biol. 2006;71:51-7
17381280
Nucleic Acids Res. 2007;35(17):5944-53
17726050
Nature. 2006 Jan 19;439(7074):283-9
16421561
RNA Biol. 2008 Jul-Sep;5(3):123-7
18698153
Science. 2004 Jan 2;303(5654):83-6
14657504
Cell. 2004 Apr 2;117(1):1-3
15066275
RNA. 2005 Mar;11(3):241-7
15701730
Cancer Res. 2008 Dec 15;68(24):10094-104
19074875
Gastroenterology. 2007 Aug;133(2):647-58
17681183
Nucleic Acids Res. 2005 Nov 27;33(20):e179
16314309
Exp Hematol. 2001 Jul;29(7):856-63
11438208
Nucleic Acids Res. 2008 Dec;36(21):e141
18927111
PLoS One. 2009;4(5):e5605
19440384
Cancer Res. 2009 Feb 1;69(3):1221-9
19141645
Nature. 2005 Jun 9;435(7043):834-8
15944708
Cell. 2007 Jun 29;129(7):1401-14
17604727
Cell. 2009 Jan 23;136(2):215-33
19167326
Leukemia. 2009 May;23(5):856-62
19151778
Nat Rev Genet. 2008 Feb;9(2):102-14
18197166
Genes Dev. 2007 May 1;21(9):1025-30
17437991
Nat Methods. 2004 Oct;1(1):47-53
15782152
Nature. 2003 Sep 25;425(6956):415-9
14508493
Stem Cells. 2008 Oct;26(10):2496-505
18583537
Cell Death Differ. 2010 Feb;17(2):215-20
19498445
Am J Pathol. 2006 Nov;169(5):1812-20
17071602
Eur J Haematol. 2008 Oct;81(4):304-10
18573170
Nat Methods. 2006 Jan;3(1):41-6
16369552
Cell Cycle. 2008 Sep 1;7(17):2643-6
18719391
Nat Neurosci. 2008 Jun;11(6):641-8
18454145
PLoS Biol. 2009 Nov;7(11):e1000238
19901979
Cell Death Differ. 2008 Apr;15(4):667-71
18219318
J Allergy Clin Immunol. 2009 Mar;123(3):603-11
19152965
Nucleic Acids Res. 2009 Jan;37(Database issue):D105-10
18996891
J Cell Mol Med. 2009 Jan;13(1):39-53
19175699
Mol Cell. 2004 Dec 22;16(6):861-5
15610730
Nat Genet. 2006 Feb;38(2):228-33
16380711
Genome Res. 2008 Oct;18(10):1602-9
18653800
Nat Struct Mol Biol. 2009 May;16(5):492-8
19377482
Exp Hematol. 2007 Apr;35(4):551-64
17379065
Semin Cell Dev Biol. 2005 Feb;16(1):129-36
15659347
Cell. 2007 Aug 24;130(4):581-5
17719533
Nucleic Acids Res. 2007;35(7):e52
17355992