Characterisation of microRNAs in Human Stem Cells
Author(s)
Chan, Elcie
Type
Thesis
Abstract
In collaboration with David Baulcombe and Attila Molnar we have generated
microRNA libraries for human embryonic stem cells (hESCs) before and after
differentiation along the neuronal lineage and also from human mesenchymal stem
cells (hMSCs). Both cell types are of medical importance and understanding how
their proliferation and differentiation is regulated by microRNAs is also of scientific
interest. The hMSC library was sequenced by 454 technology and the two subsequent
hESC libraries by Solexa sequencing.
Approximately a quarter of all currently known microRNAs were identified between
the libraries, in addition to 3 novel microRNAs and 25 annotated piRNAs. For the
hESC libraries, we verified the presence of embryonic specific microRNAs (miR-302
family) and neuronal specific microRNAs (miR-9/miR-9*), and demonstrated that
expression of these miRNAs is regulated at the transcriptional level. Additionally,
promoter assessments of miR-9 transcription revealed that multiple upstream regions
may be important in neuronal specific expression.
Almost half of all known human microRNAs are located within the introns of host
genes. We used microarrays to analyse host gene expression and found that there was
little correlation with microRNA expression, indicating that many microRNAs are not
regulated at the transcriptional level by their host promoter. Furthermore, the
expression of microRNAs from the same cluster, and also from the same hairpin
precursor, did not always correlate when compared between the stem cell libraries.
Taken together, this data indicates that microRNAs are regulated at a variety of levels
both pre- and post-transcriptionally.
Many microRNA isomers were also detected that differed in expression between
human cell types, and upon differentiation of the hMSCs through the osteoblastic
lineage. Interestingly, microRNAs and some of their isomers showed different
affinities for Argonaute proteins in pulldown assays. We also profiled mRNAs that
were immunoprecipitated with Argonaute in order to identify miRNA targets
microRNA libraries for human embryonic stem cells (hESCs) before and after
differentiation along the neuronal lineage and also from human mesenchymal stem
cells (hMSCs). Both cell types are of medical importance and understanding how
their proliferation and differentiation is regulated by microRNAs is also of scientific
interest. The hMSC library was sequenced by 454 technology and the two subsequent
hESC libraries by Solexa sequencing.
Approximately a quarter of all currently known microRNAs were identified between
the libraries, in addition to 3 novel microRNAs and 25 annotated piRNAs. For the
hESC libraries, we verified the presence of embryonic specific microRNAs (miR-302
family) and neuronal specific microRNAs (miR-9/miR-9*), and demonstrated that
expression of these miRNAs is regulated at the transcriptional level. Additionally,
promoter assessments of miR-9 transcription revealed that multiple upstream regions
may be important in neuronal specific expression.
Almost half of all known human microRNAs are located within the introns of host
genes. We used microarrays to analyse host gene expression and found that there was
little correlation with microRNA expression, indicating that many microRNAs are not
regulated at the transcriptional level by their host promoter. Furthermore, the
expression of microRNAs from the same cluster, and also from the same hairpin
precursor, did not always correlate when compared between the stem cell libraries.
Taken together, this data indicates that microRNAs are regulated at a variety of levels
both pre- and post-transcriptionally.
Many microRNA isomers were also detected that differed in expression between
human cell types, and upon differentiation of the hMSCs through the osteoblastic
lineage. Interestingly, microRNAs and some of their isomers showed different
affinities for Argonaute proteins in pulldown assays. We also profiled mRNAs that
were immunoprecipitated with Argonaute in order to identify miRNA targets
Date Issued
2009-10
Date Awarded
2009-10
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Dibb, Nick
Sponsor
BBSRC, Institute of Obstetrics and Gynaecology Trust.
Creator
Chan, Elcie
Publisher Department
Division of Surgery, Oncology, Reproductive Biology and Anaesthetics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)