Spatiotemporal expression and molecular characterisation of miR-344b and miR-344c in developing mouse brain
Mammalian brain development requires a meticulous spatiotemporal regulation of gene and protein expression. The developing brain undergoes major construction during the embryonic stage, beginning with the formation of the neural tube that eventually gives rise to a complex nervous system. Stud...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2015
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/67686/1/FPSK%202015%2067%20IR.pdf |
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| Summary: | Mammalian brain development requires a meticulous spatiotemporal regulation
of gene and protein expression. The developing brain undergoes major
construction during the embryonic stage, beginning with the formation of the
neural tube that eventually gives rise to a complex nervous system. Studies
had shown that microRNAs (miRNAs) played crucial roles in spatiotemporal
regulation of the brain development. MiRNAs are small non-coding RNAs of
about 22 nucleotides that regulate gene expression through inhibition or
repression processes during post-transcriptional or translational stages. A
recent study suggested that miR-344 was neural specific during brain
development. In this study, we characterised the expression of miR-344b and
miR-344c during the development of mouse brain. Bioinformatics analysis was
employed to identify the potential downstream target genes of miR344b and
miR-344c. Initially, miR-344b and miR-344c were found to target a total of
1,540 and 863 genes respectively. Genes that are known to be identified by
three independent bioinformatics tools and also associated with transcription
regulation and nervous system development were selected for further
screening. The genes that fulfilled these criteria and targeted by miR344b and
miR-344c were Olig2 and Otx2 respectively. Luciferase assay was performed
to validate the target genes prediction. Overexpression plasmid was cotransfected
with a 3’UTR plasmid and checked for luciferase protein inhibition.
However, both Olig2 and Otx2 were not suppressed by their respective
miRNAs. It may suggest that both Olig2 and Otx2 were not the direct targets of
miR-344b and miR-344c or a more complex mechanism is involved. Parallel to
bioinformatics study, in situ hybridisation analysis study showed that both miR-
344b and miR-344c were strongly expressed in the germinal layer during the
early developmental stages of mouse brain. MiR-344b was not expressed in
the brain from early postnatal until mature adult stage. Interestingly, miR-344c
remained expressed throughout the P1 brain and its expression was still
detectable in the mature adult brain although restricted to the olfactory bulb
only. Higher magnification on the expression of miR-344b and miR-344c
revealed that they were expressed in the nucleus. Stemloop RT-qPCR was employed to further investigate the expression level of these miRNAs in the
brain and other multiple organs. The expression of miR-344b in the developing
brain was peaked at E15.5 and decreased steadily as it progressed to
adulthood. On the other hand, miR-344c showed high expression at E15.5 and
remained steady till the adult stage. Both miR-344b and miR-344c showed
highest expression in adult pancreas when comparing with the adult multiple
organs, in line with the previous study reported that miR-344 was highly
expressed in the pancreas. In conclusion, Olig2 and Otx2 were not direct
targets of miR-344b and miR-344c respectively as previously predicted.
However, this study proves that miR-344b and miR-344c were expressed in
the developing mouse brain, especially localised to the nucleus of the neuronal
cells. In addition to whole brain, miR-344b and miR-344c were highly
expressed in pancreas and lowly expressed in muscles. |
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