When the levels of a certain microRNA are changed by stimulation (e.g., stress, infection, disease) and protein levels of its "predicted" targets are decreased by the same stimulation, how can we directly relate the change in microRNA with that in the protein? How can we confirm that the changes in protein levels are caused by that particular microRNA in vivo? Is the prediction of microRNA-mRNA interaction enough to explain the interaction between microRNA and mRNA and the effect of microRNA on protein levels? Is there any method to experimentally test if the changes in protein levels are really induced by a certain microRNA in vivo.
The paper shown below made me have this question. This paper shows changes in miR-132 levels induced by Toxoplasma infection, predicted targets of miR-132 and changes in protein levels of some predicted target genes. From these results, it would be possible to theorize that changes in miR-132 might cause the changes in protein levels of its predicted targets. However, is there any method to confirm the interaction between miR-132 and its predicted targets in cells or animals?
Xiao, J., Li, Y., Prandovszky, E., Karuppagounder, S. S., Talbot, C. C., Dawson, V. L., … Yolken, R. H. (2014). MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway. Neuroscience, 268, 128–138. https://doi.org/10.1016/j.neuroscience.2014.03.015
Hi Yuki:
As you well said, this paper showed just a possible mechanism that coul explain some of the pathogenesis of toxoplasma infection. If you want to confirm the causality of the miRNA increment with lower protein levels, you need to do a lot of work.
First, if the target mRNA is not confirmed as a some miRNA target, you need to confirm it using in vitro approaches. 3´UTR can be cloned in reporter plasmids, and transfected in cell lines. Then, using artificial ago- or antagomiRs, you can stud the direct effect on these UTRs. Another approach consist in apply these ago- or antagomiRs directly in cell culture, and then, using a 2D gel and mass spectrometry to identify the spots that change.
If you want to verify the targets in vivo (after in vitro validation), these ago- or antagomiRs can be inyected into specific organs of animals, and mRNAs and proteins can be measured.
Good luck!!!
see our paper "Exposure to Endocrine Disruptor Induces Transgenerational Epigenetic Deregulation of MicroRNAs in Primordial Germ Cells"
Dear Felipe,
Thank you for your comment.
Using 3’UTR reporter construct and artificial miRNAs would support the hypothesis that a certain miRNA and gene might be functionally related.
However, I thought that comparing the effect of miRNA on gene expression between constructs of normal 3'UTR and 3'UTR with mutation in putative binding site of the miRNA is necessary to say that a certain miRNA directly targets the gene and affects the gene expression. (Example of reporter assay with mutated 3'UTR is shown in Figure 3 of this paper;
Yamakuchi, M., Ferlito, M., and Lowenstein, C. J. (2008). miR-34a repression of SIRT1 regulates apoptosis. Proc. Natl. Acad. Sci. 105, 13421–13426. doi:10.1073/pnas.0801613105.
Is it possible to say that a particular miRNA directly suppressed the expression of a certain gene by a reporter assay using 3'UTR reporter without mutation in putative binding sites of miRNA?
Dear Yuki:
You are right. To confirm that certain miRNA can regulate the expression of some mRNA, you must to generate the construct with your 3´UTR of interest, and you must to mutate the putative binding site, in order to confirm the specificity of the supression.
Take a look of this paper, it might help you with common questions of the area:
Article Thomson, DW, Bracken, CP and Goodall, GJ. Experimental strat...
Good luck!!!!
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