Hei,
I used RT-PCR to analyze whether predicted or verified targets of a miRNA are actually targets of this miRNA in my cell lines.
For some of these genes I found either a slight but clear upregulation (1,24- 1,74 fold )in some cell lines whereas there was a clear downregulation (0,66-0,8) in other cell lines upon miRNA mimic transfection (relative expression of negative control-transfected cells were set to 1).
Right now, I am waiting for antibodies to analyze the expression of these genes by Western Blot...
MiRNA binding to mRNAs may either lead to inhibition of translation or to degradation of the mRNA.
However, might it be possible that binding of a certain mRNA may stabilize the mRNA while inhibiting translation? If so, RT-PCR would indicate higher expression of the target mRNA while analysis of protein expression would indicate decreased expression.
Are there studies showing this phenomena?
have you checked whether the predicted targets of miRNA are actually the true targets experimentally? try topflash n fopflash assays to check if thy really are. not all predictions need to be right as not all miRNAs bind at 3'UTR. before concluding anythin with RT-PCR check if the predictionas are correct.
Hi,
I had a similar result studying the targeting of CLOCK by miR-107. I recently published these resutls and I discussed how the miR targeting could lead to higher amounts of the target mRNA.
One possibility is that the miRNA inhibit protein translation and cells could respond to such inhibition by upregulating mRNA transcription that could lead to the accumulation of the mRNA. It has also been described the accumulation of mRNA-miRNA pairs in P bodies.
This is the paper in which I discuss this issue:
Mol Nutr Food Res. 2015 Sep;59(9):1865-78. doi: 10.1002/mnfr.201570094.
Dietary lipids modulate the expression of miR-107, a miRNA that regulates the circadian system.
Daimiel-Ruiz L1, Klett-Mingo M1, Konstantinidou V1, Micó V1, Aranda JF2,3, García B1, Martínez-Botas J4, Dávalos A1, Fernández-Hernando C2,3, Ordovás JM1,5.
Regards
Lidia
Thank you so much Lidia! I will read your paper!
@Sansrity: Do you mean Luciferase assays? Yes, I will do such assays. But before that, I want to be sure that the predicted targets are potential targets in my cell line. To this end, I am using RT-PCR and WesternBlot to analyze whether miRNA overexpression decreases the expression of the gene of interest.
However, I run westernBlot for one the genes on friday. But unfortunately, I could not detect the protein. I have never used the antibody before- thus the antibody is not working or my cells are expressing to little of the gene of interest....
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