Hi everyone. I am trying to validate the regulation of one miRNA over its predictive gene.
To achieve it, I have done two different experiments:
1) To do overexpression experiments in cells at two different doses.
2) To clone the predictive 3'-UTR of its target gene in a reporter expression-vector.
Results are controversial. When I transfect miRNA-mimic in cells, mRNA levels of the gene are downregulated, indicating that the miRNA affects it endogenous levels. However, when I try to validate if the miRNA binds to the 3'-UTR of the gene, no effect is observed.
How could I explain it? How is my miRNA acting? How is my miRNA regulating its predictive gene?
Thank you all
If the reporter assay is not providing expected results, then may be the 3'UTR of the gene of interest is not the direct target of the miRNA . Please check if it's binding to the 5'UTR or the promoter region of the respective gene. A miRNA can also bring out an indirect downregulating effect on a gene.
Thank you for all your suggestions.
I used miRBase algorithms (www.mirbase.org) to predict putative target genes of my miRNA. Furthermore, using TargetScan, I cloned the specific 3'-UTR downstream of the reporter gene in the expression vector.
May I consider that the prediction failed and that there is an indirect regulation of my miRNA over the gene through indirect regulating pathways?
Dear Kamil. Thank you very much for all your suggestions.
The expression vector with the insert was sequenced and confirmed that no mutations ocurred. The vector I used was the pmiGLO Dual-Luciferase miRNA Target Expression Vector, from PROMEGA. I have used this aprroach before to validate other mIRNAs and the co-transfection experiments were fine.
Maybe the only explanation I find is that the 3'-UTR sequence that is predicted to be the target site of my miRNA and that I cloned in the vector, was incorrectly predicted, so my miRNA regulates the endogenous mRNA levels of the gene, but does not bind in the sequence I cloned (maybe in another part of the 3'-UTR).
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