I did a luciferase 3'UTR reporter assay to see if one gene we have an interest as a direct target of one miRNA. I used two different cells, one is normal cell and the other is cancer cell. I got no difference of luciferase activity in normal cells, but decreased in cancer cells. Is it possible? If yes, what's the possible mechanism?
Your result is absolutely possible.
You do not have to look at the interaction within one miRNA and its target as a simple binary interaction. You can have other miRNAs targeting the same region which can compete for your miRNA for the target. At the same time, you can have other genes (or non coding RNA) which compete for the same miRNA. In this latter case, the overexpression of your mirna will be competed by the high abundance of your target. Therefore, in order to see an effect on your reporter you may increase the concentration of your construct and see what does it happen.
Hope that this may help.
Your result is absolutely possible.
You do not have to look at the interaction within one miRNA and its target as a simple binary interaction. You can have other miRNAs targeting the same region which can compete for your miRNA for the target. At the same time, you can have other genes (or non coding RNA) which compete for the same miRNA. In this latter case, the overexpression of your mirna will be competed by the high abundance of your target. Therefore, in order to see an effect on your reporter you may increase the concentration of your construct and see what does it happen.
Hope that this may help.
You can also perform and experiment with an antimicroRNA, specific to our microRNA, to see if you can restore a luciferase activity in cancer cells as in the normal ones.
I am not sure about your question: do you treat the cells with mir-inhibitor or mimic and then check for changes in 3'UTR luciferase activity? Let's assume you do then the answer by Ferlini is pretty much what I would say.
It of course depends what cell lines you compare and what the level of expression of your microRNA of interest has in both cell lines and how this relates to the concentration of inhibitor (or mimic) you expose the cells to.
Let's assume the expression level of the miRNA is the same in both cell types then the result becomes more difficult to explain. If the 3'UTR contains plenty of high quality binding sites for othe rmicroRNAs and these microRNAs are highly expressed in one cell type but not the other then you may not see an effect in the cell type with high other microRNA expression since your microRNA of interest does not contribute that much to the regulation of this 3'UTR relative to the others.
This is an interesting aspect of microRNA biology that is rarely addressed and kind of key to understanding their complex roles in gene regulation.
Some of your "problems" may be addressed by using a range of inhibitor concentrations and seeing if there is a concentration when even the second cell type starts to respond. Some 3'UTRs need much more inhibitor to show regulation than others.
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