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miRNA directed upregulation?!


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#1 banou

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Posted 03 June 2010 - 08:41 AM

Dear all!

To investigate wether my gene of interest is regulated by miRNA. I stably transfected a cell line using siRNA against Dicer and a scrambled one. I did RT-PCR of these transfected cell lines. The PCR results showed that in compare to scrambled one the Dicer has been knocked down 80% . Up to here it is ok. but when I investigated my gene I saw that in dicer knocked down cells, its expression have been down regulated. what we expected was its upregulation. ( since we proposed that it is regulated by miRNA) These results were confirmed with luciferase assay of 3'-UTR containing constructs. also I tried the differnt sizes of my gene promoter the rule out the possibility of regulation through promoter. The luciferase assay results showed no difference between the different sizes of promoter in dicer knocked down and scrambled one.

If anybody has an idea about what could be the explanation or how can I explain these results I would really appreciate it.

#2 pcrman

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Posted 03 June 2010 - 10:40 AM

The downregulation of your gene following Dicer kd could be caused by secondary effect or miR mediated gene activation (at translational, mRNA and transcriptional levels).

You can further knockdown Drosha and Agos to see if the gene will also be downregulated. If yes, try to identify miRNA targets on its 3UTR and then transfect the miRNAs to see how they regulate your gene.

#3 banou

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Posted 04 June 2010 - 05:07 AM

The downregulation of your gene following Dicer kd could be caused by secondary effect or miR mediated gene activation (at translational, mRNA and transcriptional levels).

You can further knockdown Drosha and Agos to see if the gene will also be downregulated. If yes, try to identify miRNA targets on its 3UTR and then transfect the miRNAs to see how they regulate your gene.

Thanks PCR man!
I have tried also Ago2 knockdown. ( two diffeerent shRNAs targeting different parts of Ago-2). The ago-2 knockdown was not as efficient as Dicer but even in Ago knocked dwon cells the gene expression is down regulated.

#4 pcrman

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Posted 04 June 2010 - 07:08 AM

Yes, Ago2 knockdown efficiency is expected to be low because RNAi itself depends on Ago2. You may want to knockdown Drosha to prove that the miRNA machinery is required.

If your result (upregulation) has been confirmed by luciferase assay, then your observation is novel and interesting. Although Steitz's group has shown that miRNA could activate translation but only when cells are under stressed condition.




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