I work on a tumour suppressor gene in breast cancer. The down regulation of this gene in MCF10A cells (using shRNA) caused a 12 folds decrease in the mRNA expression of PI3K catalytic subunit delta. In the mean time it caused increases in the mRNA expressions of cell cycle proteins CDK2 and 4, as well as Vimentin.
The increases in the cell cycle proteins and vimentin in response to the down-regulation of this tumour suppressor gene is expected. However, the decrease in the PIK3CD is unexplained.
If you have potential explanations to these results or if you have suggestions for follow up experiments, your help will be very much appreciated.
Thanks.
pik3cd is not usually expressed in breast tissue but has been detected in some breast cancer cell lines as a marker of increased chemotaxis (Sawyer, C et al. 2003). However, as is common with knockdowns, the effect on other genes may be a side effect, and it will be important for you to test whether knockdown of p110delta in those cells has the same effect as the knockdown of your tumor suppressor gene, and only then would you be able to say that your gene is having a direct effect on p110delta through which it has its tumor suppressor role. I would not try to do the opposite test, that is over-expressing p110delta, as that would cause transformation, as was seen by Denley et al (PNAS, 2008) with p110beta, p110delta,or p110gamma over-expression.
Hi Mohamed,
I suggest looking at PIK3CA instead of PIK3CD as PIK3CD is not common for breast tissues. PIK3CA is the gene that is mutated most in breast cancer and PI3K pathway plays a major role in breast cancer development. Also, looking at the total levels of the PIK3 proteins would not be very informative to determine the activation of PI3K pathway. I suggest looking at the levels of phospho AKT(473 and maybe 308), phospho S6K and phospho S6 for the activation of this pathway. I cannot really explain why PIK3CD goes down but I suggest only knocking down PIK3CD and looking at the levels of the same cell cycle proteins and your tumor suppressor. Also, I suggest knocking down your tumor suppressor and overexpressing PIK3CD(at the basal levels of MCF10As) at the same time to get some information whether PIK3CD downregulation is required for up-regulation of cell cycle proteins and vimentin.
Hi
look to this database, type in your gene, and look to a loot of other cancer cells how they behave in regard to your gene expression... http://www.broadinstitute.org/ccle/home
another database http://www.broadinstitute.org/gtex/.. this one tells you the gene expression in normal cells and tissues
another database http://www.ebi.ac.uk/gxa/home;jsessionid=A880D53446173BFD1E11E567199C7186 in this one you can even choose diseases and conditions
I hope they help to understand the pattern of your gene expression
I would like to thank you all (Hashem, Turgut and Hemin). your suggesions are really great. I will work on them carefully, and we may discuss this topic further (if you do not mind).
Thanks once again.
Mohamed
try also HEFALMP to see whether there is a virtual interaxction of your tumor suppressor and PIK3CD http://hefalmp.princeton.edu/
Insilico transcriptomics at www.genesapiens.org allows for correlation analyses where you can see whether there is a trend for the expression of your gene being correlated with the expression of PIK3CD
Í totally agree with Turgut Dogruluk, dat looking at the levels of PI3CA or better called as PI3Kp110 alpha subunit is best as it is the one that enhances proliferation. Moreso, PI3CD is the isoform generaly expressed and is functional in the leucocytes/immune cells.
PIK3CD is highly expressed in leukocytes and certain types of breast cancers as well as inducible by inflammatory stimuli in the endothelial cells.
Whitehead et al has published earlier on the regulation of PIK3CD mRNA regulation and that there are different transcripts that can be detected in different cells. Have a read through it is very interesting. Which tumor repress or is the one that cause such a depression?
http://www.ncbi.nlm.nih.gov/pubmed/22375552
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