Dear Aziz

I think a simple experiment may be:

Place diverse cancer cells under hypoxia (O2 around 1 per cent).

Make a PCR for HIF 1 alfa and your gene.

Compare the results with normal cells under the same conditions.

Then knock out HIF and repeat the experiment.

If the gene does not overexpress in this second experiment it would hint to HIF dependence.

I recommend you to check if this gene is under the control of HIF 1 alfa. You can find an answer to that in NCBI gene page. If it gives you no clue, you can check if the gene has in the promoter region a hypoxia responsive element nucleotide segment. You should also check what this gene normally does. If it is related somehow with metabolic pathways, it is probably one more of the aproximately 160 genes that participate in the hypoxia response pattern.

Finally I suggest you to check if it contains Sp1 bounding elements in the promoter region.

For further analysis I would have to know which gene it is.

In addition to what Dr. Koltai suggested, I would like to note that hypoxia-induced genes are also regulated in cell-type dependent manner and the extent of induction depends on a number of factors. Therefore, additional data may be helpful to make sure that the expression of gene of interest is not restricted to a particular cell type.

Hi Aziz, I did some work on hypoxia, and here refer you to one of my paper:

Article Bifurcated BACH2 control coordinates mantle cell lymphoma su...

Hope it would helpful for you!

I would suggest you take a step back and see where it fits in the big picture. Is the gene over expressed because of hypoxia or is the over expression a contributor? How many pathways lead to its over expression? How does its expression impact other findings in cancer? It is important to take a systems approach as in complexity nothing operates in isolation. See article on a systems theory and approach for cancer at https://www.frontiersin.org/articles/10.3389/fonc.2017.00198/full

Dear Tomas,

I really appreciate your suggestion. There is no information about how this gene is regulated under hypoxic condition. Do you recommend any expermint using transfection ,western blot and real time PCR to explore this?

I only have few months left for my PhD and I only have access to these techniques only.

The gene I am working on is an adaptor protein( localize in a focal adhesion) and only recently it was identified as an oncogene where it enhance migration and invasion but not proliferation . It is mainly regulated by Ras-Raf- Mapk and PI3k/AKT pathways. many growth factor such as TGF beta and GFG-2 up regulate this gene.

Dear Divaker, I will include more cell lines..thanks for your suggestion

Dear Priyankar , I will take a look at his work, thank you.

Dear Han, I will take a look at the paper thank you very much!

Dear Elizabeth,

Thank you for your reply , this is really a very good way to start. As I mentioned to Tomas this gene was identified as an oncogene where it enhance migration and invasion but not proliferation . It is mainly regukated by Ras-Raf- Mapk and PI3k/AKT pathways. many growth factor such as TGF beta and GFG-2 up regulate this gene. My question is how can I know if this over expression is a result of hypoxia or a contributor to hypoxia? the only thing that I can think about is silencing this gene under hypoxic condition and look for a down stream targets for hypoxia. I am not sure if that is the optimal approach. if it is, do you recommend any target?

Thank you very much!

Dear Aziz

I think a simple experiment may be:

Place diverse cancer cells under hypoxia (O2 around 1 per cent).

Make a PCR for HIF 1 alfa and your gene.

Compare the results with normal cells under the same conditions.

Then knock out HIF and repeat the experiment.

If the gene does not overexpress in this second experiment it would hint to HIF dependence.

The gene probably acts on the activation of metalloproteases, cathepsin B or heparanase. This is another angle to explore.

Thank you Tomas for your suggestion , I really appreciate it.

VHL, RCC,...

My former lab looked at HIF1 alpha regulation of collagen I expression; maybe this paper will give you ideas.

Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3.

Baumann B, Hayashida T, Liang X, Schnaper HW.

Kidney Int. 2016 Oct;90(4):797-808.

It can be explain briefly: Genome reorganization for adaptation

Amended on 27 February 2018

I have searched hypoxia-related proteins in my database obtained by PDMD method (please see file; HepG2 Fucoidan).

Normal fetal hepatocyte Hc and liver tissues (pseudo liver cancer, 1 tissue; LC tissues, 2 tissue specimens; HCC tissues, 2 tissues) have no such proteins.

Hepatoma HepG2 (cultured without fucoidan) has Hypoxia up-regulated 1 at 3.5 μg/mg cell protein and Hypoxia-inducible factor 1 alpha at 1.4 μg/mg cell protein. Healed HepG2 (cultured with fucoidan) has Hypoxia up-regulated 1 at 0.54 μg/mg cell protein and Hypoxia-inducible factor 1 alpha at 0.63 μg/mg cell protein. Hypoxia related proteins are detectable only one cancer among 3 cancers, and this phenomenon seems to be not general or to be not linked to cancer.

Gene expression seems to be ruled by virus. Hepatoma HepG2 (cultured without fucoidan) has uniquely Genome polyprotein (Human poliovirus; PV) at 5.1 μg/mg cell protein, and this PV seems to induce the hypoxia-related proteins.

Serum with Gait disorder (non-cancer; 32y, female) has Hypoxia-inducible factor prolyl hydroxylase 2/Transmembrane prolyl 4-hydroxylase at 6.8 μg/mg serum protein. This suggests that the gene expression of hypoxia-related proteins is very important phenomenon among brain diseases. This Hypoxia-inducible factor seems to be induced or excreted into her blood by Genome polyprotein (Yellow fever virus; YFV) at 6.1 μg/mg serum protein, which is uniquely present in her serum.

Maybe a very simple preliminary experiment could be using hypoxia mimetic like cobaltum cloride to stabilize HIF and see if your GOI is also overexpressed

To Paolo Tenti: Regarding your interesting proposal of using Cobaltum chloride to stabilize HIF, I remember that around 1976, an injectable drug was offered in Spain, to treat anemia, including some Cobalt in its formulation.

I wrote to the technical dept of Ragusant, the drug maker, to remark that Cobalt had no physiological function but being part of Vit B12, Cyanocobalamin, molecule, but in form of a salt, acting as a red-ox system poison, inducing a relative hypoxia, that in turn stimulated the release of erythropoietin, that may raise RBC, but as the tissues have difficulties in using the oxygen, the final situation was unchanged or worse.

They answered: 'You are right!', but I still ignore how long did it take for the formulation being changed, if they did, and why the HRAs didn't notice the danger upon filing for drug registration.

Regards. Gesund +

Very interesting suggestions, especially Dr. Tomas and Dr. Kou.

Aziz, You have to start soon. It may be promising.

Good luck