hi i need advise on the matter
i performed RNA-seq after exposing cells to mechanical stress. I had designed a device to deliver this stress to cells. Looking at the RNA-seq data, i see some upregulation in genes associated with hypoxia, like glut-1, hexokinase, KDM, VEGFA etc
But there is no overexpression of HIF-1A transcript (decreased in fact but statistically not significant - definitely not increased).
Is there possibly a condition whereby there is hypoxia induced in my device condition. Apart from measuring O2 concentration in media, what arguments can i use to support that hypoxia is not causing some of these genes to be induced.
Hi Iwan, the increased HIF-1a expression under hypoxia is mainly due to the stabilization of HIF-1a protein but not the upregulated mRNA levels. The HIF-1a protein can be degraded rapidly under normoxic conditions because of PHD-VHL-dependent ubiquitination and proteasomal degradation. Under hypoxic conditions, PHD activity is inhibited, which results in HIF-1a stabilization. Therefore, it's reasonable not to have elevated mRNA levels of HIF-1a in your RNA-seq analysis.
I am not sure I understand your Q completely but I will speculate anyway.
If you meant to say that you see expression of hypoxia associated genes but not HIF-1a, it could be that the mRNA for HIF-1a has a shorter half life and was degraded by the time you collected RNA. Try collecting at earlier time points. Better still, do a kinetic study so you can get a clearer picture.
There are also three different isoforms. It might help to measure all of them.
Hi Iwan, the increased HIF-1a expression under hypoxia is mainly due to the stabilization of HIF-1a protein but not the upregulated mRNA levels. The HIF-1a protein can be degraded rapidly under normoxic conditions because of PHD-VHL-dependent ubiquitination and proteasomal degradation. Under hypoxic conditions, PHD activity is inhibited, which results in HIF-1a stabilization. Therefore, it's reasonable not to have elevated mRNA levels of HIF-1a in your RNA-seq analysis.
A good indicator of hypoxia is carbonic anhydrase IX. It is not quickly inactivated. You should test it, because I agre with Han. HIF 1 is rapidly inactivated under normoxia.
Why do not you try in parallel just normal RNA and protein isolation in a small scale after your experimental condition and test for the expression levels of transcript of Hif1a as well protein. This will confirm your result why you do not see increase in mRNA of HIF1a.
i agree with Han, no mRNA different will seen under normoxia and hipoxia condition, to see it you have to measure prteoin level if you want to demostrate that your changea are because of HIF-1alfa you can try culture your cell with CoCl2 or try with Hif-1alfa that have it VHL deleted
good luck
The oxygen sensing HIF system is a little more complex than just on/off hypoxia. There are a number of conditions that need to be considered, cellular activation status and the generation of superoxide anions, nitric oxide, all can influence the oxygen partial pressure in the media and increase HIF stability. The mechanical insult may also increase the expression of other arkyrin-repeat domains such as NOTCH, FIH has a far greater affinity for NOTCH than HIF1. This is a graded interaction that may sequester bio-available FIH allowing for greater HIF1 activity in a higher oxygen environment. The identification of arkyrin-repeat domains should be easy with your RNAseq data set. I would measure the oxygen tension in the media before and after the mechanical stress. If there is a substantial shift in oxygen consumption, is this through ROS generation. There are a number of commercial kits that can be used to measure ROS. Is the shift toward a glycolytic state a real phenomenon, if you have a sea-horse measure the glycolytic status of the cells following mechanical stem, or measure the pH of the media.
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