A miRNA switch in regulation of PI3K activity controls B cell maturation and survival

Introduction:

PI3K-AKT is the main pathway which controls B-cell development and survival. During development in the bone marrow, the regulation of PI3K is dependent on miR17-92 cluster through PTEN phosphatase. However, with maturation expression of miR17-92 decreases, while PI3K activity increases. Hence, we hypothesize that B cell maturation is accompanied with miRNA switch controlling of PI3K. We show that regulation of PI3K switches from miR17-92 in developing cells to miR29 in mature cells, and this is critical for B cell maturation and survival.

Material and methods:

Mice: Dicer F/F and PTEN F/F mice crossed with Mx-CRE transgenic mice.

Apoptosis quantification: cells were stained for 7AAD.

Luciferase assay: used to quantify binding of miR29 to PTEN 3’UTR.

qPCR analysis: using Taqman probes.

Results and discussion:

1. Using mice where Dicer can be inducibly ablated, we show that miRNAs are critical to regulate PI3K activity and mature B cell survival. Loss of one PTEN allele restores PI3K activity in Dicer ablated cells and promotes their survival.
2. Analysis of miRNA in B cells suggests miR-29 is preferentially expressed in mature B cells. We validated this by qPCR of B cell subsets and demonstrated its binding to PTEN 3’UTR using luciferase.
3. To test the interrelationship between miR17-92 and miR29, we applied overexpression and knockdown in-vitro. Splenic B cells treated with miR29a antisense oligos expressed decreased levels of miR19b, whereas overexpression of miR29a in WEHI cells resulted in a decrease in miR17 and miR19. These two findings indicate a control loop between miR29 and miR17-92 cluster, whose mechanism is still unknown.
4. Using miR29 deficient mice we are able to show that loss of miR29 results in impaired maturation of splenic B cells, increased apoptosis, increased PTEN levels and reduced PI3K activity.

Conclusion: These findings suggest that PI3K regulation by miRNAs is developmentally regulated and guided by a specific switch from miR17-92 during development to miR29 in mature. Since PI3K activity is critical for B cell survival, we speculate that this miRNA switch may have major role in B cell pathologies. Further studies exploring this are now conducted in the lab.