טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBenhamou David
SubjectMicroRNAs Regulation of PI3K Pathway Determines Lymphocyte
Development and Differentiation
DepartmentDepartment of Medicine
Supervisor Professor Doron Melamed
Full Thesis textFull thesis text - English Version


Abstract

microRNAs are small RNA molecules that regulate gene expression and play critical roles in hematopoietic cell fate decisions, and play a crucial role in B cells development function survival and homeostasis.

Earlier studies have shown that fate decision of B cells can be altered through modification of intracellular signals. The major intracellular pathway which controls B cells fate and development is the PI3K pathway. In the first part of this work we focus on PI3K regulation by miRNAs. For this propose we used an inducible system to ablate in vivo the expression of Dicer in hematopoietic cells. Dicer is one of the enzymes responsible for processing miRNAs to their mature form and ablation of Dicer prevents miRNAs expression. We found that Dicer ablated immature B cells undergoing enhanced apoptosis. Biochemical analysis revealed that absence of miRNAs leads to suppressed PI3K activity due to increased expression of (Pten, the PI3K’s main antagonistic phosphatase. However, the extent of feedback regulation between PI3K activity and Pten expression during B cell development is unclear. To resolve this, based on our finding we tested here the hypothesis that PI3K controls Pten expression via microRNA. To confirm the physiological relevance of this mechanism, we tested whether reducing expression of Pten can reconstitute PI3K activity to rescue Dicer-ablated transitional B cells from apoptosis. Indeed, upon deletion of one Pten allele we found that PI3K activity was increased and facilitated survival of Dicer-ablated immature cells. Further biochemical studies uncovered a c-myc/miR17-92/Pten axis autostimulatory axis which controls PI3K activity in B lineage cells. Using a genetic approach of deletion and complementation, we show that the miR17-92 regulation on Pten expression  critically controls PI3K activity and the survival of immature B cells.


Lifelong blood cell production is governed through homeostasis of hematopoietic stem cells (HSC), which is a tightly regulated process, controlled by intrinsic and extrinsic signals. Here we investigated whether miRNAs regulate intrinsic signals to promote HSC to self-renew and differentiation. Since in first part of this work we showed that miRNAs regulate PI3K through repressing Pten expression and since the PI3K controls HSC functions, we next tested whether miRNAs regulate HSC survival and differentiation. To address this globally we used our inducible system to ablate expression of Dicer. We found aplastic bone marrow outcome in Dicer ablated mice and limited survival of the mice. We found that HSCs are present in Dicer-ablated mice but in reduced numbers due to failure of self-renewal and increased apoptosis due to reduced PI3K activity Enhancing PI3K in hematopoietic cells restores normal HSC progenitor homeostasis in Dicer deficient mice as evidenced by a normal cell growth of all cell lineages in the bone marrow.


Overall our findings support that miRNAs regulate hematopoiesis and B cells fate decision through the PI3K pathway. Furthermore, our results suggest that miRNAs control lymphocyte development and homeostasis of mature B cells through a synergy between PI3K and Notch signaling. Lastly, we identified miRNAs responsible to maintain B cells phenotypic difference between mature and immature B cells.