טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentPolonsky Inna
SubjectRole of MicroRNAs in Regulation of Apoptosis in B
Lymphocytes
DepartmentDepartment of Medicine
Supervisor Professor Doron Melamed


Abstract

microRNAs are small non-coding post-transcriptional regulatory molecules that control gene expression. They are transcribed as primary transcripts and undergo processing both in the nucleus and cytoplasm by the endonucleases Drosha and Dicer, respectively, to form mature transcripts. They act by binding to a complementary sequence in the 3’ untranslated region of their mRNA target gene to repress translation or promote degradation of the targeted mRNA. Numerous studies have demonstrated that miRNAs are critical regulators of B cell development and function. Previous bioinformatic analysis in our lab revealed three miRNAs that are differentially expressed in immature versus mature B cells. In this work we sought out to determine the role of these miRNAs in regulating B cell development and fate decisions.

We first studied the role of miR181a in immature and mature B cells. miR181a is highly expressed in immature B cells and downregulated in mature B cells. Since the transition of immature to mature is limited by negative selection mechanisms, we speculated that miR181a regulates expression of genes controlling cell fate. Here we show that the expression of the anti-apoptotic protein Bcl-2 is regulated by miR181a throughout B cell development and activation. Ectopic expression of miR181a in the immature B cell WEHI 231 model resulted in downregulation of Bcl-2 and increased apoptosis imposed by B cell receptor (BCR) ligation. In mature splenic B cells, we found that miR181a is upregulated following stimulation of the BCR, and Bcl-2 protein is subsequently downregulated. Our studies fail to show that miR181a is involved in FAS-mediated apoptosis.

Our second aim was to determine the role of miR29a in mature B cells. Based on published miRNA data sets and miRNA target prediction software, we hypothesized that with B cell maturation, miR29a replaces miR19b in controlling expression of Pten and the consequential activation of PI3K, which is the main signalling pathway controlling B cell survival. We first show that loss of Dicer sensitizes splenic B cells to BCR-mediated apoptosis, demonstrating that miRNAs are important for mature B cell survival. In further experiments we validate that miR19b decreases with B cell maturation, while miR29a is upregulated. We show that inhibition of endogenous miR29a in splenic B cells, elevates Pten expression and downregulates PI3K activity. Yet, we were unable to show that inhibition of miR29a sensitizes splenic B cells to BCR-mediated apoptosis.

In the third part of our study, we aimed to determine a target gene for miR150 in B cells. miR150 is highly expressed in mature B cells, but not in their progenitors. Previous studies have demonstrated that premature expression of miR150 blocks B cell development via targeting the transcription factor c-Myb. We suggest here an additional mechanism by which miR150 expression blocks B cell development through targeting the transcription factor Foxp1 and suppressing V(D)J recombination. We show that Foxp1 is downregulated in 38c-13 cells overexpressing miR150 and demonstrate that Foxp1 overexpression rescues B cell development from miR150 mediated block.

Overall, our study demonstrates the importance of these three miRNAs in regulating B cell fate decisions.