טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentDai Isabelle
SubjectThe Role of MicroRNA in Resistance to Antibody-Induced
Cell Death in B Lineage Cells
DepartmentDepartment of Medicine
Supervisor Professor Doron Melamed
Full Thesis textFull thesis text - English Version


Abstract

Activation-induced cell death (AICD) is an important mechanism to eliminate self-reactive immature B cells. Resistance to AICD has been implicated with autoimmunity as well as with development of resistant B cell neoplasms.  One frequent malignancy of the B cell lineage is chronic lymphocytic leukemia (CLL). A commonly used drug for CLL is the monoclonal antibody against CD20, also known as rituximab, which is followed by deletion of the B cells. Some patients develop resistance to the drug and to AICD induced by it. A major mechanism by which resistance to AICD develops is by enhanced activation of the pro-survival PI3K pathway.

We aimed to understand how microRNAs are involved in determination of cells' fate. Aberrant expression of miRNAs is found in many malignancies, one important miRNAs is the oncogenic miR 17-92 cluster that has been found aberrantly expressed in many cancers. Overexpression of this cluster in B cells leads to malfunction in the normal cell cycle pathway and impairs PI3K activity.  Our goal in this work was to define whether microRNAs play a role in regulating AICD.

Our results show that miRNAs have a role in AICD. To prove this we used apoptosis-resistant cell line 38c-13 and apoptosis-sensitive wehi-231. We used shRNA to knock down Dicer in these cells, the key enzyme in the biogenesis of microRNA. We tested whether this would alter resistance to AICD upon anti-BCR stimulation. The results show an increase in apoptotic cells in both cell lines thus, proving the role of miRNAs in AICD. After that we focused on the miR 17-92 cluster since its known involvement in many cancerous diseases. To test the effect of miRNAs from this cluster on resistance to apoptosis upon anti-BCR or rituximab stimulation, we used BL-2 and 38c-13 cell lines which are refractory to stimulations. We used sponge system vectors which reduce the levels of miRNAs from this cluster and stimulated the cells with either anti-BCR or with rituximab. Our results indicate that miRNAs from the miR 17-92 are involved in acquiring resistance to AICD. In a reciprocal experiment we introduced a miR17-92 overxpression vector to wehi-231 cells and found that upon anti-BCR stimulation there was a significant reduction in apoptosis. These experiments directly showed that the miR 17-92 cluster have a role in AICD. We then looked for the mechanism by which miR17-92 controls the PI3K activity and the sensitivity to AICD, since PI3K has been shown to be the most important pathway controlling cells' fate. Our analysis revealed that Pten is the main antagonistic of the PI3K pathway and its levels are in inverse correlation with the levels of miR17-92. Besides this, we tested if miRNAs in general and miRNAs from the miR 17-92 cluster affect the levels of the prognostic marker, CD38. We tested CD38 levels in cell cultures and in extracted B cells from the bone marrow and spleen from knock out Dicer mice and stained the cells with anti-CD38 antibody. Our results show that miRNAs from the miR17-92 cluster do not regulate CD38 level.