Uncovering miR-142 Targets Regulating B-cell Expansion in NHL

Non-Hodgkin’s Lymphoma (NHL) is the fifth most prevalent type of cancer in Canada. Diffuse Large B-cell Lymphoma (DLBCL) accounts for 90% of aggressive NHL cases. The microRNA miR-142 is mutated in 20% of DLBCL cases. Additionally, miR-142 -/- B-cells display increased expansion and elevated B-cell Activating Factor Receptor (BAFF-R) expression. However, it remains unclear whether loss of miR-142 directly leads to elevated BAFF-R expression, leading to increased expansion. Mutating the miR-142 target site on BAFF-R and expanding B-cells could help elucidate the regulatory mechanism. If miR-142 target site KO B-cells expand more, it shows that miR-142 directly regulates BAFF-R expression. The miR-142 target site will be deleted using CRISPR. A gRNA targeting the miR-142 binding site on BAFF-R will be cloned into the PX458 plasmid, which contains the cas9 expression cassette. The resulting plasmid will then be transfected into B-cells. The transfected B-cells will then be cultured in vitro under conditions that promote B-cell expansion. The frequency of miR-142 target site mutations will be quantified via sequencing before and after expansion. If mutations are enriched in the expanded population, it would suggest that miR-142 directly regulates BAFF-R. Thus, in miR-142 -/- mice, the lack of regulation by miR-142 would be directly leading to increased levels of BAFF-R, driving increased B-cell expansion. A broader screen targeting all miR-142 targets could help uncover other critical regulators of B-cell expansion. These targets could then be studied further to develop therapeutic strategies for treating DLBCL, given that microRNAs are difficult to mimic therapeutically.