P765: mib2/suz12 regulated the proliferation of pnh clone

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: On the basis of our previous studies, we found that SUZ12 participate in the proliferation of PNH clone by regulating H3K27me3. And then we used immunoprecipitation and mass spectrometry to identify that MIB2 interacting with SUZ12. Aims: We explore the mechanism of MIB2 regulating the proliferation of PNH clones. We hope to improve the pathogenesis of PNH and explore a new target. Methods: We have constructed a PIGA gene knock-out K562 cell line(K562-KO) by CRISPR/cas9. Immunoprecipitation and mass spectrometry were used to identify SUZ12 interacting proteins. And then, COIP was used to verify their interaction. Next, immunofluorescence was used to explore subcellular localization of SUZ12 and MIB2 protein. Furthermore, RNA-seq was used to explore signaling pathway. Finally, colony formation assays was used to clarify proliferative abilities of cells and western blot was used to test protein level changes and FCS was used to test apoptosis level of cells. Results: We found that MIB2 protein exists in cell membrane, cytoplasm and nucleus, MIB2 and SUZ12 are co-located in nucleus. GSEA analysis of the RNA-seq results in the control and siMIB2 groups showed that PRC2-related target genes were up-regulated in siMIB2 group. What’s more, we observed that SUZ12 and H3K27me3 expression level, cell proliferation ability was significantly decreased after MIB2 knockdown, while cell apoptosis level was significantly increased when knockdown MIB2. But in the SUZ12 plasmid rescue group after MIB2 knockdown, we found that SUZ12 protein level, H3K27me3 level, cell proliferation ability and the level of apoptosis were significantly restored. Summary/Conclusion: We found that MIB2 and SUZ12 were co-located in cell nucleus, MIB2 intervened SUZ12 protein level and the function of PRC2 complex, which regulating the proliferation of PNH clone. Our results showed the mechanism of MIB2 in PNH clone proliferation and might provide a new target for PNH.Keywords: Paroxysmal nocturnal hemoglobinuria (PNH), Clonal expansion, Proliferation