Roles Of Adenylate Cyclase9and IκBα Cleavage In Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly malignant disease with abnormalproliferation of myeloid precursor cells in hematopoietic system as its character.According to FAB (French-American-British) criteria, AML is mainly divided intoM0-M7subtypes. At present, except all-trans retinoic acid (ATRA), which has achievedgreat success in treating acute promyelocytic leukemia (APL), traditional chemotherapyis still the basal theatment for other AML subtypes. Therefore, the molecular mechanismof AML occurrence and treatment has been one of the hot spots in the research ofdomestic and foreign scholars.In the first part of this thesis, we extensively explored the role and the expressionregulation of adenylate cyclase9(AC9) during ATRA-induced APL cell differentiation.It was reported that cAMP/PKA pathway was an indispensable event duringATRA-induced maturation of APL cells. AC could catalyze the conversion of ATP tocAMP and regulate the level of intracellular cAMP to affect cell proliferation,differentiation and apoptosis. Currently, nine membrane-associated members of humanAC have been identified. It was shown by cDNA chip and RT-PCR that AC9isoform wasexpressed with a relatively high level in APL cell line NB4. We found that AC9couldaffect intracellular cAMP level and the transactivity of retinoic acid receptor α (RARα)by overexpression and knockdown experiments. Knockdown of AC9in NB4cells couldobviously inhibit ATRA-induced differentiation, which indicated the importance of AC9in ATRA-induced APL cell differentiation. MicroRNA (miRNA) has been found to be one of the most important posttranscriptional regulators fine-tuning gene expression. Bymicroarray and bioinformatics analysis, we predicted miR-181a as a candidate miRNAprobably pairing with the complementary sites in the3’UTR of AC9mRNA andpresenting a relatively large difference in NB4and NB4-R1cells. Luciferase reporterassay showed that miR-181a could indeed decrease AC9expression by targeting3’UTRof AC9mRNA. We also found that CEBP protein considered as a key factor for normalgranulopoiesis could effectively bind to the promoter of miR-181a gene and dramaticallyinhibit miR-181a expression. In this study, we also detected the expression of miR-181aand AC9in primary bone marrow cells from APL patients. A significant inversecorrelation between miR-181a and AC9expression was observed. In addition, bothmiR-181a and AC9expression were correlated well with the leukemogenesis of APL,implying that the level of miR-181a or AC9in primary bone marrow cells might functionas biomarker for use in diagnosis and prognosis of APL.In the second part of this thesis, we explored the specific cleavage of IκBα proteinin AML cells. Nuclear factor-κB (NF-κB) is an important transcription factor, whichcould regulate expression of genes to inhibit cell apoptosis and promote cell proliferation.Sustained NF-κB activation could be observed in many cancers including AML. IκBαprotein could interact with NF-κB dimers and inhibit its trans-regulatory activity. Thus,the degradation or cleavage of IκBα protein could lead to the activation of NF-κB. In thiswork, we observed a specific cleavage of IκBα protein in AML cells, which generated aN-terminal and a C-terminal fragment. Compared to the full-length IκBα protein, theN-terminal IκBα fragment generated by cleavage exhibited a weaker binding ability toNF-κB protein and a decreased inhibition of NF-κB transactivity. The C-terminal IκBαfragment formed by cleavage could be degradated by proteasome. Interestingly, ATRA,IFNα and RIG-G (retinoic acid-induced gene G) were able to inhibit the cleavage ofIκBα protein in AML cells. Moreover, we found that the cleavage of IκBα wassignificantly different between AML and ALL patients. The ratio of full-length IκBα protein to the N-terminal IκBα protein formed by cleavage was lower in AML patientsthan that in ALL patients. However, the mechanism inducing the specific cleavage ofIκBα in AML cells remained unclear, which would be worth exploring in our future work.Our studies revealed for the first time the specific cleavage of IκBα protein in AML cells,which promoted the transactivity of NF-κB, providing new experimental basis for themolecular mechanism of NF-κB activation in AML cells.