1. The Effect Of FHL2 And IASPP Interaction On Leukemia Cell Proliferation And Apoptosis 2. Identification And Amplification Of MLL-MYH11 Fusion Gene

Objective:iASPP is an inhibitory member of apoptosis-stimulating proteins of p53(ASPP)family,which inhibits p53-dependent apoptosis.iASPP was highly expressed in acute leukemia,inhibited leukemia cells apoptosis and promoted leukemogenesis.In order to clarify its mechanism,a yeast two-hybrid screen was performed and FHL2 was identified for the first time as one of the binding proteins of iASPP.Moreover,the roles and mechanisms of interaction between FHL2 and iASPP in proliferation and apoptosis of leukemia cells were investigated.Methods:A yeast two-hybrid screen was performed to identify FHL2 as a novel binding partner of iASPP.The expression of FHL2 in leukemia patients and leukemia cell lines was detected by real-time PCR.Immunofluorescence,immunoprecipitation(Co-IP)and immunoblotting analysis were used to confirm the communication between FHL2 and iASPP.Full-length cDNA and different truncation mutants of FHL2 were cloned into pcDNA3.1-myc expression vector,co-transfecting into HEK293T cells with pcDNA3.1-iASPP-flag respectively.Then,Co-IP and immunoblotting analysis were performed to detect which domian of FHL2 interacts with iASPP.Knockdown of FHL2 or iASPP in K562 and Kasumi-1 cells by lentiviral,MTT assay and flow cytometry were performed to detect the proliferation,cell cycle distribution and apoptosis rate of leukemic cells,meanwhile the expression level of cell cycle regulators and anti-apoptotic proteins were analyzed by Western blot assay.Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax or p21 when iASPP and FHL2 were overexpressed or FHL2 was knocked down.Results:FHL2 was highly expressed in patients with acute erythroleukemia than others with acute myelocytic leukemia subtypes,and in K562 and Kasumi-1 cells than other leukemia cell lines.FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm.The first 3 and a half LIM domains(L1/2-3)of FHL2 were required for the interaction with iASPP.Either FHL2 or iASPP silenced could reduce cell proliferation,induce cell cycle arrest at G0/G1 phases,and increase cell apoptosis.Western blot analysis showed the level of p21 and p27 increased,CDK4,E2F1,Cyclin E and anti-apoptotic proteins Bcl-2 reduced.Interestingly,when FHL2 was knocked down,the protein expression level of iASPP also decreased.Similarly,the expression of FHL2 would reduce when iASPP was silenced.Dual luciferase assay suggested that iASPP could reduce the transcriptional activity of p53 on Bax,furthermore,when FHL2 was knocked down at the same time,the transcriptional activity of p53 was rescued.Conclusions:FHL2 was highly expressed in patients with acute erythroleukemia than others with acute myelocytic leukemia subtypes.The interaction between FHL2 and iASPP in AML was observed for the first time.Cell proliferation reducing,cell cycle arresting at G0/G1 phases,and cell apoptosis increasing occurred in either FHL2 knockdown or iASPP knockdown.Meanwhile,the level of p21 and p27 increased,CDK4,E2F1,Cyclin E and anti-apoptotic proteins Bcl-2 reduced.In addition,the expression correlation was found between FHL2 and iASPP.Moreover,iASPP and FHL2 participated in the regulation of the transcriptional activation function of p53.These results indicated that FHL2 might be a novel potential target for AML treatment.Objective:In the study of an intractable case encountered in our daily clinical work,we discovered a new t(11;16)(q23;p13)chromosomal translocation.A novel MLL fusion gene MLL-MYH11 was found and identified by transcriptome sequencing and PCR technique,which has never been reported.In this study,the coding region of MLL-MYH11 was amplified and the protein structure was analyzed to supplement the knowledge of MLL fusion genes.Methods:The transcriptome sequencing technique was performed to find MYH11 as the rival gene of MLL.PCR was used to identify whether the patient really carried the novel fusion gene MLL-MYH11.MLL-MYH11 fusion gene was separated into 6 parts amplified by PCR,then linked by overlap PCR or enzymes restriction and adapter ligation.The MLL-MYH11 lentiviral vector was constructed and was transfected into HEK293T cells by PEI reagent.The mRNA level of MLL-MYH11 was detected by real-time PCR.Results:In our study of an intractable case with a new t(11;16)(q23;p13)chromosomal translocation,a novel MLL fusion gene MLL-MYH11 was discovered.The full length of MLL-MYH11 fusion gene is 9948bp,encoding a fusion protein with 3315 amino acid residues.Similar to other MLL fusion genes,MLL-MYH11 contains the exon 7 and its 5' sequence of MLL.MLL-MYH11 fusion protein contains the AT hooks,SNL1 and SNL2 nuclear localization signals,RD1 and RD2 transcriptional repression domains from MLL and the whole sequence from MYH11.When MLL-MYH11 was transfected into HEK293T cells,the mRNA expression level of MLL-MYH11 increased significantly,but the fusion protein was not detected.Conclusions:In this study,we discovered a novel MLL fusion gene MLL-MYH11.The whole coding sequence of MLL-MYH11 was amplified and the structure of the fusion protein was analyzed.Its role in leukemogenesis remains to be further studied.