TWIST2Plays A Role Of Tumor Suppressor Gene Function In Human Myeloid Leukemia

Objective: Twist protein is a transcription factor that contains basic helix loop-helixstructure, it is highly conserved during evolution process, including Twist1and Twist2.Twist2is66%identical to Twist1and identity increases to98%in the basic and HLHregions of the proteins, but their function is not consistent. The role of Twist proteinsshowed potential carcinogenicity in variety solid tumors. Twist can regulate theexpression of proto-oncogene AKT2and promote cell epithelial mesenchymaltransition (EMT), Twist involved in cancer migration and metastasis. In blood diseases,Twist-2function was different to solide tumor. In Twist-2-deficient mice, it shows themyeloid cell was proliferation, it has the manifestations of myeloproliferative disease(MPD). Twist-2was a negative regulator of myeloid lineage development. So we thinkTWIST2plays a suppress role in myeloid Leukemia, To further verified our hypothesis andfirst reveal the function about TWIST2in myeloid leukemia, We carried out a series ofbasic research, hoping to provide new method to the treatment of myeloid leukemia.Methods: After informed consent was given, we collected bone marrow from AMLpatients and healthy donor, mononuclear cells were obtained by centrifugation onfioll-hypaque medium, we also collected Raji、Dami、THP-1、SHI-1cells, Genomic DNAwas extracted, then Combined Bisulfite Restriction Analysis detected the methylation ofthese DNA samples; Direct sequencing detected the methylation of the13CpG sites aboutTWIST2promoter; The CD34+cells was purified by Immunomagnetic beads, includeAML30samples, CML14samples, healthy People6samples, Quantitative real-timepolymerase chain reaction (Q-RT-PCR) detected the expression of TWIST2;To constructedTWIST2lentiviral vector, calcium phosphate precipitation prepared the VENUS and TWIST2lentiviruses, we detected the cell proliferation ability about AML cell line THP-1and Dami and CML cell line MEG-01and K562, these cell lines was over-expression theVENUS and TWIST2gene; Enrichment of AML and CML patients CD34+cells, CD34+cells was transduced with VENUS and TWIST2virus respectively, detected the influenceabout VENUS and TWIST2to the hematopoietic stem cells; Overexpression VENUS andTWIST2in THP-1cells, we injected a certain amount of cells to nude mice subcutaneously,then detection the cell tumorigenic ability in vivo; In order to investigate the mechanism ofTWIST2inhibit cell proliferation, FACS detection cell cycle progression in two groupsabout four cell lines, For the further study about TWIST2gene’s functional domain, we usepiont mutation and PCR method to generated a series of TWIST2mutants. ContainingTWIST2△N (1～28),TWIST2△C(140～160),TWIST2b-,TWIST2F86P; Detection thecell proliferation and colony forming ability about TWIST2and its mutants in K562cells,FACS detected the influence on the cell cycle, and found a possible functional domains;Separation CD34+cells from4CML patients, TO further validate the importance of HLHfunctional domains, we detected colony-forming cell (CFC) in primary hematopoietic stemcells that were transduced with VENUS,TWIST2,TWIST2F86P letivirals; To study theinteraction about TWIST2and imatinib, we detected the CFC numbers in K562cells; Tofurther analysis the target gene of TWIST2, we first compare the differentially expressedgenes in THP-1/VENUS cells and THP-1/TWIST2cells, Second, Q-RT-PCR detect partlygenes associate with tumor in K562cells and CML CD34+cells. Third, we detected thetarget gene with CHIP, then clone P21promoter, luciferase dual reporter gene detection thetranscriptional activity of P21;Results:①We collected four AML cell lines and20cases bone marrow mononuclearcells of patients with acute myeloid leukemia, COBRA detect the methylation of theseSamples, It was interesting observe that the degree of TWIST2promoter methylation inAML was higher than healthy donor; The abnormal methylation leads to the silencing ofgene transcription, we purified CD34+cells from30AML cases and14CML patients,Q-RT-PCR detected the expression of TWIST2, The results show that the expression of TWIST2in AML and CML group was lower than normal control group, the AML andCML group were compared with normal controls have a statistically significant difference(P <0.05);②We construct the lentiviral vector to overexpressing TWIST2, CML cell linesMEG-01and K562and AML cell lines Dami and THP-1were transduced with Venus andTWIST2viruses respectively, compare the proliferation of VENUS and TWIST2group,The results shown that in the four cell lines TWIST2can inhibit cell growth, there was astatistically significant difference between VENUS and TWIST2(P <0.05); CFCexperiments also observed the same phenomenon; For the CML and AMLCD34+cells,TWIST2also inhibited their colony formation, CML group has statisticallysignificance between the VENUS and TWIST2(P <0.05);③In vitro experiments we haveconfirmed TWIST2inhibit cell proliferation，in vivo the test showed that VENUS group ofnude mice has more tumor than TWIST2group, there is a statistically significantdifference between the two groups (P <0.01);④FACS detected the influence aboutTWIST2to cell cycle, the results were displayed in the four cell lines TWIST2can inhibitcell G1-S phase conversion, cells was arrest in the G0/G1phase, three independentexperiments was show that the two groups has a statistically significant difference (P<0.05);⑤To understand the molecular mechanisms and functional domains of TWIST2,we generated a series of TWIST2mutants, cell proliferation and CFC experiment resultsshow that TWIST2inhibit the growth of K562cells depended bHLH domains, N-and C-isnot necessary; We enrichment CMLCD34+cells, then VENUS,TWIST2,TWIST2F86Plentivirus transfected cells. We inoculated cells to the semi-solid methylcellulose mediumthat contain cytokine, The CFC experiment result show TWIST2can inhibit various colonyformation about CMLCD34+cells, TWIST2F86P group has more colonies. there is astatistically significant difference about VENUS and TWIST2F86P compared withTWIST2(P <0.05), HLH domain was important functional domains about TWIST2inhibition of cell growth;⑥FACS detected the influence to cell cycle about TWIST2andits mutants, the results suggest TWIST2F86P and VENUS compared with TWIST2hassignificant difference (P <0.05), The G0/G1phase arrest was disappeared in TWIST2F86Pgroup;⑦K562cells were transfected with TWIST2and its mutantslentivirus, different concentrations of IM in K562cells, the results suggest thatoverexpression TWIST2can enhance the sensitivity of K562cells to IM;⑧To furtherexplore TWIST2mechanism, Microarray analysis the differences gene expression betweenTHP-1/VENUS and THP-1/TWIST2,662genes were detected, Q-RT-PCR validation15tumor-related genes have consistent result to Microarray in THP-1cell, overexpressionTWIST2in K562and CMLCD34+cells, Q-RT-PCR detected the differentially expressedgenes, P21and ID2expression was increased and TERT and CCND1expression wasreduced; CHIP detected the TWIST2target gene, the result show that TWIST2group hasenrichment of TD2, P21,CCND1; We clone the promoter of the cell cycle related gene P21,the transcriptional activity about P21in TWIST2group was significantly higher than theVENUS and TWIST2F86P group, TWIST2target genes is P21,TWIST2regulatetranscriptional activity of P21Conclusions:In summary,The promoter methylation of TWIST2was obvious in acute myeloidleukemia, while the healthy donors was no methylation.The expression of TWIST2inAML and CML CD34+cells was lower than healthy donors.TWIST2inhibit theproliferation of myeloid cell lines and primary CD34+cell, cell cycle is blocked, the cellsarrest in G0/G1phase.We generated a series of TWIST2mutants,TWIST2inhibit thegrowth of K562cells and primary CD34+cell dependent bHLH domains, N-and C-is notnecessary.TWIST2can enhance the sensitivity of K562cells to IM.The target gene ofTWIST2is P21, CCND1, ID2in CML,TWIST2can enhance the transcriptional activity ofP21...