The Four And A Half LIM-only Protein2Interacts With IASPP And Exhibits Aberrant Biological Behaviors In Leukemic Cells Study Of FHL2Interacting With MiR-340and Influence On Leukemic Cells

Objective:To clarify the mechanism of iASPP as an oncogene that inhibited apotosis of leukemic cells, the amino terminus of iASPP was used as a bait in a yeast two-hybrid system, and the four and a half LIM-only protein2(FHL2) was indentified as one of the binding proteins. In this study, the effects of FHL2and iASPP interaction on the proliferation and apoptosis of K562cells and its mechanism were investigated.Methods:Real time PCR and Western blot was used to detect the expresion of FHL2in leukemia cell lines and newly diagnosed acute leukemia patients at mRNA and protein level. The immunofluroescence analysis was used to clarify the subcellular localization of FHL2and iASPP. Lentiviral vectors pLKO.1-puro containing the FHL2-shRNA or the control-shRNA were constructed and transfected into leukemic cells to down-regulate FHL2expression, and to analyze its biological effects. The expression of FHL2after shRNA treatment in the K562cells was detected by real-time PCR and Western blot. Effect of FHL2-shRNA on the proliferation of K562cells was examined by MTT method. Flow cytometry was used to analyze cell cycle and apoptosis. pGL3-basic vector containing FHL2promoter was constructed and dual-luciferase reporter assay was used to test the regulation among FHL2, iASPP and P53.Results:1. FHL2is expressed in most leukemic cells, its expression level in K562, Nalm6, Raji and KG1a cell lines was higher than in other leukemic cell lines.2. FHL2is highly expressed in primary acute erythroid leukemia patients.3. FHL2interacts with iASPP in vivo in K562cells. Decreasing the expression level of iASPP can inhibit the expression of FHL2in K562cells, while knocking down FHL2expression can significantly reduce iASPP expression. Over expression of iASPP in K562cells can also increase the expression level of FHL2. The expression of FHL2and iASPP correlates with each other.4. There are P53binding sites on the upstream promoter of FHL2, the transcription of FHL2can be suppressed by P53. FHL2enhances the transcriptional activation of P21. 5. Knocking down of FHL2in K562cells can result in the decreased cell proliferation, cell cycle arrest in G0/G1stage and increased cell apoptosis induced by chemotherapy drugs.Conclusion:FHL2is highly expressed in acute erythroid leukemia patients. In leukemic cells FHL2interacts with iASPP, and their expression levels are in positive correlation with each other. FHL2exhibits important biological function, it can induce decreased cell proliferation, cell cycle arrest in G0/G1stage and increased cell apoptosis induced by chemotherapy drugs when knocking down FHL2expression. The transactivation of p21by P53was mediated by FHL2, meanwhile P53could inhibit the transcription of FHL2. In a word, FHL2and iASPP take part in leukemogenesis and progression possibly through regulating P53/P21pathway. It suggests that disrupting FHL2signaling may provide a thearapeutic strategy for AML treatment. Objective:To study the mechanism of differential expression of FHL2in mRNA level and protein level in U937cellsMethods:FHL2expression in different leukemic cells was detected by real time PCR and western blot respectively. Possible microRNA molecules that regulate FHL2transplanation process after transcription was found by bioinformatic softwares. They were miR-340and miR-124. Furthemore, the expression of miR-340and miR-124was checked by real time PCR in different leukemic cells, so as to clarify the interaction among FHL2and those two miRNAs. The luciferase reporter vector containing3’-untranslated regions (3’UTR) of FHL2was constructed and cotransfected with miR-340and miR-124precursors. U937cells treated with miR-340inhibitor were analyzed by Western blot to observe the change of FHL2protein and the biological function.Results:FHL2was expressed highly in U937cells at mRNA level, but nearly no protein could be observed. FHL2expression might be regulated by miR-340and,miR124at the post transcriptional process. But no significant changes on the biological function could be found in U937cells treated with miR-340inhibitor.Conclusion:miR-340and miR124might participate in the regulation of FHL2at the post-transcriptional process, and influence the expression of FHL2. When the function of miR-340was inhibited, the tolerance for the chemotherapy drugs in U937cell was increased, and cell apoptosis was reduced.