The Function Of ETV6 In Chronic Myeloid Leukemia And Its Mechanism Of Action

Background: Chronic myeloid leukemia(CML)is one of the hematological malignancies and is a clonal myeloproliferative disorder of a pluripotent stem cell,most of which contain oncogenic BCR-ABL fusion gene.K562 is a BCR-ABL-positive cell line and is an ideal cell model for studying CML.Annexin A11(ANXA11)is a member of Annexin family.Its abnormal expression affects the malignant behaviors such as proliferation,migration,invasion and apoptosis of cancer cells,it not reported in CML.ETV6 is a member of the ETS transcription factor family,its chromosomal translocation produces carcinogenic fusion genes widely studied in hematological malignancies,and its effect on the malignant behavior of CML has not been reported.Abnormal mi RNA expression is involved in tumorigenesis by targeting tumor suppressor genes or oncogenes,and mi R-4521 is underexpressed in kidney cancer,lung cancer and chronic lymphocytic leukemia(CLL).The related mechanism has not been reported.The results of our group of experiments indicate that mi R-4521 is underexpressed in CML samples,and overexpression of mi R-4521 can inhibit the malignant behavior of K562 cells.Objective: 1.To detect the expression levels of ETV6 and ANXA11 in CML samples,and to analyze the correlation between ETV6 and ANXA11 and mi R-4521.2.To explore the regulatory relationship between ETV6 and ANXA11 and mi R-4521 in K562 cells.3.To study the effect of ETV6 down-regulation on the malignant phenotype of K562 cells and its molecular mechanism.Methods: 1.qRT-PCR detects the expression levels of ETV6 and ANXA11 in CML samples,and to analyzes the correlation between ETV6 and ANXA11,mi R-4521;2.Gene chip combined with bioinformatics analysis to predict the relationship of ETV6 and ANXA11,mi R-4521;WB,co-immunoprecipitation(Co-IP)and chromatin immunoprecipitation(Ch IP)assay were used to detect the relationship between ETV6 and ANXA11,mi R-4521 in K562 cell line;3.WB detect the efficiency of ETV6 in K562 cells that transiently transfected with si ETV6;Trypan blue counting method and methylcellulose cell in vitro clone formation assay to detect the effect of ETV6 down-regulation on the proliferation of K562 cells;Transwell assay to detect the migration and invasion of K562 cells after ETV6 downregulation;q RT-PCR and WB detect the expression levels of MMP-2,MMP-9,TIMP-1 m RNA and protein in K562 cells that were changed after transfected with si ETV6;Results: 1.Compared with the Normal group,ETV6 and ANXA11 are up-regulated in CML initial patient samples;In paired CML samples(initial-CR),after complete remission(CR),the expression levels of ETV6 and ANXA11 were recovery compared with the initial stage;In the initial CML samples,there was a positive correlation between ETV6 and ANXA11,while ETV6 showed a negative correlation with mi R-4521 expression,and there was a negative correlation trend between ANXA11 and mi R-4521;2.ANXA11 and ETV6 can directly bind with each other,and ANXA11 regulates the expression of ETV6;Up-regulation of ETV6 expression can decrease the expression level of mi R-4521,ETV6 binds to the promoter region of mi R-4521,and inhibit its transcription,the gene chip and bioinformatics prediction results are same with the experiments' results;3.After interfered transfection of si ETV6,the expression level of ETV6 is down;the downregulation of ETV6 inhibited the proliferation and clonality of K562 cells in vitro,si ETV6 inhibited the expression of MMP-2,MMP-9 and promoted TIMP-1 m RNA and protein level,thereby inhibiting the migration and invasion ability of K562 cells in vitro.Conclusion:1.In CML initial patient samples,ETV6 and ANXA11 are up-regulated,after complete remission(CR),the levels were recovery;There was a positive correlation between ETV6 and ANXA11,while ETV6 showed a negative correlation with mi R-4521 expression,and there was a negative correlation trend between ANXA11 and mi R-4521;2.ANXA11 decreased ETV6 expression and increased mi R-4521 expression in K562-sh ANXA11,ANXA11 can directly bind and regulates with ETV6;ETV6 binds to the promoter region of mi R-4521,and inhibit its transcription;3.The downregulation of ETV6 inhibited the proliferation and clonality of K562 cells,si ETV6 inhibited the expression of MMP-2,MMP-9 and promoted TIMP-1 m RNA and protein level,thereby inhibiting the migration and invasion ability of K562 cells in vitro.