The Effects Of Endocan Expression Silencing On Chemosensitivity In Myeloid Leukemia Cells

Objective: To investigate the effects of Endocan expression silencing on the proliferation,cell cycle,apoptosis and chemotherapeutic drug sensitivity in human myeloid leukemia cell lines,and explore its potential mechanism.Methods: Two human myeloid leukemia cell lines(U937 and K562)with relatively higher expression of Endocan were selected by RT-PCR and Western blot.LentivirusEndocan-shRNA was transfected into U937 and K562 leukemia cell lines,and the expression of Endocan was detected by RT-PCR and Western blot.The effects of Endocan expression silencing on the proliferation,cell cycle and apoptosis of U937 and K562 cells were detected by CCK-8,Western blot and flow cytometry.The IC50 value of 5-FU was selected by the CCK-8 to deal with U937 and K562 cells,flow cytometry was used to detect the effect of Endocan expression silencing on sensitivity of chemotherapy drugs to U937 and K562 cells.Western blot was used to detect apoptosis related proteins.A mouse model of leukemia was established with stably transfected K562 leukemia cells to further study the effect of Endocan expression silencing on the tumor.Observe the tumor growth in mice.HE staining and TUNEL method were used to observe the apoptosis and necrosis.Immunohistochemistry was used to analyze the expression of PCNA and Cyclin D1.Western blot was used to detect apoptosis related proteins.Western blot was used to detect NF-κB、P65、p-P65、NF-κB inhibitor alpha(IκBα)、p-IκBα、nuclear P65.The DNA binding activity of NF-κB was determined using the EMSA.Results: The expression level of Endocan in human myeloid leukemia cell lines U937 and K562 was relatively high,and lentivirus Endocan-shRNA was successfully established,which can effectively silence Endocan gene expression.Endocan expression silence,can make PCNA and Cyclin D1 expression decreased,inhibit cell proliferation,promote apoptosis and led to the cells G 1 phase arrest in U937 and K562 cells.Western blot analysis indicated that the expression of anti-apoptotic protein Bcl-2 was decreased,pro-apoptotic protein Bax,caspase-3,caspase-9,cleaved PARP was increased.Endocan expression silencing,which can increase the sensitivity of U937 and K562 cells to chemotherapy drugs.In animal experiments of leukemia,Endocan silencing can enhance the inhibitory effect of 5-FU on tumor growth.HE staining and TUNEL showed that Endocan silencing and 5-FU increased apoptosis,and the combination of both showed that apoptosis was the most serious.Immunohistochemistry showed that Endocan silencing and 5-FU could decrease the expression of PCNA and Cyclin D1,and the combination of both showed that the expression of PCNA and Cyclin D1 was the lowest.Endocan silencing enhanced the regulation of 5-FU on apoptosis related proteins.Furthermore,we demonstrated that the knockdown of Endocan inhibited nuclear factor kappa B(NF-κB)activity,as evidenced by the increased NF-κB inhibitor alpha(IκBα)expression,decreased p-IκBα,p-P65 and nuclear P65 expression,and reduced NF-κB DNA-binding activity.Conclusion: Endocan expression silencing,inhibits cell growth and induces cell apoptosis,which is accompanied by the inhibition of NF-κB activity in myeloid leukemia cells,indicating that Endocan is a promising therapeutic target in leukemia.