TRIB2 Knockdown As A Regulator Of Chemotherapy Resistance And Proliferation Via The ERK/STAT3 Signaling Pathway In Human Chronic Myelogenous Leukemia K562/ADM Cells

Objective:To investigate the change of Adriamycin resistance in human chronic myelogenous leukemia(K562/ADM cells)after knocking down the expression of TRIB2 gene,to explore the diagnostic value of TRIB2 knockdown in children CML treatment,and to provide experimental basis for the further study of the role of TRIB2 knockdown in CML therapy.Methods:1.A pair of vector sequences targeting TRIB2 were generated and transfected.Transfection efficiency was determined using fluorescence microscopy and the non-transfected K562/ADM cells were used as the control.After 48h of transfection,G418 was added to the medium to obtain stable positive clones.Then Western blot assay,RT-PCR and RT-qPCR analyses were performed to test the expression of TRIB2 gene.2.Both K562/ADM-TRIB2 cells which have successfully knocked down the TRIB2 expression and their parental cells were cultured in 37 ℃,5%CO2 incubator,then proliferation of the cells were detected by CCK-8 assay after 24h,48h and 72h,respectively.Similarly,total of 5 μM ADM was applied to the wells.After incubation for 1 h.The cell associated mean fluorescence intensity(MFI)of ADM was detected using a FACSCalibur flow cytometer.3.The total RNA or protein was extracted from K562/ADM-TRIB2 cells and parental cells.The RT-PCR and RT-qPCR was also performed to test the expression of relative drug-resistance gene,such as MDR1 and MRP1.Simultaneously,Western blot assay was used to detect the difference of translation level of above genes.4.The Western blot assay was used to verify the differential expression of ERK pathway in K562/ADM-TRIB2 cells and negative groups,including p-ERK and STAT3 protein.Then we used the ERK-specific blocker U0126 to demonstrate this phenomenon.Results:1.After 4 weeks of G418 selection,we successfully obtained stable positive clones.RT-PCR,RT-qPCR and Western blot analyses revealed that TRIB2 expression was markedly decreased in the K562/ADM-TRIB2 group at the transcription and translation levels,compared with that in the K562/ADM group.Then we found that the proliferation of cells knocked down of TRIB2 was markedly inhibited,while their parental cells exhibited no significant difference.All values were statistically significant(*P<0.05).2.CCK-8 assay shows that a reduction in the IC50 value was obvious in the K562/ADM-TRIB2 group,with a reversal fold of 12.12(**P<0.01).Flow cytometry demonstrate that the intracellular ADM accumulation in K562/ADM-TRIB2 cells was considerably higher than that in the K562/ADM cells(**P<0.01).3.Notably,Western blot assay,RT-PCR and RT-qPCR analyses illustrated that the expression levels of MDR1 and MRP1 were lower in the K562/ADM-TRIB2 cells,compared with levels in the control cells(*P<0.05).This indicated that TRIB2 may be involved in key steps of MDR development in children CML.4.Results suggested that the expression of p-ERK and STAT3 was clearly downregulated in the K562/ADM-TRIB2 cells.After treatment with U0126,the expression of p-ERK and STAT3 in K562/ADM-TRIB2 cells was significantly decreased(*P<0.05),indicating that TRIB2 knockdown may act by blocking ERK pathway activity.These results suggest that downregulation of TRIB2 affects cell resistance by altering the expression of p-ERK and STAT3 in CML K562/ADM cells.Conclusions:The results showed that the expression of MDR1 and MRP1 was significantly reduced in K562/ADM cells knocked down of TRIB2.Due to the downregulation of MDR1 and MRP1,the intracellular accumulation of ADM was increased in the transfected cells compared with that in the parental K562/ADM cells.Therefore,the sensitivity of the K562/ADM cells to ADM was enhanced and proliferation was inhibited.Our research revealed that protein expression of the ERK signaling pathway was inhibited by downregulating TRIB2,indicating that the ERK pathway was involved in cell drug resistance and proliferation.In summary,our research suggested that knockdown of TRIB2 could slow cell growth and reverse resistance,implying that TRIB2 is a potential therapy target for resistant children CML.