Overexpression Of S100A9 Induces Chemotherapy Resistance In HL-60 Cell Line

Part I. Manipulation of a S100A9 overexpressed lentiviral vector and package of lentivirusObjective: We aim to manipulate a S100A9 overexpressed lentiviral vector and subsequently package the lenitvirus, laying foundation for exploring the role of S100A9 in AML chemotherapy resistance,Methods: Full length of S100A9 c DNA was cloned in the template of BMMC c DNA from a normal donor and then inserted into p MD19-T vector. The bacterial colony was verified by PCR, restriction enzyme reaction and sequencing as well. The colony, the sequence of which was 100% matching the record in NCBI database, was involved in following experiments. We ligated the cloned S100A9 fragment to the lentiviral vector p LVX-IRES-puro and then verified the bacterial colony by PCR, restriction enzyme reaction and sequencing. To produce lentivirus, 293 T cells were co-transfected with the S100A9 overexpression vector and package plasmids by the method of calcium phosphate. Supernatant containg lentiviral particle was collected at two time points including 24 h and 48 h post transfection respectively.Results: The band size of PCR and restriction enzyme reaction products were similar to the size we expected after ligating the cloned S100A9 to p MD19-T vector. The sequences of the five colonies were all 100% matching the result in NCBI database. Comparably, the band size of PCR and restriction enzyme reaction products was also similar to the size we expected after ligating cloned S100A9 to p LVX-IRES-puro. The sequence of the chosen colony was also 100% matching the data in NCBI database. S100A9 protein was strongly expressed in 293 T cells transfected with p LVX/S100A9, compared to the empty vector control.Conclusion: We successfully manipulate the S100A9 overexpressed lentiviral vector and packaged the lenivirus as well.Part II. Overexpression of S100A9 selectively induces AML cell line HL-60 resistance to chemotherapyObjective: To explore whether overexpression of S100A9 is associated with the resistance to chemotherapy including DNR, VP-16, Ara-C and HHT in acute myeloid leukemia cell line HL-60.Methods: We transduced HL-60 cells with p LVX/S100A9 lentivirus in order to set up a stable S100A9 overexprssing AML cell line. HL-60 cells were also transduced with p LVX as empty vector control. Cell viability was determined by CCK 8 assay after the treatments of four clinical-grade chemotherapy drugs including DNR, VP-16, Ara-C and HHT at incremental concentrations for 24 hours. Apoptotic cells were assessed by flow cytometry after the transduced cells were exposed to the four chemotherapy drugs at IC50 for 24 hours. Protein expression of c-PAPR was also determined by western blot after the treatments with DNR, VP-16, Ara-C and HHT at 0, IC25 and IC50 concentrations for 24 hours.Results: Exogenous S100A9 coding sequence was intergrated into the genomic DNA of HL-60 cells. m RNA and protein expression of S100A9 were both elevated in HL-60 cells transduced with S100A9 in comparison to the empty vector control. Constitutive overexpression of S100A9 sustained cell viability and protected cells from apoptosis caused by exposure to DNR, VP-16 and Ara-C, but not HHT. Concomitantly, c-PARP expression was minimal in S100A9 overexpressing cells treated with DNR, VP-16 and Ara-C, but pronounced in cells exposed to HHT.Conclusion: Enforced S100A9 expression is highly correlated with the poor response to chemotherapy including DNR, VP-16 and Ara-C.Part III. Overexpression of S100A9 is sensitive to Mcl-1 inhibitor in acute myeloid leukemia cell line HL-60Objective: We aim to further study the molecular mechanism of poor response to chemotherapy in S100A9 transduced HL-60 cells and also to discover an approach to attenuate its resistance.Methods: RNA-Seq was applied to determine total RNA expressions of HL-60/p LVX and HL-60/S100A9. Based on data analysis, the candidate protein was confirmed by western blot. YM-155, a Mcl-1 inhibitor, was employed to treat HL-60/p LVX and HL-60/S100A9 cells. Cell viability and Mcl-1 protein expression were both assessed by MTT assay and western blot after YM-155 exposure.Results: RNA-Seq analysis data implied that m RNA expression of S100A9 and Mcl-1 were both elevated in HL-60/S100A9 in comparison to the empty vector control. Bcl-2 and Mcl-1 protein expression levels increased in S100A9 transduced cells, and were not affected by treatment with DNR, VP-16 and Ara-C. In comparison, HHT visibly decreased Mcl-1 protein expression in S100A9 transduced cells, although Bcl-2 expression remained unaffected. Cell viability and the chemoprotective effects of S100A9 were also reduced when cells were exposed to another Mcl-1 inhibitor, YM-155.Conclusion: Overexpression of S100A9 increases Mcl-1 protein level and Mcl-1 is a target for attenuating chemoresistance induced by S100A9.