Study On The Mechanism Of SPARC To Enhance The Sensitivity Of SKM-2 Cells To Ara-C

Background:Secreted protein acidic and rich in cysteine（SPARC）has a complicated and pleiotropic biological role in cellular senescence during disease.However,the role of SPARC in acute myeloid leukemia（AML）post-myelodysplastic syndromes（MDS）patients（secondary AML,sAML）is not yet fully understood.In the present study,we performed an analysis of proteins and genes at two different levels.Methods:SKM-1 cells were infected with the pGC-GV-SPARC vector to construct a SPARC overexpression model.A high-efficiency HEK293F expression system was established to purify recombinant human SPARC（rh-SPARC）.RNA sequencing was employed to examine differential gene expression after gene alteration in the cells.Auto VP-capillary differential scanning calorimetry,CCK-8 kit,flow cytometry,PCR and western blot analyses were used to assess the characteristics and bioactivity of SPARC.Isothermal titration calorimetry was performed to observe whether there was a direct interaction between rh-SPARC and cytarabine（Ara-C）.Results:The SPARC overexpression and rh-SPARC protein suppressed the proliferation of SKM-1 cells in a concentration-dependent manner and suppressed the SKM-1 cell cycle in the G₀/G₁ phase,with a more significant observed when SPARC overexpression/rh-SPARC and Ara-C were used in combination.However,ITC shows that rh-SPARC has no direct interaction with Ara-C.Through the corresponding mRNA expression profile microarray analysis,we obtained four high-potential SPARC pathways for SKM-1 cells:SPARC-lncHNF1A-CASC2-P21;SPARC-LTF-PAK6;SPARC-PRKACB-PKA;SPARC-PHACTR4-STAT3.In addition,we obtained targets and downstream pathways that may be involved in the action of SPARC sensitizing chemicals.SPARC may enhance the sensitivity of SKM-1 cells to Ara-C by down-regulating MLLT3 gene or down-regulating the drug resistance gene ABCB9 or decreasing AKT phosphorylation.The PCR results indicated that some candidate genes were consistent with the chip results,indicating that the mRNA chip with the difference multiple and P value as the statistical basis has a high guiding value for our basic and clinical research to narrow the research scope.Conclusions:The results indicated that SPARC has a selective inhibition activity towards leukemia cells,as it could suppress the sAML cell line SKM-1 in the G₀/G₁ phase in a concentration-dependent manner by signaling pathway.Through the corresponding mRNA expression profiling,we obtained four high-potential SPARC pathways against SKM-1cells and possible target genes or downstream pathways associated with SPARC-enhanced SKM-1 cells sensitivity to chemotherapeutic drugs.But in order to clarify the role of these signaling pathways in the biological function of SPARC in the conversion of MDS to AML and its interaction with chemotherapeutic drugs,further research and exploration are needed.