The Role Of Granulocyte Colony-Stimulating Factor Regulating MicroRNA-146a/CXCR4 Expression In Priming Chemotherapy Regiment

ObjectiveWith the progress of diagnosis and treatment, the response rate to chemotherapy has been improved obviously, but the outcome of acute myeloid leukemia in older, relapsed/refractory, secondary patients is dismal. The cytarabine, aclarubicin and G-CSF(CAG) protocol has been widely used in these patients and has been achieved dramatic therapeutic efficacy, while the underlying mechanisms are still not very clear. Here we investigated the potential ability of G-CSF to overcome stromal-mediated drug resistance and analyzed the underlying possible mechanism. Design and MethodsTwo kinds of HS-5/HL-60 co-culture models have been established to imitate the interactions between stromal cells and leukemia cells. Then we assessed the migration and adhesion abilities of HL-60 cells through trans-well assay and co-culture system, respectively. Cell viability was measured by Cell Counting Kit-8(CCK-8). Apoptosis was assessed by flow cytometry using the Annexin V Apoptosis Kit. Surface CXCR4 and total CXCR4 protein were detected by flow cytometry and Western blotting, respectively. Real-time quantitative PCR was used to analysis mRNA expression. We wondered whether stromal cells could protect HL-60 cells from apoptosis, whether G-CSF could overcome the stromal-mediated protective effect and what might be the underlying possible signal pathway. Results Part 1HL-60 cells could be attracted and adhered to HS-5 cells, while these effects were partially blocked by G-CSF and/or AMD3100, the CXCR4 antagonist. When co-cultured with HS-5 supernatant(indirect contact) or HS-5 cells(direct contact), HL-60 cell viability was increased and apoptosis ratio was reduced. Theses protective effects were partially blocked by G-CSF and/or AMD3100, and both result in synergy. The protection effects could be more easily found when HL60 cells were cultured with HS-5 cells than with HS-5 supernatant. Meanwhile, G-CSF and/or AMD3100 did not have a significantly effect on viability or apoptosis when HL-60 cells were cultured with medium alone. Part 2We verified that G-CSF clearly reduced the levels of HL-60 cell surface CXCR4 protein, total CXCR4 protein and CXCR4 mRNA expression, along with up-regulated micro RNA-146a(mi R-146a) expression. CXCR4 mRNA was negatively correlated with mi R-146 a. However, AMD3100 only reduced the surface CXCR4 expression, but not total CXCR4, CXCR4 mRNA or mi R-146 a expression. Part 3We also observed a significant up-regulation of NF-?B p50, p65 mRNA expression when treated with G-CSF, along with down-regulated CXCR4 expression and up-regulated mi R-146 a expression. NF-?B inhibitor sanguinarine showed an opposite effect when co-administrated with G-CSF, while it did not when treated alone. At the same time, we observed a significant up-regulation of NF-?B p50, p65 and phosphorylated I?B protein when treated with G-CSF. ConclusionsG-CSF overcomes stromal-mediated drug resistance in HL-60 cell line by interfering with the CXCR4/SDF-1? axis, and G-CSF reduces CXCR4 expression mainly through up-regulating mi R-146 a expression which may depend on NF-?B signal pathway activation.