G-CSF Breaks The Bone Marrow Microenvironment Protection On Acute Myeloid Leukemia Cells

Objectives To investigate the role of G-CSF on leukemia cells proliferation,cell cycle distribution and migration.Human AML cell lines HL-60 and THP-1 have been chosen to construct a leukemia research model in vitro to elucidate the molecular mechanisms of chemosensitizing acute myeloid leukemic cells through miR-146a/CXCR4 and miR-146a/Smad4 axis via G-CSF treatment.We further analyzed the effects of G-CSF on overall survival(OS)and relapse free survival(RFS)of AML patients and discussed the possibility of miR-146 a and CXCR4 acting as clinical molecular markers and their predictive significance in evaluating prognosis.Methods and Results Part OneMiR-146 a over-expressing and Knock-down plasmids were transfected to human acute myeloid leukemic cells HL-60 and THP-1.Cells were pre-cultured with100ng/ml G-CSF for 48 hours or with normal medium as negative control.RT-qPCR was applied to detect miR-146 a and CXCR4 mRNA expression,western blotting and flow cytometry were used to detect total and surface CXCR4 protein levels,PI and Annexin V were used to estimate apoptosis rate.HS-5/HL-60(THP-1)co-culture model was further constructed to simulate interaction between AML cells and bone marrow microenvironment.Migration rates were compared in different groups by introducing transwell chambers.We found that G-CSF down-regulated CXCR4 expression by up-regulating miR-146 a levels,thus enhancing migration activities of leukemia cells and promoting their sensitivities to cytarabine eventually.Part TwoMiR-146 a over-expressing and Knock-down plasmids were transfected to human acute myeloid leukemic cells HL-60.Cells were pre-cultured with 100ng/ml G-CSFfor 48 hours or in normal medium as negative control.RT-qPCR was applied for Smad4 mRNA detection,western blotting was used to detect total Smad4 protein levels.Cell Counting Kit-8(CCK8)was applied to cell viability measurement.Flow cytometry(FCM)was used for cell cycle detection.PI and Annexin V were used to estimate apoptosis rate.Our results confirmed that G-CSF down-regulated Smad4 expression by up-regulating miR-146 a levels leading more quiescent leukemia cells into cell cycle,with an increase in proliferative activities,ultimately these cells became more sensitive to cytarabine treatment.Part ThreeWe gathered 97 cases of newly-diagnosed AML patients in last ten years,and divided them into two groups according to G-CSF application conditions.Kaplan-Meier survival curves were used to describe the deciding effect of G-CSF in OS and RFS of AML patients,and univariate and multivariate Cox regression analysis were applied to define prognostic significance of the observed correlation between disease outcome and G-CSF application as well as the prognostic relevance of other parameters.Primary leukemic BM samples from 8 patients were isolated.We analyzed CXCR4 and miR-146 a expression by applying WB and RT-qPCR methods.We pre-cultured 4 primary AML cells with 100ng/ml G-CSF for 48 h and measured their miR-146 a and CXCR4 levels.Finally we used RT-qPCR to detect miR-146 a and CXCR4 mRNA variation range of primary AML cells before and after receiving G-CSF treatment,and further discussed their correlation with long-term outcomes.The analysis results indicated that G-CSF could extended overall survival period of AML patients,while their role in predicting prognosis did not reach statistical significance.The expression of miR-146 a and CXCR4 in primary AML cells were negatively correlated,and G-CSF could down-regulated CXCR4 expression by means of up-regulating miR-146 a levels.Furthermore,we found a longer OS and RFS rates in those patients who had a significant decrease of CXCR4 or increase of miR-146 a levels after G-CSF treatment,and patients who had high CXCR4 expression at diagnosis would behave well if their CXCR4 levels decreased sharply upon G-CSF application.ConclusionG-CSF could affect CXCR4 and Smad4 expression by regulating miR-146a/CXCR4 and miR-146a/Smad4 axis separately,which promoted acute myeloid leukemia cells into peripheral blood circulation from bone marrow niche and stimulated quiescent cells into proliferative stage.Breaking the protection of bone marrow microenvironment made leukemia cells become more sensitive to cytarabine treatment,and eventually led to a prolonged survival period.