Reversal of glucocorticoid resistance in Acute Lymphoblastic Leukemia cells by miR-145.

Sili Long,Fangfang Zhong,Xiang Qin,Danwei Ren,Yana Niu,Dan Mu,Wenjun Liu

doi:10.7717/peerj.9337

Abstract

ObjectiveTo analyze the expression levels of miR-145 in ALL children and their effects on the prognosis of ALL and to explore the mechanism of miR-145 in reversing the resistance of ALL cells to glucocorticoids.MethodsA GEO database dataset was used to analyze the expression levels of miR-145 in ALL children. The association between miR-145 and childhood prognosis was analyzed by the TARGET database data. The expression levels of miR-145 in the glucocorticoid-resistant ALL cell line CEM-C1 were increased by lipofectamine 2000-mediated transfection. Cell proliferation inhibition experiments were performed to detect the effect of miR-145 on the response of CEM-C1 cell line to glucocorticoids. The expression levels of the apoptotic, autophagic and drug resistance-associated genes and proteins were detected by qPCR and western blot analysis.ResultsThe expression levels of miR-145 were decreased in ALL patients (P < 0.001) and the prognosis of ALL in children with high miR-145 expression was significantly improved (P < 0.001). Increased miR-145 expression can improve the sensitivity of CEM-C1 cells to glucocorticoids. The expression levels of the proapoptotic and the anti-apoptotic genes Bax and Bcl-2 were increased and decreased, respectively, whereas the expression levels of the autophagicgenes Beclin 1 and LC were increased. In addition, the expression levels of the drug resistance gene MDR1 were decreased.ConclusionThe expression levels of miR-145 in ALL children were decreased and they were associated with disease prognosis. The data indicated that miR-145 can reverse cell resistance by regulating apoptosis of CEM-C1 cells and autophagy.

Highlights

Acute lymphoblastic leukemia (ALL) is the most common malignant tumor in children and is associated with malignant proliferation of immature T or B lymphocytes (Pui et al, 2015)
Association between miR-145 levels and prognosis of children with ALL The expression of miR-145 in bone marrow samples of 179 children with ALL was determined using the TARGET database and the log-rank survival analysis. These results were combined with clinical data showing that the prognosis of children with ALL in the high expression miR-145 group was significantly higher than that of the low expression group (P < 0.001) (Fig. 2)
We found that the expression levels of miR-145 in childhood acute lymphoblastic leukemia were lower than those noted in healthy controls, suggesting that miR-145 played a regulatory role in the development of childhood ALL (Fig. 1)

Summary

INTRODUCTION

Acute lymphoblastic leukemia (ALL) is the most common malignant tumor in children and is associated with malignant proliferation of immature T or B lymphocytes (Pui et al, 2015). The miR-145 inhibitor and the miR NC control sequences were transfected into the CEM-C1 cells They were divided into the following groups: miR-145 mimic: MM, miR-145 mimic NC: MMN, miR-145 inhibitor: MI, miR-145 inhibitor NC: MIN. CCK8 detects miR-145 affects CEM-C1 cell responsiveness to dexamethasone Following transfection, CEM-C1 cells were incubated for 48 h and the cell density was adjusted to 1 × 105 cells per well. Flow cytometry for miR-145 detection of CEM-C1 cell apoptosis CEM-C1 cells were collected 48 h following transfection, washed with pre-cooled PBS, and mixed with 500 ml binding Buffer. Acridine orange staining indicates the effects of miR-145 on the induction of CEM-C1 cell apoptosis CEM-C1 cells were collected 48 h following transfection, washed twice with PBS and added to a final concentration of 100 mg/ml acridine orange stain. The difference was statistically significant at P < 0.05

RESULTS

DISCUSSION

CONCLUSIONS