MiR-199a-5p Affects Sensitivity of Acute Myeloid Leukemia to Adriamycin by Targeting DRAM1 / 中国实验血液学杂志

ABSTRACT

OBJECTIVE@#To compare the expression of miR-199a-5p between ADM-resistant AML cell (K562/ADM)and ADM-sensitive AML cell (K562), and to investigate the effect of miR-199a-5p on regulating AML drug resistance as well as its molecular mechanism.@*METHODS@#MTT method was used to detect the proliferation inhibition effect of ADM on K562 and K562/ADM cells, the IC was calculated. miR-199a-5p expression in cell lines (K562 and K562/ADM) and bone marrow sample (refractory/relapsed AML patients and complete remission AML patients) was detected by RT-qPCR. K562/ADM and K562 cells were transfected by miR-199a-5p mimic and miR-199a-5p inhibitor respectively to ensure that miR-199a-5p expression in K562/ADM cells was increased and that in K562 cells was decreased. Then proliferation inhibition effect of ADM on both cells was detected by CCK-8 and mRNA and protein DRAM1 expression in both cells was measured by real time RT-PCR and Western blot respectively. Dual luciferase reporter assay was used to detect wether there were direct binding sites between miR-199a-5p and DRAM1 3' UTR. CCK-8 was used to measure the proliferation inhibition effect of ADM on K562/ADM cells when DRAM1 was downregulated by siRNA.@*RESULTS@#The IC of ADM for K562/ADM and K562 cells was 146.14±0.079 and 3.08±0.056 μg/ml respectively. As compared with patients in complete remission group, MiR-199a-5p expression in refractory/ relapsed AML patients significantly decreased, and the MiR-199a-5p expression in K562/ADM cells was also dramatically downregulated, compared with K562 cells (P＜0.05). When the expression of miR-199a-5p was upregulated in K562/ADM cells, the proliferation inhibition effect of ADM on cells elevated and both DRAM1 mRNA and protein expressions decreased. Conversely, when miR-199a-5p expression was downregulated in K562 cells, the proliferation inhibition effect of ADM on cells obviously reduced and both DRAM1 mRNA and protein expression increased (P＜0.05). Dual luciferase reporter Assay showed a direct interaction between miR-199a-5p and its binding site within DRAM1 mRNA. Both DRAM1 mRNA and protein expression in K562/ADM were markedly higher than those in K562 cells (P＜0.05). The ADM chemosensitivity of K562/ADM cells was improved significantly when DRAM1 expression was downregulated (P＜0.05).@*CONCLUSION@#miR-199a-5p is downregulated in chemoresistant AML cells. miR-199a-5p expression plays an important role in regulating the sensitivity of AML cells to ADM treatment. DRAM1 is a functional target gene for miR-199a-5p modulating AML chemoresistance.