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[MiR-199a-5p Affects Sensitivity of Acute Myeloid Leukemia to Adriamycin by Targeting DRAM1]
Yang Li1, Ying Sun1, Miao Miao1
1Department of Hematology, Shengjing Hospital of China Medical University, Shenyang 110022, Liaoning Province, China.
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 IC50 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 IC50 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.
Insights
MicroRNA-199a-5p (miR-199a-5p) is downregulated in acute myeloid leukemia (AML) cells resistant to Adriamycin (ADM). Restoring miR-199a-5p levels increases ADM sensitivity by downregulating DRAM1, offering a potential therapeutic strategy for AML.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Acute myeloid leukemia (AML) poses significant treatment challenges due to drug resistance.
- MicroRNAs (miRNAs) are increasingly recognized for their roles in cancer progression and drug resistance.
- Adriamycin (ADM) is a key chemotherapeutic agent used in AML treatment, but resistance limits its efficacy.
Purpose of the Study:
- To compare miR-199a-5p expression in ADM-resistant versus ADM-sensitive AML cells.
- To investigate the functional role of miR-199a-5p in modulating AML drug resistance.
- To elucidate the molecular mechanism by which miR-199a-5p affects ADM sensitivity, focusing on its interaction with DRAM1.
Main Methods:
- Quantitative real-time PCR (RT-qPCR) to assess miR-199a-5p and DRAM1 expression in cell lines and patient samples.
- Cell viability assays (MTT, CCK-8) to evaluate ADM's effect on cell proliferation.
- Transfection with miR-199a-5p mimics and inhibitors to manipulate its expression.
- Western blotting to determine DRAM1 protein levels.
- Dual-luciferase reporter assays to confirm the direct binding of miR-199a-5p to DRAM1 3' UTR.
- siRNA-mediated knockdown of DRAM1 to assess its impact on ADM sensitivity.
Main Results:
- miR-199a-5p expression was significantly downregulated in ADM-resistant K562/ADM cells and in refractory/relapsed AML patient samples compared to sensitive counterparts.
- Upregulating miR-199a-5p in K562/ADM cells enhanced ADM-induced cell death and decreased DRAM1 expression.
- Downregulating miR-199a-5p in K562 cells reduced ADM sensitivity and increased DRAM1 expression.
- Dual-luciferase assays confirmed DRAM1 as a direct target of miR-199a-5p.
- Knockdown of DRAM1 in K562/ADM cells significantly restored sensitivity to ADM.
Conclusions:
- miR-199a-5p is significantly downregulated in chemoresistant AML, suggesting its role as a tumor suppressor.
- miR-199a-5p directly targets DRAM1, modulating AML cell sensitivity to ADM.
- Targeting the miR-199a-5p/DRAM1 axis represents a potential therapeutic strategy to overcome ADM resistance in AML.
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