Abstract
MicroRNA (miR)-381-3p is the newly discovered tumor-associated miRNA, which is frequently associated with diverse human malignancies; but, it is still unknown about its effect on acute myeloid leukemia (AML) in children. This work focused on exploring miR-381-3p’s effect on childhood AML and identifying the possible mechanisms facilitating new treatment development. Using qRT-PCR analysis, miR-381-3p expression remarkably reduced in pediatric AML patients and AML cell lines (HL-60 and U937). Following transfection of miR-381-3p mimic or inhibitor into HL-60 and U937 cells, we conducted MTT assay to evaluate cell proliferation, flow cytometry (FCM) to measured cell apoptosis and cell cycle, whereas Transwell assays to detect cell invasion and migration. Our results demonstrated that miR-381-3p overexpression remarkably repressed cell growth, invasion and migration; additionally, miR-381-3p overexpression resulted in arrest of cell cycle and enhanced cell apoptosis. In contrast, miR-381-3p knockdown led to an opposite effect. Moreover, we predicted miR-381’s target gene and validated it by luciferase reporter assay and TargetScan, separately. We identified miR-381-3p’s binding site in ROCK1 3′-UTR. As revealed by Western-blot (WB) assay, miR-381-3p overexpression notably suppressed ROCK1 level. Moreover, restoring ROCK1 expression abolished miR-381-3p’s inhibition on cell proliferation, invasion and migration. Data in this work indicated the role of miR-381-3p as the tumor suppressor within pediatric AML by targeting ROCK1. Therefore, miR-381-3p might serve as a potential therapeutic target for the treatment of pediatric AML.
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Funding
This study was funded by grants from Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (2020KY254) and Ningbo Natural Science Foundation (2018A05).
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QY and QY were responsible for designing and performing experiments. QD and CL were in charge of analyzing data. GX played a role in enrolling cases and measuring RNA expression within clinical samples. QY was in charge of study initiation and manuscript writing. The authors approved the eventual manuscript.
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The Ethics Committee of Ningbo First Hospital of Zhejiang University approved human tissue utilization. All patients provided informed consents.
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ESM 1
Figure S1. miR-381-3p knockdown promotes cell growth, and inhibits apoptosis and cell cycle arrest. miR-381-3p inhibitor or inhibitor NC was transfected into HL-60 and U937 cells. (A) MTT assay was conducted to test cell viability. (B) FCM was conducted to analyze cell cycle distribution. (C) Cell apoptosis was measured by FCM. Results were expressed as means ± SD from 3 separate assays. *P < 0.05, **P < 0.01 compared with inhibitor NC group. (PNG 421 kb)
ESM 2
Figure S2. miR-381-3p knockdown promotes cell migration and invasion. miR-381-3p inhibitor or inhibitor NC were transfected into HL-60 and U937 cells. Transwell assays were conducted to assess (A) cell migration and (B) invasion. Results are displayed as means ± SD from 3 separate assays. **P < 0.01 relative to inhibitor NC group. (PNG 2015 kb)
ESM 3
Figure S3. ROCK inhibitor Y-27632 inhibits AML cell migration and invasion. miR-381-3p mimics or ROCK inhibitor Y-27632 were transfected into HL-60 and U937 cells, respectively. (A) MTT assay was conducted to test cell viability. Transwell assays were conducted to assess cell migration (B) and invasion (C). Results are displayed as means ± SD from 3 separate assays. **P < 0.01 relative to NC group. (PNG 1368 kb)
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Ye, Q., Ying, Q., Dai, Q. et al. Tumor-suppressing effects of miR-381-3p in pediatric acute myeloid leukemia via ROCK1 downregulation. Funct Integr Genomics 23, 43 (2023). https://doi.org/10.1007/s10142-022-00950-9
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DOI: https://doi.org/10.1007/s10142-022-00950-9