[miR-212 Is Involved in AML Pathogenesis by Regulating WT1 Expression Through Methylation Modification]

To investigate the underlying mechanism of WT1 gene overexpression in patients with acute myeloid leukemia (AML) and to clarify the role of miR-212 in the pathogenesis of AML.

Stem-loop reverse transcription was used to synthesize miR-212 cDNA, and RT-qPCR was used to detect the expression levels of miR-212 and WT1 in bone marrow samples from de novo AML patients, bone marrow transplant donors, and patients with iron deficiency anemia (IDA). The correlation between miR-212 and WT1 expression was further analyzed. A AML cell model with miR-212 overexpression was constructed by infecting Kasumi-1 cells with lentivirus. WT1 protein expression level was detected by Western blot, cell proliferation capacity was detected by CCK-8 assay, and cell cycle distribution and apoptosis were detected by flow cytometry. Dual-luciferase reporter gene assay was used to verify the direct targeted binding relationship between miR-212 and WT1 . Bisulfite sequencing PCR (BSP) was used to analyze the methylation status of MIR212 gene promoter.

The expression level of miR-212 in de novo AML patients was significantly lower than that in the control group, and it was negatively correlated with WT1 mRNA expression (r =-0.77). Overexpression of miR-212 significantly decreased the expression of WT1 mRNA and protein in Kasumi-1 cells, inhibited cell proliferation, reduced the proportion of cells in the S and G₂/M phases, and increased the proportion of early apoptotic cells. The dual-luciferase reporter gene assay revealed that the luciferase activity ratio in 293T cells cotransfected with the WT1 wild-type vector and miR-212-3p mimic was significantly lower than that in the control group, indicating a direct targeted binding relationship between miR-212 and WT1 mRNA. The sequencing results showed that the average methylation rate of CpG sites in the MIR212 promoter region of Kasumi-1 cells was 96.7%.

In AML, promoter hypermethylation leads to the downregulation of miR-212, and such downregulation relieves the targeted inhibition of WT1 , resulting in the upregulation of WT1 expression and thus contributing to the pathogenesis of AML. miR-212 may be a new therapeutic target for AML patients with high WT1 expression.