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. 2022 Aug 29;13(8):748. doi: 10.1038/s41419-022-05169-x

Fig. 4. EZH2 inhibition induces miR-124-3p level and regulates CCL2 expression.

Fig. 4

A Differentially expressed miRNAs in MDA-MB-231 EZH2 siRNA or inhibitor-treated cells screened by miRNA-seq (part of the result), aligned according to their expression levels, red showing upregulation in MDA-MB-231 siEZH2 cells. B The miR-124-3p level was analyzed by RT-qPCR in MDA-MB-231 cells treated with EZH2 inhibitors or siRNAs for 48 h. U6 was included as a control. n = 3. C Relationship between has-miR-124 expression level and overall survival of patients with breast cancer (http://kmplot.com). D The miR-124-3p and CCL2 level were analyzed by RT-qPCR and western blot in MDA-MB-231 cells treated with miR-124-3p mimics or con-mimics for 48 h. U6 was included as a control. E Western blot showed miR-124-3p mimics repressed CCL2 level induced by EZH2 inhibitors in MDA-MB-231 and 4T1 cells. F The miR-124-3p and Ccl2 level were analyzed by RT-qPCR and western blot in MDA-MB-231 cells treated with miR-124-3p inhibitor or con-inhibitor for 48 h. U6 was included as a control. G Western blot showed miR-124-3p inhibitor induced CCL2 level repressed by EZH2 knockdown in MDA-MB-231 and 4T1 cells. The CCL2 concentration was tested by ELISA in MDA-MB-231 and 4T1 cells treated with EPZ-6438 and miR-124-3p mimics H, or siEZH2 and miR-124-3p inhibitor I. RT-qPCR tested the expression of M2-type markers in THP-1-derived M2 macrophages after being co-cultured with MDA-MB-231 cells treated with EPZ-6438 and miR-124-3p mimics J, or siEZH2 and miR-124-3p inhibitor K. The shown were representative of replicates and the experiments were repeated three times. Statistical significance was addressed using unpaired, two‐tailed Student’s t‐test, *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant. The statistical significance was calculated compared to the negative control (NC) in each group.