Figure 5. Downregulation of miR-92a-1-5p, a negative MLKL regulator, mediates shikonin-induced necroptosis in CML cells. (A) Heat map of miRNA expression profiles from control (PBS-treated) and shikonin-treated (20 μM, 1 h) K562 cells. The expression of each miRNA corresponds to the average of 3 replicates. Asterisks indicate miRNAs in the miR-17-92 cluster. Also shown is the correlation of the log fold expression changes of the 1,342 miRNAs with differential expression between the two groups. For the shikonin group, transcripts with >2-fold decrease in are shown in green, while those with >2-fold increase are shown in red. (B) Validation of NGS data by miRNA-specific qRT-PCR. Values are presented as the mean ± SD of 3 independent experiments. *p < 0.05; **p < 0.01. (C) Analysis of relative miR-92a-1-5p expression in CML cell lines and HUVECs (i); Relative miR-92a-1-5p expression in CML patient samples (ii). (D) Sequence alignment of the 3’UTR of human (hsa) MLKL mRNA, highlighting an 8-mer binding site for miR-92a-1-5p. (E) Dual-luciferase reporter assay demonstrating the interaction between miR-92a-1-5p and MLKL mRNA. HEK-293T cells were co-transfected with 50 nM miR-92a-1-5p mimics or NC-mimics and a dual-luciferase vector containing either the wild-type (WT) or mutant (MT) 3’UTR of MLKL. Relative firefly luciferase activity (normalized to Renilla luciferase activity) was determined 48 h after transfection. Data are presented as the mean ± SD of three independent experiments. **p < 0.01. (F) qRT-PCR analysis of miR-92a-1-5p expression in K562 cells transduced with lentiviral vectors encoding miR-92a-1-5p. Western blot images exemplify the decrease in MLKL expression induced by miR-92a-1-5p overexpression. All experiments were repeated at least three times.