We used YT cells because they expressed the lowest endogenous level of miR-223 relative to NK92, NKL, and K562 cells. qRT-PCR analysis identified significantly increased level of miR-223 in YT cells Mdm2 inhibitor transfected with the miR-223 mimic compared to the negative control (Figure 7A, P < 0.001). The expression level of the PRDM1α protein decreased to 54.44% in YT cells treated with ectopic miR-223 relative to YT cells treated with the negative control (Figures 7B and C, P = 0.008); however,

there was no significant difference in the mRNA level of PRDM1α between these 2 groups (Figure 7D), demonstrating that PRDM1α protein expression may be directly downregulated by miR-223 via the inhibition of translation but not by the degradation of PRDM1α mRNA. Vistusertib Figure 7 Endogenous PRDM1 protein expression is affected by increased miR-223 or decreased miR-223. A miR-223 mimic or mimic negative control (NC) was transfected into YT cells by electroporation.

(A) qRT-PCR analysis revealed a significantly increased level of miR-223 in YT cells transfected with miR-223 mimic compared to NC. The results were confirmed in 3 independent experiments with data presented as mean ± SE (※ P < 0.001). (B) Western blot showed that PRDM1α protein level was markedly diminished (54.44% relative to YT-NC, normalised to β-actin) in YT cells transfected with miR-223 mimic. YT-NC was adjusted to 100%. Results were quantified by densitometry in 3 independent experiments (mean ± SD) NVP-BSK805 (# P = 0.008). (C) A representative image of PRDM1α protein expression in YT cells as detected by western blot. (D) RT-PCR and agarose gel electrophoresis showed ectopic expression of miR-223 with no effect on PRDM1α transcript. NK92, NKL, and

K562 cells were transfected with miR-223 inhibitor or NC with HiPerFect Transfection Reagent. (E) Compared to NC, the level of endogenous miR-223 was significantly decreased in NK92, NKL, and K562 cells by qRT-PCR analysis. The data are presented as mean ± SE of 4 independent experiments (∆ P = 0.026, ∆∆ P = 0.017, and ∆∆∆ P = 0.044). (F) Semi-quantitative analysis by densitometry demonstrated that the PRDM1α protein was restored to 220% and 234% by miR-223 Isoconazole inhibition in NKL and K562 cells, respectively, compared to NC, but the level of PRDM1α protein in NK92 cells was not significantly affected. The data are presented as mean ± SD of 4 independent experiments (§ P = 1.000, §§ P = 0.040, and §§§ P = 0.022). (G) Representative western blot images of PRDM1α protein levels in NK92, NKL, and K562 cells are shown. Restoration of PRDM1 expression by reducing miR-223 To test the effect of miR-223 reduction on PRDM1 protein in NK92, NKL, and K562 cells, a miR-223 inhibitor was transfected into cells to reduce the endogenous expression of miR-223. qRT-PCR revealed that the miR-223 inhibitor reduced the levels of endogenous miR-223 in NKL and K562 cells to 40.12% (P = 0.017) and 45.10% (P = 0.