Mimic Negative Control was used as a negative control (NC). Firefly luciferase activity was normalised relative to Renilla luciferase activity. Transfection of the miR-223 mimic resulted in a marked decrease in luciferase activity in the WT group compared to the NC group (48.08%). Mutations in each of the putative target sites or combined mutations restored luciferase activity to varying degrees: 74.87% for Mut1, 85.21% for Mut2, 74.84% for Mut3, 90.76% for Mut1 + 2, 87.55% for Mut1 + 3, 81.15% for Mut2 + 3, and 94.51% for Mut1 + 2 + 3. Data are presented as mean ± SE of 4 independent experiments.

(C) Two nucleotides in the middle of #PD-1/PD-L1 Inhibitor 3 cost randurls[1|1|,|CHEM1|]# each target site were mutated to generate different mutant luciferase reporters. The expression of PRDM1 in EN-NK/T-NT correlates with miR-223 To investigate the association between PRDM1 and miR-223 in EN-NK/T-NT cases, we performed a correlative analysis between PRDM1 immunostaining and miR-223 ISH. As shown in the scatter diagram (Figure 6A), there is a significant

inverse correlation APR-246 supplier between the levels of PRDM1 expression and miR-223 expression in EN-NK/T-NT cases (P < 0.001). Only 2 cases exhibited similar expression levels of miR-223 and PRDM1. Figure 6B shows one representative case of this inverse correlation in which ISH revealed strong positive expression of miR-223, and IHC indicated no PRDM1 expression in EN-NK/T-NT. Figure 6 Correlation of the expression of PRDM1 and miR-223 in extranodal NK/T-cell lymphoma, nasal type (EN-NK/T-NT). (A) The Isoconazole expression of PRDM1 and miR-223 in EN-NK/T-NT cases were analysed by immunohistochemistry (IHC) and in situ hybridisation (ISH), respectively, and the result is shown

as a scatter diagram. As described in the Materials and Methods section, these results were semi-quantitatively scored into 3 grades according to the number of positive tumour cells. In this figure, the numbers of ordinate are as follows: “1” indicates negative (0% to <10% positive cells), “2” indicates weak (10% to ≤50% positive cells), and “3” indicates strong (>50% to 100% positive cells). Statistically, a significantly opposing correlation was observed between the levels of PRDM1 protein and miR-223 expression in 31 EN-NK/T-NT cases (P < 0.001); only 2 cases had the same relative expression levels of PRDM1 and miR-223. (B) One representative case of EN-NK/T-NT was negative for PRDM1 by IHC but strongly positive for miR-223 by ISH (400×). (C) qRT-PCR analysis revealed much lower levels of miR-223 in YT cells than in NK92, NKL, and K562 cells (mean ± SE of 3 independent experiments).