Down-regulation of proapoptotic miR-342 is mediated by miR-199a*, which targets the miR-342-encoding gene EVL. Levels of (A) miR-342 and (B) EVL in SzS samples (n = 17) and CD4⁺ T-cell (n = 7) and CD3⁺ T-cell (n = 6) controls measured by qRT-PCR. Data shown as box-whisker plots. (C) CpG-island methylation status of EVL in SzS samples (n = 17) and CD4⁺ T-cell controls (n = 6) measured by methylation-specific PCR. Universally methylated and unmethylated DNA were used as controls. Colorectal cell lines RKO and VACO400 were methylated and unmethylated, respectively, as previously reported.⁹  (D) Levels of miR-199a* in SzS samples (n = 17) and CD4⁺ T-cell (n = 7) and CD3⁺ T-cell (n = 6) controls measured by qRT-PCR. (E) Predicted binding site for miR-199a* within the 3′UTR sequence of EVL gene. (F) Transfection of miR-199a* in HeLa cell line–suppressed EVL 3′-UTR luciferase reporter activity compared with vector only control or Scramble-miR-199a* sequence. (G) Inhibition of miR-199a* in SeAx cells resulted in increased levels of miR-342 measured by qRT-PCR. Fold change levels shown are relative to Scramble-miR-199a* transfected control (ie, ΔΔC_t). (H) Transfection of Jurkat cell line with miR-199a* resulted in decreased levels of miR-342 measured by qRT-PCR. Fold change levels shown are relative to Scramble-miR-199a* transfected control (ie, ΔΔC_t). (I) Expression of miR-342 or inhibition of miR-199a* in SeAx cell line resulted in an increase in apoptosis levels compared with mock-transfected control. Values shown are mean values from 3 experiments. (J) Schematic diagram of proposed pathway of miR-342 regulation in SzS.