Figure 2

Nef target region downregulated reporter (luciferase) activity at a post-transcriptional level (A and B) while LNA modified anti-miRNA restored reporter activity (C, D and E). (A) A construct containing the nef target region cloned into the MCS (3'UTR) of pMIR-REPORT™ Luciferase vector was co-transfected into HeLa cells along with pMIR-REPORT™ β-gal vector. After 24 h, luciferase activity was measured and normalized to beta-galactosidase levels. Relative Luciferase activity was calculated with respect to cells transfected with pMIR-REPORT™ vector alone. Data represent mean ± SEM of three independent experiments performed, each in triplicates. (B) Post-transcriptional regulation of Nef. Northern blot using luciferase probe to show transcript levels of luciferase in luc and luc-nef transfected HeLa cells. β-actin was used as a loading control (lower panel). (C) Design of LNA-modified anti-miRNA molecules for hsa-miR-29a and 29b. Red asterisks indicate positions of LNA-modification in the backbone of the anti-miRNA molecule. (D and E) LNA-modified anti-miRNA against hsa-miR-29a (D) and hsa-miR-29b (E) restored reporter activity from the luc-nef transfected cells. Varying concentrations (0 nM–40 nM) of LNA-modified anti-miRNA molecules were co-transfected with luc-nef clone and control pMIR-REPORT™ β-gal vector into HeLa cells. Luciferase activity was measured after 24 hours and normalized to beta-galactosidase levels. Luciferase activity relative to vector (luc) was plotted. Mean ± SEM from three replicate experiments are shown. LNA, locked nucleic acid; luc, pMIR-REPORT™ vector; and luc-nef, nef target cloned in 3'UTR of pMIR-REPORT™.