Figure 5.

Transcriptional repression of miR-23b by Myc. (a) U266 cells were transfected with a firefly luciferase construct containing the miR-23b promoter, together with an Sp1 or c-myc expression constructs or the corresponding empty vectors; pCMV-RL was used for normalizing firefly luciferase activity. Results are expressed as the ratio between firefly and renilla luciferase of three independent experiments performed in triplicate. *P<0.01. (b) MM1S cells transfected using different concentrations of myc siRNA or scramble siRNA (Scr). Quantitative PCR (qPCR) analyses confirmed reduction in c-myc mRNA levels and increased miR-23b levels following transient transfection of MM1S cells with c-myc siRNA compared with cells transfected with control scrambled siRNA. (c) Stable c-myc overexpression was achieved in U266 cell line using Precision LentiORFs GFP-tagged. qPCR analyses confirmed increased in c-myc mRNA levels and decreased miR-23b levels following myc overexpression in U266. (d) Inverse correlation between mRNA levels of c-myc and miR-23b was evaluated in a cohort of 12 MM patients by qPCR, Pearson correlation and linear regression analysis. R=regression coefficient. (e) The effect of Sp1 knockdown on miR-23b in MM1S and MWCL1 cells transfected with Sp1 or control siRNA was assessed by qPCR and presented as change relative to control cells.