Figure 3.

Modulation of Both miR-125b Targets and miR-34a Expression in MM Cells after Ectopic Expression of miR-125b or Its Modified Analogs

(A) Analysis of basal miR-34a levels by qRT-PCR in SKMM-1, RPMI 8226, U266, OPM-2, and KMS-12 cell lines. Each experiment was repeated at least three times and data are shown as mean ± SD. (B) miR-34a levels in U266 MM cell lines after ectopic expression of miR-125b mimics or its modified analogs. Analysis of miR-34a levels by qRT-PCR is shown in the U266 cell line after 48 h from transfection with 100 nM miR-125b, miR-NC, miR-125b-Omet (Omet), miR-125b-LNA (LNA), or miR-125b-2′F (F). Each experiment was repeated at least three times and data are shown as mean ± SD. *p ≤ 0.05, **p ≤ 0.01. (C and D) Western blot analysis of U266 cells transfected with 100 nM miR-125b, miR-NC, miR-125b-Omet (Omet), miR-125b-LNA (LNA), or miR-125b-2′F (F). After 24 h (C) and 48 h (D) from transfection, the cells were collected for western blotting analysis. Subsequently, the expressions of IL6-R, cRaf, Akt, Bcl-2, Mcl1, STAT3, pSTAT3, EIF5A, 4EBP1, p4EBP1, and p53 were evaluated after blotting with specific antibodies, as described in the Materials and Methods. The actin protein was used as a loading control. (E) Scheme representing the molecular mechanism of miR-125b-induced oncosuppressive effect. Bcl-2, Mcl-1, IL-6R, and STAT3 are miR-125b validated targets, as we have herein demonstrated. STAT3 has a great impact in transcriptional activation of pro-survival mediators Bcl-2 and Mcl-1 and, in turn, Bcl-2 can induce STAT3 activation.7, 89 STAT3 silencing by miR-125b inhibits Bcl-2 and Mcl-1 expressions, already repressed by miR-125b itself. In addition, the miR-125b-mediated simultaneous inhibition of IL-6R and STAT3 induces a clear upregulation of the tumor suppressor miR-34a, which, in turn, can further down-modulate IL-6R expression.