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Effects of miR-34b/c Over-Expression on c-Myc Stability in LNCaP Cells Post CDDP Treatment

This study investigates the expression levels of miR-34b and miR-34c, along with c-Myc, in LNCaP cells post CDDP (cisplatin) exposure. Utilizing qRT-PCR and western blot analysis, it was found that miR-34b/c expression diminishes shortly after CDDP treatment, allowing c-Myc stimulation. Despite the over-expression of miR-34b/c, c-Myc stabilization remains partially intact, suggesting alternative regulatory mechanisms are at play. These findings underscore the complexity of post-transcriptional regulation of c-Myc in response to drug-induced stress.

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Effects of miR-34b/c Over-Expression on c-Myc Stability in LNCaP Cells Post CDDP Treatment

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  1. Supplementary Figure 7 a LNCaP LNCaP 0 0 2 2 4 4 12 12 Relative miR-34c expression Relative miR-34b expression  Vector  Vector ● pUSP2aWT + miR-34c  pUSP2aWT + miR-34b  pUSP2aWT + miR-Ctr  pUSP2aWT + miR-Ctr CDDP (hrs) CDDP (hrs) b CDDP (hrs) 0 2 4 12 c-Myc -actin + miR-Ctr + miR-34b + miR-34c LNCaP_pUSP2aWT Supplementary Figure 7 Exogenous miR-34b/c over-expression does not completely counteract c-Myc stabilization induced by CDDP (2 µg/ml). Evaluation of the expression levels of (a) miR-34b and miR-34c, and (b) c-Myc, carried out by qRT-PCR (miRscript kit by Qiagen) and western blot analysis, respectively, in the reported transfectants following CDDP and miR mimic molecule pre-loading. The relative abundance of specific miRs was calculated by normalizing to small nucleolar RNAs using the 2-ΔΔCt method.19 Results: As reported in SFXa, miR-34b/c expression goes down in LNCaP cells (Vector) at 2 hrs following drug exposure, to allow c-Myc stimulation in response to stress (as reported in Figure 4b). At 4 hrs time, miR expression starts to rise back to basal levels, suggesting the involvement of post-transcriptional stabilization in the sustained c-Myc induction still observed at 12 hours following CDDP (see Figure 4b). In the untreated USP2aWT over-expressing cells (pUSP2aWT + miR-Ctr), miR-34b/c are kept at low levels to allow Myc release and GSH increase. Upon CDDP exposure, miR-34 levels undergo a very small decrease and are thereafter maintained under control by USP2a regulation, up to 24 hours following the drug stimulus. When pre-loaded with mimic molecules (pUSP2aWT + miR-34b/c), USP2a-dependent control on miR-34 expression is reverted, c-Myc expression falls down, but still a limited amount of protein is stimulated by CDDP at 2 hours (SFXb). In the following hours, c-Myc protein induction rapidly decreases by 4 hours time. These data seem thus to suggest that alternative pathways control the early Myc stimulation triggered by drug in this experimental system, and that, as expected, c-Myc stabilization is not dependent on miR regulation.

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