Research Paper Volume 12, Issue 3 pp 2777—2797

TP53/miR-34a-associated signaling targets SERPINE1 expression in human pancreatic cancer

Figure 5. miR-34a targets SERPINE1. (A, B) To determine the cytotoxic effect of miR-34a mimic and inhibitor, MIA-PaCa2+pLXSN cells were transfected with different concentrations of miR-34a mimic and inhibitor. At 24 h post transfection, MTT was added to each well and the absorption was measured. Percentage of cell death was monitored for miR-34a mimic (miR-mimic) (A) and miR-inhibitor (B) compared with 0.01% DMSO as control. (C, D) miR-34a mimic significantly decreased expression of SERPINE1 and PAI-1 in MIA-PaCa-2+pLXSN cells. MIA-PaCa-2+pLXSN cells were untransfected, mock transfected, or transfected with miR-34a mimic or miR-NC. At the end of 24h of incubation at 37°C, the cells were lysed, RNA extracted (panel C), cDNA synthesized, and SERPINE1 expression monitored by qRT-PCR. In another set of experiments, the cells were lysed were probed for PAI-1 expression by Western blotting (panel D). (E) The relative expression of SERPINE1 and miR-34a in MIA-PaCa-2 target cells was monitored by qRT-PCR. The expression was measured in terms of cycle threshold value (Ct) and normalized to expression of β-actin and snRNA RNU6B, respectively. The x-axis denotes the cell type and y-axis denotes fold change in expression of SERPINE1 and miR-34a. The R2 values for the miRNA expression are provided. (F) In another set of experiments, the above cells were lysed and probed for PAI-1 expression by Western blotting (panel F). Bars (AC, E) represent average ± s.d. of five individual experiments. Student t test was performed to compare groups. Two-tailed P value of 0.05 or less was considered statistically significant. **p,0.01; ***p < 0.001; NS-not significant.