Research Paper Volume 12, Issue 3 pp 2777—2797

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

Figure 3. miR-34a expression in MIA-PaCa-2+pLXSN cells. (A) qRT-PCR was conducted to determine the miR-34a expression in MIA-PaCa-2+WT-TP53 and MIA-PaCa-2+pLXSN cells. Briefly, approximately 500 ng of RNA was reverse transcribed in a 25 μl reaction volume using the All-in-one miRNA qRT-PCR detection kit (GeneCopoeia, Rockville, MD). The synthesized cDNAs were used in the PCR reaction. The expression levels of miR-34a were measured employing the SYBR green detection and specific forward primer for the mature miRNA sequence and the universal adaptor reverse primer (GeneCopoeia, USA). Two-tailed P value of 0.05 or less was considered statistically significant; ***p < 0.001. (B) The putative targets of miR-34a that were significantly altered in MIA-PaCa-2+pLXSN and MIA-PaCa-2+WT-TP53 cells when the cells were treated with BBR and MBBR. A select few of the miR-34a target proteins that were significantly altered by treatment of MIA-PaCa-2 cells with BBR and NAX060 are projected. The data represent average of three individual experiments. (C) qRT-PCR was conducted to determine the expression of miR-34a-target genes in MIA-PaCa2+pLXSN and MIA-PaCa-2+WT-TP53 cells. qRT-PCR was performed to monitor expression of the different miR-34a-putative target genes in untreated MIA-PaCa-2+pLXSN cells and MIA-PaCa-2 expressing WT-TP53 or those treated with BBR and MBBR, respectively, using specific primers and SYBR green detection as per standard protocols. Bars represent average ± s.d. of three individual experiments.