Heat shock factor 1 represses Ras-induced transcriptional activation of the c-gene

Heat shock factor 1 represses Ras-induced transcriptional activation of the c-gene. exhibited the greatest G1 to S transition, a process known to be inhibited by PKC, and the effect PSC-833 (Valspodar) was mitigated by specific miRNA inhibitors. These results demonstrate that exposure to clinically relevant hypothermia or hyperthermia modifies expression of a narrow subset of miRNAs and impacts expression of at least one signaling protein involved in multiple important cellular processes. 0.01 versus 37C. = 3 for microarray and 4 for nCounter. TABLE 1. Microarray analysis of the effect of incubation temperature on expression levels of pre- and mature miRNAs Open in a separate window Confirmation of selected miRNA by quantitative PCR The six miRNAs (hsa-miR-18b, hsa-miR-27a-5p, hsa-miR-27b-5p, hsa-miR-92a-1-5p, hsa-miR-181a-3p, and hsa-miR-1260a) shown to exhibit consistent patterns of temperature-dependent expression by microarray and nCounter assays were further analyzed by qRT-PCR. Five of these miRNAs (hsa-miR-27b-5p, hsa-miR-92a-1-5p, hsa-miR-27a-5p, hsa-miR-181a-3p, and hsa-miR-1260a) showed similar patterns of temperature-responsiveness by qRT-PCR and microarray or NCounter analysis (Fig. 2ACE). The identities of each qRT-PCR product were confirmed to be the miRNA of interest by cloning and sequencing; the chromatograms for each are shown in Figure 2. The absolute expression level of each of the five temperature-responsive miRNAs was estimated from the Nanostring data and is shown on the right-hand 0.05 versus 37C; (?) 0.05 versus 39.5C; () 0.05 versus 37C with TNF; (?) 0.05 versus 39.5C with TNF, = 4. The temperature-responsive miRNAs were cloned into T-Vector and sequenced (= 4. The 0.05, (?) 0.04 versus untreated 37C and 39.5C Rabbit polyclonal to PHF7 cells and miRNA inhibitor-treated 32C cells, (?) 0.02 versus untreated 39.5C cells; = 4. To PSC-833 (Valspodar) further demonstrate the specificity of hsa-miR-92a-1-5p, hsa-miR-27b-5p, and hsa-miR-1260a for the PKC 3 UTR, multiple potential binding sites for these three miRNAs were identified in the 3 PSC-833 (Valspodar) UTR of PRKCA gene using PITA (Kertesz et al. 2007) and incorporated into pmirGLO dual luciferase miRNA reporter plasmid (Fig. 5A,B). pmirGLO-PKC-WT contained three binding sites each for hsa-miR-92a-1-5p and hsa-miR-27b-5p and one binding site for hsa-miR-1260a and pmirGLO-PKC-Mut contained an identical sequence except inactivating mutations were introduced into each of the putative miRNA binding sequences. HEK 293T cells were transfected with miRNA mimics for hsa-miR-92a-1-5p, hsa-miR-27b-5p, and hsa-miR-1260a or with scrambled mimic and with pmirGLO-aPKCa-WT or pmirGLO-PKC-Mut and were PSC-833 (Valspodar) lysed and analyzed for luciferase activities 48 h later (Fig. 5C). The miRNA mimics reduced firefly luciferase levels in cells transfected with pmirGLO-PKC-WT by 49.5% compared with scrambled mimic. In contrast, the miRNA mimics had no effect on luciferase levels in cells transfected with pmirGLO-PKC-Mut. Open in a separate window FIGURE 5. Functional analysis of the PKC 3 UTR binding sites for the temperature-sensitive miRNAs. ( 0.05, = 4. Downstream biological consequences for changes in temperature To understand the potential biological impact of altered PKC protein levels, we analyzed cell-cycle progression in growth-factor starved hSAECs incubated for 24 h at 32C, 37C, and 39.5C in serum-containing growth medium (Fig. 6A). Human SAECs incubated at 32C exhibited relatively fewer cells in G1 phase and more cells in S phase compared with 37C and 39.5C cells. To evaluate the contribution of the temperature-responsive PKC-targeting miRNAs, we transfected HEK 293T cells with miRNA inhibitors against hsa-miR-92a-1-5p, hsa-miR-27b-5p, and hsa-miR-1260a 24 h prior to growth factor PSC-833 (Valspodar) starvation at 32C or 37C, then replaced the medium with serum-containing growth.