Shifts in temperature within the physiologic range modify strand-specific expression of select human microRNAs

Abstract

Previous studies have revealed that clinically relevant changes in temperature modify clinically relevant gene expression profiles through transcriptional regulation. Temperature dependence of post-transcriptional regulation, specifically, through expression of miRNAs has been less studied. We comprehensively analyzed the effect of 24 h exposure to 32°C or 39.5°C on miRNA expression profile in primary cultured human small airway epithelial cells (hSAECs) and its impact on expression of a targeted protein, protein kinase C α (PKCα). Using microarray, and solution hybridization-based nCounter assays, with confirmation by quantitative RT-PCR, we found significant temperature-dependent changes in expression level of only five mature human miRNAs, representing only 1% of detected miRNAs. Four of these five miRNAs are the less abundant passenger (star) strands. They exhibited a similar pattern of increased expression at 32°C and reduced expression at 39.5°C relative to 37°C. As PKCα mRNA has multiple potential binding sites for three of these miRNAs, we analyzed PKCα protein expression in HEK 293T cells and hSAECs. PKCα protein levels were lowest at 32°C and highest at 39.5°C and specific miRNA inhibitors reduced these effects. Finally, we analyzed cell-cycle progression in hSAECs and found 32°C cells exhibited the greatest G1 to S transition, a process known to be inhibited by PKCα, and the effect 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.

Keywords: hyperthermia; hypothermia; lung epithelium; microRNA.

© 2015 Potla et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Figures

FIGURE 1.

Microarray and nCounter analysis of the effects of incubation temperature on miRNA expression pattern in human SAECs. (A) HEATMAP of selected miRNAs detected by microarray in hSAECs following 24 h incubation at 32°C, 37°C, or 39.5°C in the absence or presence of 1 ng/mL rhTNFα. (B,C) Microarray data for seven miRNAs exhibiting temperature-dependent expression and nCounter data for miR1260a expression expressed as fold-change versus 37°C cells in the absence (B) or presence (C) of TNFα. Mean ± SEM. (*) P < 0.01 versus 37°C. N = 3 for microarray and 4 for nCounter.

FIGURE 2.

Quantitative RT-PCR confirmation of temperature effects on miRNA expression pattern in human SAECs. Human SAECs from two lots were incubated for 24 h at 32°C, 37°C, or 39.5°C without or with 1 ng/mL rhTNFα. Total RNA was collected and levels (A) hsa-miR-92a-1-5p, (B) hsa-miR-27b-5p, (C) hsa-miR-1260a, (D) hsa-miR-27a-5p, (E) hsa-miR-181a-3p, and (F) hsa-miR-18b were analyzed by qRT-PCR as described in Materials and Methods and expressed as a fold-change versus 37°C cells without TNFα. The right-hand axis displays the total expression level in copies per cell, which was calculated as described in Materials and Methods. Mean ± SEM. (*) P < 0.05 versus 37°C; (†) P < 0.05 versus 39.5°C; (§) P < 0.05 versus 37°C with TNFα; (¶) P < 0.05 versus 39.5°C with TNFα, n = 4. The temperature-responsive miRNAs were cloned into T-Vector and sequenced (inset).

FIGURE 3.

Quantitative RT-PCR analysis of temperature effects on pri-miRNA, pre-miRNA, and opposite strand expression pattern in human SAECs. Human SAECs from two lots were incubated for 24 h at 32°C, 37°C, or 39.5°C without rhTNFα. Total RNA was collected and levels of the temperature-responsive strand, the sister strand, the pri-miRNA, and the pre-miRNA for (A) hsa-miR-92a-1, (B) hsa-miR-27b, (C) hsa-miR-27a, and (D) hsa-miR-181a were analyzed by qRT-PCR as described in Materials and Methods and are expressed relative to expression of each in 37°C hSEAECs. Mean ± SEM, n = 4. The P-values from one-way ANOVA are indicated.

FIGURE 4.

Clinically relevant temperature change-induced expression of miRNAs alters the PKCα protein levels. (A) Human SAECs were incubated at 32°C, 37°C, or 39.5°C for 24 h and cell lysates were immunoblotted for PKCα. Representative of four similar blots and quantified band densities for PKCα normalized to β-tubulin and expressed as percent of levels in 37°C cells. (B) HEK 293T cells were transfected either with a combination of miRNA inhibitors against hsa-miR-27b-5p, hsa-miR-1260a, and hsa-miR-92a-1-5p or an equal concentration of a non-targeting scrambled-sequence inhibitor control. Transfected and untransfected cells were incubated at 32°C, 37°C, or 39.5°C for 24 h and immunoblotted for PKCα. A representative immunoblot and quantified band densities from four similar blots normalized to ß-tubulin and expressed as percent of levels in untreated 37°C cells are shown. Mean ± SEM. (*) P < 0.05, (†) P < 0.04 versus untreated 37°C and 39.5°C cells and miRNA inhibitor-treated 32°C cells, (¶) P < 0.02 versus untreated 39.5°C cells; n = 4.

FIGURE 5.

Functional analysis of the PKCα 3′ UTR binding sites for the temperature-sensitive miRNAs. (A) The putative binding sequences for hsa-miR-27b-5p, hsa-miR-1260a, and hsa-miR-92a-1-5p in PKCα 3′ UTR are shown. (B) Sequence of pmirGLO-PKCα-WT or pmirGLO-PKCα-Mut generated by cloning repeats of the putative miR binding sites or mutated sequences, respectively, into pmirGLO. The mutated nucleotides in each miR target sequence is indicated by black text for pmirGLO-PKCα-Mut. (C) HEK 293T cells were transfected with a combination of synthetic miRNA mimics for hsa-miR-27b-5p, hsa-miR-1260a, and hsa-miR-92a-1-5p or an equal concentration of non-targeting scrambled control mimic and pmirGLO-PKCα-WT (wild-type) or pmirGLO-PKCα-Mut (mutant), incubated at 37°C for 24 h, and dual luciferase assays were performed. Mean ± SEM. (*) P < 0.05, n = 4.

FIGURE 6.

Effect of incubation temperature on cell-cycle progression. (A) Human SAECs were serum-starved for 24 h at 37°C, then incubated at 32°C, 37°C, or 39.5°C for an additional 24 h in serum-containing growth medium, stained with propidium iodide, and the proportion of cells in G1, S, and G2 phases was determined using flow cytometry. (B) HEK 293T cells were transfected with a combination of synthetic miRNA inhibitors for hsa-miR-27b-5p, hsa-miR-1260a, and hsa-miR-92a-1-5p or an equal concentration of non-targeting scrambled control inhibitor. After 24 h recovery, the cells were serum-starved for 24 h at 37°C, then incubated in serum-containing growth medium at 32°C or 37°C for 24 h, and cell-cycle analysis performed and the G1:S ratio calculated. Mean ± SEM, n = 5, (*) P < 0.05 in A. P-values are indicated in B.