Differential microRNA profiling in a cellular hypoxia reoxygenation model upon posthypoxic propofol treatment reveals alterations in autophagy signaling network

Abstract

Recent studies indicate that propofol may protect cells via suppressing autophagic cell death caused by excessive reactive oxygen species induced by hypoxia reoxygenation (H/R). It is established that gene expression patterns including autophagy-related genes changed significantly during the process of H/R in the presence or absence of propofol posthypoxia treatment (P-PostH). The reasons for such differences, however, remain largely unknown. MicroRNAs provide a novel mechanism for gene regulation. In the present study, we systematically analyzed the alterations in microRNA expression using human umbilical vein endothelial cells (HUVECs) subjected to H/R in the presence or absence of posthypoxic propofol treatment. Genome-wide profiling of microRNAs was then conducted using microRNA microarray. Fourteen miRNAs are differentially expressed and six of them were validated by the quantitative real-time PCR (Q-PCR) of which three were substantially increased, whereas one was decreased. To gain an unbiased global perspective on subsequent regulation by altered miRNAs, predicted targets of ten miRNAs were analyzed using the Gene Ontology (GO) analysis to build signaling networks. Interestingly, six of the identified microRNAs are known to target autophagy-related genes. In conclusion, our results revealed that different miRNA expression patterns are induced by propofol posthypoxia treatment in H/R and the alterations in miRNA expression patterns are implicated in regulating distinctive autophagy-related gene expression.

Figure 1

Build the H/R and P-PostH model. The culture media were replaced by glucose and serum free DMEM balanced in normal incubator in 30 minutes, and these HUVEC cells were then placed in hypoxic conditions which were created by a small enclosed humidified plexiglass chamber filled with 94% N₂, 5% CO₂, and 1% O₂ at 37°C for 12 h. After hypoxia, the medium was immediately washed off, and the HUVECs were returned to the maintenance medium (NCS-DMEM) in normal incubator for 4 h. At the same time, prepared propofol was added to the medium to different concentrations (25 μmol/L–150 μmol/L).

Figure 2

Propofol increased the viability and reduced the apoptosis of the H/R induced injury in HUVECs. (a) The normal HUVECs were treated with different concentrations (0–150 μmol/L) of propofol for 4 h. (b) The cells were postconditioned with increasing concentrations of propofol (0–150 μmol/L) after 12 h of hypoxia and 4 h of reperfusion. Cell viability was determined by CCK-8 assay, as previously described. Values are represented as the percentage of viable cells; vehicle-treated cells were considered as 100% viable. The data represented are mean percentage of viable cells + SD of three independent experiments. *P < 0.001, compared with control group, ^▼ P < 0.05, compared with the H/R group. (c) Detection of apoptosis with Annexin V-FITC and propidium iodide staining. Every group of cells with Annexin V and propidium iodide staining was measured by flow cytometry. Histogram representing the percentage of early apoptotic cells and late apoptotic cells. The data represent the mean + SD of three independent experiments. *P < 0.01, compared with the control group, and ^▼ P < 0.05 with H/R group. (d) Expression of apoptosis-related proteins in normal group, H/R injury group, and propofol posthypoxia treatment groups. Data are representative WB from 3 independent experiments (n = 2).

Figure 3

Propofol suppresses the H/R-induced autophagy. (a) The expression of LC3 was determined in normal cells, H/R injury cells, and propofol posthypoxia treatment cells (25–150 μmol/L). (b) The expression of LC3 was determined in control cells and cells treated by normoxia with propofol (25–150 μmol/L). (c) The expression of LC3 was determined in normal cells group, H/R injury cells group, and DMSO posthypoxia treatment groups (25–150 μmol/L). Data are representative WB from 3 independent experiments (n = 3). (d) Autophagosomes and mitochondria were probed by anti-LC3 and anti-Tim23 in normal cells, H/R injury cells, and propofol posthypoxia treated cells. Bar, 20 μm.

Figure 4

miRNA profiles differentiate the propofol group from the H/R group. (a) Hierarchical clustering of 14 miRNAs whose expression was significantly altered (fold change >2, P < 0.01, FDR > 0.05) in the propofol and HR groups. The color stands for the intensity of the signal (n = 3). (b) In the scatter diagram, red spots stand for the miRNAs expressed higher in the HR group, the green ones stand for the ones higher in the propofol groups, and the black ones with no significant changes.

Figure 5

Validation of selected microarray data by qRT-PCR. Statistically significant difference between propofol and H/R is indicated by *P < 0.05 (n = 4).

Figure 6

miRNA-gene interaction networks of autophagy and mTOR pathway. Red box nodes represent miRNA that are upregulated in propofol group. Blue represent downregulated ones and green cycle nodes represent mRNA. Edges show the inhibitory effect of miRNA on mRNA.