| BackgroundOxidative stress and high levels of reactive oxygen species (ROS) are associated with the drug-and noise-induced, and age-related hearing injury and loss. Cisplatin, aminoglycosides and continual noise can promote high levels of ROS production in hair cells. Furthermore, increased levels of antioxidants support hair cell survival and function. Previous studies have revealed that high levels of ROS regulate the expression of epigenetic and transcriptional factors. Gentamicin and cisplatin can promote high levels of ROS production and modulate the transcription of a large number of genes in auditory hair cells. Exposure of auditory cells to ROS modulates the activation and signal transduction of oxidation-sensitive signal pathways. Given that post-transcriptional regulation is crucial for gene expression and cell survival, there are only a few studies of the ROS-related gene expression at the transcriptional level, but no study on auditory cells. Hence, discovery of the pathways involved in the pathogenesis of ROS-related hair cell cytotoxicity and illustration of molecular regulators of the pathogenic process will be of great significance. MicroRNAs (miRNAs) are endogenous, small, non-coding RNAs that can negatively regulate gene expression by degradation and translational inhibition of their target mRNAs. An individual miRNA can regulate the expression of its multiple target genes, and several miRNAs can also synergistically act on one target gene, regulating cell differentiation, proliferation/growth, mobility, and apoptosis. MiRNAs also play an important role in the development and maturation of sensory epithelia in mouse inner ear, and may be pivotal regulators of the process of hearing loss. Recent studies have shown that a mutation in miRNA may be a causative factor for the development of progressive hearing loss in humans and mice. However, the effects of oxidative stress and high levels of ROS on the expression of miRNAs and their potential roles in the ROS-mediated gene regulation and biological functions in auditory cells have not been explored. Furthermore, little is known about how the expression profiles of miRNAs and mRNAs contribute to the regulatory networks in the ROS-related auditory cell injury and death.Object(1) This study aimed at determining the effects of treatment with tert-butyl hydroperoxide (t-BHP) on the production of ROS, apoptosis, and the survival of House Ear Institute-Organ of Corti 1(HEI-OC1)cells. Established oxidative damage model of cochlea hair cells.(2) Characterizing the ROS-related expression of miRNAs and mRNAs in HEI-OC1 cells in vitro.(3) Furthermore, we analyzed the potential interaction of differentially expressed miRNAs with the targeted mRNAs in the process of ROS-induced auditory cells. Hence, our findings may provide new insights into understanding the regulation of miRNAs on the oxidative stress-related auditory cell injury and death. Methods(1) Cell viability assay The viability of HEI-OC1 cells responding to t-BHP treatment was measured using Cell Counting Kit-8 (CCK-8), according to the manufacturer's instruction. Briefly, cells were treated with different concentrations (0,25,50,100,200, or 400μM) of t-BHP for 12 h, and exposed to CCK-8, followed by measuring at 450 nm on a microplate reader. Additionally, the cells were treated with 100μM of t-BHP for varying periods (0,3,6,12,24 and 48 h).(2) Measurements of apoptosis The t-BHP-induced HEI-OC1 cell apoptosis was analyzed by FACS analysis using the FITC Annexin V Apoptosis Detection Kit I, according to the instructions from the manufacturer. Briefly, HEI-OC1 cells were treated with t-BHP (0,25,50,100,200, or 400μM) for 12 h. Subsequently, the cells were stained with FITC-Annexin V and PI, and examined by FACS analysis on a Becton Dickinson FACScan flow cytometer.(3) Detection of intracellular ROS The contents of intracellular ROS were determined using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), according to the manufacturer's instructions. Briefly, HEI-OC1 cells were treated with different concentrations (0-400μM) of t-BHP for 12 h. DCF fluorescence was detected by FACS analysis on a Becton Dickinson FACScan flow cytometer.(4) MicroRNA microarray Individual RNA samples were labeled using the miRCURY Hy3 labeling kit. After evaluating the labeling efficiency, the labeled RNA samples were hybridized on the miRCURY LNATM (locked nucleic acid, LNA) Array (v.11.0). The resulting signals were scanned using the GenePix 4000B scanner and the values of signal intensity were normalized to per-chip median values, and then used to obtain geometric means and standard deviations for each miRNA using the GenePix Pro V6.0 software. (5) mRNA microarray Individual RNA samples were amplified and labeled using the Quick Amp labeling kit and hybridized in triplicate with the Agilent Whole Mouse Genome 4×44 k Oligo Microarray Kit format, according to manufacturer's instructions. After hybridization and washing, the processed slides were scanned with the Agilent microarray scanner using settings recommended by Agilent Technologies.(6) Quantitative RT-PCR The levels of individual gene transcripts were determined by qRT-PCR using the qRT-PCR mRNA detection kit, according to the manufacturer's instruction. Briefly, total RNA were reversely transcribed into cDNA and used as templates for qRT-PCR analysis of the mmu-miR-29a, mmu-miR-203, ATF7IP, and CCND2 expression, respectively.(7) Integrated Analysis of miRNA and mRNA The relationships of differentially expressed miRNAs and mRNAs were further analyzed. The potential mRNA targets of individual differentially expressed miRNAs were predicted using the TargetScan version 5.1. Their potential ontology, pathway and networks were analyzed using the DAVID Bioinformatics Resources 6.7 and the Osprey 1.2.0, respectively.(8) Statistical analysis All data were expressed as mean±standard deviation (SD) from at least three independent experiments. The difference among groups was analyzed by one-way ANOVA using SPSS 16.0. A value of P<0.05 was considered as statistically significant.Results(1) Cytotoxicity of t-BHP on HEI-OC1 cells Treatment with lower concentrations (≥25μM) of t-BHP reduced the proliferation of HEI-OC1 cells (P＜0.05). Furthermore, treatment of HEI-OC1 cells with 100μM t-BHP more than 6h reduced the proliferation of HEI-OC1 cells (P＜0.01).(2) Apoptosis of HEI-OC1 cells induced by t-BHP To assess whether t-BHP could induce HEI-OC1 cell apoptosis, HEI-OC1 cells were treated with 0-400μM of t-BHP for 12 h. Apparently, treatment with higher concentrations (≥50μM) of t-BHP significantly promoted HEI-OC1 cell apoptosis (P＜0.05).(3) Effect of t-BHP on production of intracellular ROS in HEI-OC1 cells HEI-OC1 cells were treated with different concentrations (0-400μM) of t-BHP, and the generated ROS was measured using fluorescent dye DCFH-DA and observed by FACS analysis. Treatment with 50μM of t-BHP induced significantly higher levels of ROS production (P<0.05) and treatment with a higher concentration of t-BHP further elevated the levels of ROS in HEI-OC1 cells. Apparently, t-BHP promoted the formation of ROS in a dose-dependent manner.(4) Treatment with t-BHP modulates the relative levels of miRNA expression in HEI-OC1 cells In comparison with that in unmanipulated control cells, treatment with t-BHP (50,100, and 200μM) significantly increased the transcription levels of 35 miRNAs, but decreased the expression of 40 miRNAs. Treatment with 50,100, or 200μM of t-BHP up-regulated the relative levels of 21, 19, and 21 miRNAs, but down-regulated the transcription levels of 30,17, and 33 miRNAs, respectively.(5) Treatment with t-BHP modulates the relative levels of mRNA expression in HEI-OC1 cells in vitro In comparison with that of untreated control HEI-OC1 cells, treatment with 50,100, and 200μM of t-BHP modulated the transcription of 2656 genes by up-regulating 2076 and down-regulating 580 gene transcriptions. Notably, treatment with 50,100, and 200μM of t-BHP significantly up-regulated the transcription of 62,1803, and 533 genes, but down-regulated the expression of 26,298, and 367 genes, respectively. (6) Validation of miRNA and mRNA expression by real-time qRT-PCR Obviously, treatment with 100 and 200μM of t-BHP significantly reduced the transcription of mmu-miR-203 (P＜0.05), while treatment with 200μM of t-BHP increased the expression of mmu-miR-29a (P＜0.05). Analysis of the relative levels of mRNA transcripts revealed that treatment with 50,100, or 200μM of t-BHP significantly reduced the relative levels of CCND2 transcripts (P＜0.001). Furthermore, treatment with 100 or 200μM of t-BHP also up-regulated the expression of ATF7IP (P＜0.01). Collectively, these data indicated that the expression profiles of these miRNAs and mRNAs were consistent with that observed in the microarray assays.(7) Integrated analysis of the miRNA and mRNA expression profiles in the t-BHP-treated HEI-OC1 cells Interestingly, the relative levels of 5,20, and 63 potential miRNA-targeted mRNA transcripts were down-regulated, while 4,121, and 65 potential miRNA-targeted mRNA transcripts were up-regulated in the 50, 100, or 200μM of t-BHP-treated cells, respectively. Analysis of the identified miRNAs revealed that 11 out of 35 up-regulated miRNAs had 81 down-regulated mRNA targets, while 15 out of 40 down-regulated miRNAs had 180 up-regulated target mRNAs in the t-BHP-treated HEI-OC1 cells (50,100, and 200μM).(8) GO and Pathway analysis GO analyses of 180 up-regulated target mRNAs revealed that these mRNAs belonging to 97 GO category, and "cellular process" was the top GO category, associated with down-regulated miRNAs. GO analyses of 81 down-regulated target mRNAs revealed that these mRNAs belonging to 153 GO category, both "regulation of biological process" and "biological regulation" were the top GO category, associated with up-regulated miRNAs. Pathway analyses of differentially expressed mRNAs revealed that 180 up-regulated target mRNAs belonging to 6 Pathway category, while 81 down-regulated target mRNAs belonging to 14 Pathway category.Conclusions(1) In this study, we employed an in vitro cellular model of oxidative stress in auditory cells and found that treatment with t-BHP promoted the production of ROS in a dose-dependent manner. Furthermore, treatment with t-BHP inhibited HEI-OC1 cell proliferation, which was associated with inducing HEI-OC1 cell apoptosis.(2) Further microarray analyses revealed that treatment with t-BHP increased the transcription of 35 miRNAs, but decreased the expression of 40 miRNAs. In addition, treatment with t-BHP up-regulated the transcription of 2076 mRNAs, but down-regulated the levels of 580 mRNA transcripts.(3) Analysis of the identified miRNAs revealed that 11 up-regulated miRNAs had 81 down-regulated mRNA targets, while 15 down-regulated miRNAs had 180 up-regulated target mRNAs in the t-BHP-treated HEI-OC1 cells (50,100, and 200μM). Importantly, these differentially expressed mRNAs belonged to different GO and Pathway categories, forming a network participating in the oxidative stress-related process in HEI-OC1 cells. |
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