耳蜗毛细胞氧化损伤microRNA与mRNA表达谱及其调控网络研究
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摘要
背景
氧化损伤和活性氧(也称氧自由基,Reactive Oxygen Species, ROS)与药物性耳聋、噪声性耳聋、以及老龄性耳聋密切相关。顺铂、氨基糖甙类药物和持续的噪声均可使耳蜗毛细胞产生高浓度的ROS而导致毛细胞损伤。此外,抗氧化剂有助于耳蜗毛细胞的存活及维持其正常功能。已有研究表明ROS可以调节基因转录水平的表达,庆大霉素和顺铂可通过产生高浓度的ROS而调节耳蜗毛细胞中基因的转录,以及氧化损伤相关的信号转导通路。然而,转录后调控在基因表达和细胞存活中起着重要的作用,仅有研究发现在心肌细胞和血管平滑肌细胞中ROS可以通过转录后调控影响基因的表达,但未见有关耳蜗毛细胞氧化损伤相关的基因转录后调控方面的报道。因此,探寻ROS对耳蜗毛细胞基因转录后的调控及其调控机制,对于阐明耳蜗毛细胞氧化损伤的机制具有重要意义。
MicroRNAs(miRNAs)是一种内源的、非编码的小RNA,可通过对靶基因mRNAs的降解作用和翻译抑制作用而负向调控基因的表达。一种miRNA可调控多个靶基因,而若干miRNAs也可协同作用于一个靶基因,从而调控细胞的分化、增殖/生长、迁移和凋亡。MiRNAs在小鼠内耳感觉上皮的发育和成熟过程中具有重要作用,也是听力损失的重要调控因素。最近的研究显示miRNA异常可能是人类和小鼠进行性听力损失的病因。然而未见有关耳蜗毛细胞氧化损伤相关的miRNA表达及其在基因调控方面的报道。因此,研究耳蜗毛细胞氧化损伤和高浓度ROS对miRNAs表达的影响,以及miRNAs在ROS介导的基因调控中的作用及其生物功能,将对深入了解耳蜗毛细胞损伤和听力损失的机制具有重要意义。
研究目的
(1)利用有机氧化剂叔丁基过氧化氢(tert-Butyl Hydroperoxide, t-BHP)染毒耳蜗毛细胞(House Ear Institue-Organ of Corti 1, HEI-OC1),通过检测t-BHP对细胞增殖、细胞凋亡和ROS产生的影响,建立耳蜗毛细胞氧化损伤模型；
(2)通过miRNA表达芯片和全基因组mRNA表达芯片检测,研究耳蜗毛细胞氧化损伤miRNA和mRNA表达谱；
(3)通过生物信息学分析耳蜗毛细胞氧化损伤差异表达miRNA与mRNA,研究耳蜗毛细胞氧化损伤miRNA与mRNA表达调控网络及其生物功能,为耳蜗毛细胞氧化损伤的基因表达及其转录后调节提供更直接的科学线索。
研究方法
(1)细胞增殖检测HEI-OC1细胞经t-BHP(0μM、25μM、50μM、100μM、200μM、400μM)染毒12h,以及100μM t-BHP染毒0h、3h、6h、12h、24h、48h后,分别用Cell Counting Kit-8 (CCK-8)检测不同浓度和不同时间t-BHP染毒后细胞增殖能力的改变。
(2)细胞凋亡检测HEI-OC1细胞经t-BHP(0μM、25μM、50μM、100μM、200μM、400μM)染毒12h,用膜联蛋白V(Annexin V)与碘化丙啶(Propidium Iodide, PI)双标记后,通过流式细胞术检测不同浓度t-BHP染毒对HEI-OC1细胞凋亡的影响。
(3)胞内ROS检测设置0μM、25μM、50μM、100μM、200μM、400μM6个浓度t-BHP染毒HEI-OC1细胞组,经DCFH-DA探针标记后,通过荧光倒置显微镜观察胞内ROS生成情况,并用流式细胞术定量检测胞内ROS水平。
(4) microRNA芯片检测HEI-OC1细胞经0μM、50μM、100μM、200μMt-BHP染毒12h后,利用Exiqon LNA探针标记细胞总RNA,经浓缩、杂交、图像扫描后,分析耳蜗毛细胞氧化损伤miRNA表达谱。
(5)全基因组表达谱芯片检测HEI-OC1细胞经0μM、50μM、100μM、200μM t-BHP染毒12h后,利用Agilent 4×44K小鼠全基因组表达谱芯片检测细胞氧化损伤后mRNA表达谱。
(6)实时定量RT-PCR检测miRNA和mRNA表达t-BHP (0μM、50μM、100μM、200μM)染毒HEI-OC1细胞12h后,利用实时定量RT-PCR检测各浓度t-BHP染毒组细胞中mmu-miR-29a、mmu-miR-203、CCND2、ATF7IP的表达水平,并分别以U6和GAPDH为内参,用2-ΔΔCT法进行相对定量。
(7)生物信息学分析通过Targetscan 5.1预测耳蜗毛细胞氧化损伤差异表达miRNA的靶基因,并结合差异表达mRNA进行整合分析,通过Osprey 1.2.0构建miRNA与mRNA调控网络；并利用DAVID对差异表达miRNA调控的表达上调(和下调)的靶基因进行GO分析和Pathway分析。
(8)统计分析所有实验结果以均数±标准差(x±SD)表示,并根据实验数据的性质利用SPSS16.0软件进行方差分析。P<0.05为有显著性差异。
研究结果
(1) t-BHP对耳蜗毛细胞增殖能力的影响不同浓度t-BHP对HEI-OC1细胞染毒12h后各组细胞生长变化率有统计学差异(F=79.445,P<0.001),且25μM以上浓度的t-BHP染毒12h后可抑制HEI-OC1细胞的增殖(P<0.05)；100μMt-BHP染毒HEI-OC1细胞不同时间后各组细胞生长变化率有统计学差异(F=16.056,P<0.001),且100μM t-BHP染毒HEI-OC1细胞6h以上可抑制细胞增殖(P<0.01)。
(2) t-BHP对耳蜗毛细胞凋亡的影响各浓度t-BHP组早期细胞凋亡率无统计学差异(F=1.416,P=0.287)；各浓度t-BHP组细胞凋亡率有统计学差异(F=8.372,P=0.001),50μM以上浓度t-BHP致HEI-OC1细胞凋亡率增高(P<0.05),且主要为晚期细胞凋亡(P<0.05)的增加所致。
(3) t-BHP对耳蜗毛细胞胞内ROS水平的影响荧光显微镜下可见ROS分布于胞内,不同浓度t-BHP染毒组间细胞内荧光有极大差别。流式细胞术定量检测胞内ROS结果显示,各浓度t-BHP组荧光细胞率有统计学差异(F=347.897,P<0.001),未染毒对照组荧光细胞仅为3.45%,25μM t-BHP组荧光细胞为4.26%(P=0.651); 50μM t-BHP组荧光细胞为7.59%(P<0.05)；100μM、200μM、400μM t-BHP组荧光细胞增多(P<0.001),分别为17.26%、27.90%、59.85%；以上结果表明50μM以上浓度t-BHP可致HEI-OC1细胞氧化损伤,并致胞内ROS生成量明显增多。
(4)耳蜗毛细胞氧化损伤microRNA表达谱以0μM t-BHP作为对照组,50μM、100μM、200μM t-BHP染毒组共有40个miRNA表达上调,35个miRNA表达下调。相比未染毒对照组,50μM t-BHP组有21个miRNA表达上调、30个miRNA表达下调；100μMt-BHP组有19个miRNA表达上调、17个miRNA表达下调；200μM t-BHP组有21个miRNA表达上调、33个miRNA表达下调。
(5)耳蜗毛细胞氧化损伤全基因组mRNA表达谱以0μM t-BHP作为对照组,50μM、100μM、200μM t-BHP染毒组共有2076个mRNA表达上调,580个mRNA表达下调。相比未染毒对照组,50μM t-BHP组有62个mRNA表达上调、26个mRNA表达下调；100μM t-BHP组有1803个mRNA表达上调、298个mRNA表达下调;200μM t-BHP组有533个mRNA表达上调、367个mRNA表达下调。
(6)实时定量RT-PCR验证miRNA和mRNA芯片结果经实时定量RT-PCR检测,200μM t-BHP组mmu-miR-29a表达上调(P<0.05),100μM、200μM t-BHP组mmu-miR-203表达下调(P<0.05); 100μM、200μM t-BHP组ATF7IP表达上调(P<0.01),50μM、100μM、200μM t-BHP组CCND2表达下调(P<0.001)；与miRNA和mRNA表达芯片检测结果基本一致,表明miRNA和mRNA芯片实验结果可信。
(7)耳蜗毛细胞氧化损伤miRNA与mRNA调控网络分析各浓度t-BHP组差异表达miRNA靶基因筛选结果表明,50μM t-BHP染毒HEI-OC1细胞组中3个表达上调的miRNA有5个表达下调的靶基因,4个表达下调的miRNA有4个表达上调的靶基因。100μM t-BHP染毒HEI-OC1细胞组中5个表达上调的miRNA有20个表达下调的靶基因,8个表达下调的miRNA有121个表达上调的靶基因。200μM t-BHP染毒HEI-OC1细胞组中8个表达上调的miRNA有63个表达下调的靶基因,11个表达下调的miRNA有65个表达上调的靶基因。整合3个浓度t-BHP染毒HEI-OC1细胞差异表达miRNA靶基因筛选结果,表明11个表达上调的miRNA有81个表达下调的靶基因,15个表达下调的miRNA有180个表达上调的靶基因。
(8)GO分析和Pathway分析GO分析结果显示,受下调miRNA调控的表达上调的180个靶基因属97个GO (Biological Process)分类,其中"cellular process"分类中基因数最多,为105个基因；受上调miRNA调控的表达下调的81个靶基因属153个GO (Biological Process)分类,其中"regulation of biological process"和"biological regulation"分类中基因数最多,均为42个基因。Pathway分析结果显示,受下调miRNA调控的表达上调的180个靶基因属6个Pathway分类,受上调miRNA调控的表达下调的81个靶基因属14个Pathway分类。
结论
(1)50μM以上浓度的t-BHP染毒HEI-OC1细胞12h后可抑制细胞增殖、诱导细胞凋亡率增高、并致胞内ROS生成增多,从而确定了通过50μM、100μM、200μM t-BHP染毒HEI-OC1细胞12h,建立轻、中、重度的耳蜗毛细胞氧化损伤模型。
(2)相比未染毒对照组,50μM、100μM、200μM t-BHP染毒HEI-OC1细胞组共有40个miRNA表达上调、35个miRNA表达下调；共有2076个mRNA表达上调、580个mRNA表达下调,构建出耳蜗毛细胞氧化损伤miRNA和mRNA表达谱。
(3)生物信息学整合分析表明,耳蜗毛细胞氧化损伤中11个表达上调的miRNA有81个表达下调的靶基因,属97个GO (Biological Process)分类、6个Pathway分类；15个表达下调的miRNA有180个表达上调的靶基因,属153个GO (Biological Process)分类、14个Pathway分类；明确了耳蜗毛细胞氧化损伤miRNA与mRNA调控网络及其生物功能。
Background
Oxidative 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.
氧化损伤和活性氧(也称氧自由基,Reactive Oxygen Species, ROS)与药物性耳聋、噪声性耳聋、以及老龄性耳聋密切相关。顺铂、氨基糖甙类药物和持续的噪声均可使耳蜗毛细胞产生高浓度的ROS而导致毛细胞损伤。此外,抗氧化剂有助于耳蜗毛细胞的存活及维持其正常功能。已有研究表明ROS可以调节基因转录水平的表达,庆大霉素和顺铂可通过产生高浓度的ROS而调节耳蜗毛细胞中基因的转录,以及氧化损伤相关的信号转导通路。然而,转录后调控在基因表达和细胞存活中起着重要的作用,仅有研究发现在心肌细胞和血管平滑肌细胞中ROS可以通过转录后调控影响基因的表达,但未见有关耳蜗毛细胞氧化损伤相关的基因转录后调控方面的报道。因此,探寻ROS对耳蜗毛细胞基因转录后的调控及其调控机制,对于阐明耳蜗毛细胞氧化损伤的机制具有重要意义。
MicroRNAs(miRNAs)是一种内源的、非编码的小RNA,可通过对靶基因mRNAs的降解作用和翻译抑制作用而负向调控基因的表达。一种miRNA可调控多个靶基因,而若干miRNAs也可协同作用于一个靶基因,从而调控细胞的分化、增殖/生长、迁移和凋亡。MiRNAs在小鼠内耳感觉上皮的发育和成熟过程中具有重要作用,也是听力损失的重要调控因素。最近的研究显示miRNA异常可能是人类和小鼠进行性听力损失的病因。然而未见有关耳蜗毛细胞氧化损伤相关的miRNA表达及其在基因调控方面的报道。因此,研究耳蜗毛细胞氧化损伤和高浓度ROS对miRNAs表达的影响,以及miRNAs在ROS介导的基因调控中的作用及其生物功能,将对深入了解耳蜗毛细胞损伤和听力损失的机制具有重要意义。
研究目的
(1)利用有机氧化剂叔丁基过氧化氢(tert-Butyl Hydroperoxide, t-BHP)染毒耳蜗毛细胞(House Ear Institue-Organ of Corti 1, HEI-OC1),通过检测t-BHP对细胞增殖、细胞凋亡和ROS产生的影响,建立耳蜗毛细胞氧化损伤模型；
(2)通过miRNA表达芯片和全基因组mRNA表达芯片检测,研究耳蜗毛细胞氧化损伤miRNA和mRNA表达谱；
(3)通过生物信息学分析耳蜗毛细胞氧化损伤差异表达miRNA与mRNA,研究耳蜗毛细胞氧化损伤miRNA与mRNA表达调控网络及其生物功能,为耳蜗毛细胞氧化损伤的基因表达及其转录后调节提供更直接的科学线索。
研究方法
(1)细胞增殖检测HEI-OC1细胞经t-BHP(0μM、25μM、50μM、100μM、200μM、400μM)染毒12h,以及100μM t-BHP染毒0h、3h、6h、12h、24h、48h后,分别用Cell Counting Kit-8 (CCK-8)检测不同浓度和不同时间t-BHP染毒后细胞增殖能力的改变。
(2)细胞凋亡检测HEI-OC1细胞经t-BHP(0μM、25μM、50μM、100μM、200μM、400μM)染毒12h,用膜联蛋白V(Annexin V)与碘化丙啶(Propidium Iodide, PI)双标记后,通过流式细胞术检测不同浓度t-BHP染毒对HEI-OC1细胞凋亡的影响。
(3)胞内ROS检测设置0μM、25μM、50μM、100μM、200μM、400μM6个浓度t-BHP染毒HEI-OC1细胞组,经DCFH-DA探针标记后,通过荧光倒置显微镜观察胞内ROS生成情况,并用流式细胞术定量检测胞内ROS水平。
(4) microRNA芯片检测HEI-OC1细胞经0μM、50μM、100μM、200μMt-BHP染毒12h后,利用Exiqon LNA探针标记细胞总RNA,经浓缩、杂交、图像扫描后,分析耳蜗毛细胞氧化损伤miRNA表达谱。
(5)全基因组表达谱芯片检测HEI-OC1细胞经0μM、50μM、100μM、200μM t-BHP染毒12h后,利用Agilent 4×44K小鼠全基因组表达谱芯片检测细胞氧化损伤后mRNA表达谱。
(6)实时定量RT-PCR检测miRNA和mRNA表达t-BHP (0μM、50μM、100μM、200μM)染毒HEI-OC1细胞12h后,利用实时定量RT-PCR检测各浓度t-BHP染毒组细胞中mmu-miR-29a、mmu-miR-203、CCND2、ATF7IP的表达水平,并分别以U6和GAPDH为内参,用2-ΔΔCT法进行相对定量。
(7)生物信息学分析通过Targetscan 5.1预测耳蜗毛细胞氧化损伤差异表达miRNA的靶基因,并结合差异表达mRNA进行整合分析,通过Osprey 1.2.0构建miRNA与mRNA调控网络；并利用DAVID对差异表达miRNA调控的表达上调(和下调)的靶基因进行GO分析和Pathway分析。
(8)统计分析所有实验结果以均数±标准差(x±SD)表示,并根据实验数据的性质利用SPSS16.0软件进行方差分析。P<0.05为有显著性差异。
研究结果
(1) t-BHP对耳蜗毛细胞增殖能力的影响不同浓度t-BHP对HEI-OC1细胞染毒12h后各组细胞生长变化率有统计学差异(F=79.445,P<0.001),且25μM以上浓度的t-BHP染毒12h后可抑制HEI-OC1细胞的增殖(P<0.05)；100μMt-BHP染毒HEI-OC1细胞不同时间后各组细胞生长变化率有统计学差异(F=16.056,P<0.001),且100μM t-BHP染毒HEI-OC1细胞6h以上可抑制细胞增殖(P<0.01)。
(2) t-BHP对耳蜗毛细胞凋亡的影响各浓度t-BHP组早期细胞凋亡率无统计学差异(F=1.416,P=0.287)；各浓度t-BHP组细胞凋亡率有统计学差异(F=8.372,P=0.001),50μM以上浓度t-BHP致HEI-OC1细胞凋亡率增高(P<0.05),且主要为晚期细胞凋亡(P<0.05)的增加所致。
(3) t-BHP对耳蜗毛细胞胞内ROS水平的影响荧光显微镜下可见ROS分布于胞内,不同浓度t-BHP染毒组间细胞内荧光有极大差别。流式细胞术定量检测胞内ROS结果显示,各浓度t-BHP组荧光细胞率有统计学差异(F=347.897,P<0.001),未染毒对照组荧光细胞仅为3.45%,25μM t-BHP组荧光细胞为4.26%(P=0.651); 50μM t-BHP组荧光细胞为7.59%(P<0.05)；100μM、200μM、400μM t-BHP组荧光细胞增多(P<0.001),分别为17.26%、27.90%、59.85%；以上结果表明50μM以上浓度t-BHP可致HEI-OC1细胞氧化损伤,并致胞内ROS生成量明显增多。
(4)耳蜗毛细胞氧化损伤microRNA表达谱以0μM t-BHP作为对照组,50μM、100μM、200μM t-BHP染毒组共有40个miRNA表达上调,35个miRNA表达下调。相比未染毒对照组,50μM t-BHP组有21个miRNA表达上调、30个miRNA表达下调；100μMt-BHP组有19个miRNA表达上调、17个miRNA表达下调；200μM t-BHP组有21个miRNA表达上调、33个miRNA表达下调。
(5)耳蜗毛细胞氧化损伤全基因组mRNA表达谱以0μM t-BHP作为对照组,50μM、100μM、200μM t-BHP染毒组共有2076个mRNA表达上调,580个mRNA表达下调。相比未染毒对照组,50μM t-BHP组有62个mRNA表达上调、26个mRNA表达下调；100μM t-BHP组有1803个mRNA表达上调、298个mRNA表达下调;200μM t-BHP组有533个mRNA表达上调、367个mRNA表达下调。
(6)实时定量RT-PCR验证miRNA和mRNA芯片结果经实时定量RT-PCR检测,200μM t-BHP组mmu-miR-29a表达上调(P<0.05),100μM、200μM t-BHP组mmu-miR-203表达下调(P<0.05); 100μM、200μM t-BHP组ATF7IP表达上调(P<0.01),50μM、100μM、200μM t-BHP组CCND2表达下调(P<0.001)；与miRNA和mRNA表达芯片检测结果基本一致,表明miRNA和mRNA芯片实验结果可信。
(7)耳蜗毛细胞氧化损伤miRNA与mRNA调控网络分析各浓度t-BHP组差异表达miRNA靶基因筛选结果表明,50μM t-BHP染毒HEI-OC1细胞组中3个表达上调的miRNA有5个表达下调的靶基因,4个表达下调的miRNA有4个表达上调的靶基因。100μM t-BHP染毒HEI-OC1细胞组中5个表达上调的miRNA有20个表达下调的靶基因,8个表达下调的miRNA有121个表达上调的靶基因。200μM t-BHP染毒HEI-OC1细胞组中8个表达上调的miRNA有63个表达下调的靶基因,11个表达下调的miRNA有65个表达上调的靶基因。整合3个浓度t-BHP染毒HEI-OC1细胞差异表达miRNA靶基因筛选结果,表明11个表达上调的miRNA有81个表达下调的靶基因,15个表达下调的miRNA有180个表达上调的靶基因。
(8)GO分析和Pathway分析GO分析结果显示,受下调miRNA调控的表达上调的180个靶基因属97个GO (Biological Process)分类,其中"cellular process"分类中基因数最多,为105个基因；受上调miRNA调控的表达下调的81个靶基因属153个GO (Biological Process)分类,其中"regulation of biological process"和"biological regulation"分类中基因数最多,均为42个基因。Pathway分析结果显示,受下调miRNA调控的表达上调的180个靶基因属6个Pathway分类,受上调miRNA调控的表达下调的81个靶基因属14个Pathway分类。
结论
(1)50μM以上浓度的t-BHP染毒HEI-OC1细胞12h后可抑制细胞增殖、诱导细胞凋亡率增高、并致胞内ROS生成增多,从而确定了通过50μM、100μM、200μM t-BHP染毒HEI-OC1细胞12h,建立轻、中、重度的耳蜗毛细胞氧化损伤模型。
(2)相比未染毒对照组,50μM、100μM、200μM t-BHP染毒HEI-OC1细胞组共有40个miRNA表达上调、35个miRNA表达下调；共有2076个mRNA表达上调、580个mRNA表达下调,构建出耳蜗毛细胞氧化损伤miRNA和mRNA表达谱。
(3)生物信息学整合分析表明,耳蜗毛细胞氧化损伤中11个表达上调的miRNA有81个表达下调的靶基因,属97个GO (Biological Process)分类、6个Pathway分类；15个表达下调的miRNA有180个表达上调的靶基因,属153个GO (Biological Process)分类、14个Pathway分类；明确了耳蜗毛细胞氧化损伤miRNA与mRNA调控网络及其生物功能。
Background
Oxidative 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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