耳蜗毛细胞氧化损伤microRNA与mRNA表达谱及其调控网络研究

发布时间：2018-02-28 01:30

  本文关键词： 耳蜗毛细胞 氧化损伤 活性氧 微小RNA 表达谱 微阵列芯片　出处：《南方医科大学》2010年博士论文　论文类型：学位论文

【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。P0.05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,P0.001),且25μM以上浓度的t-BHP染毒12h后可抑制HEI-OC1细胞的增殖(P0.05)；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,P0.001),且100μM t-BHP染毒HEI-OC1细胞6h以上可抑制细胞增殖(P0.01)。 (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(P0.05),且主要为晚期细胞凋亡(P0.05)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,P0.001),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%(P0.05)；100μM、200μM、400μM t-BHP组荧光细胞增多(P0.001),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(P0.05),100μM、200μM t-BHP组mmu-miR-203表达下调(P0.05); 100μM、200μM t-BHP组ATF7IP表达上调(P0.01),50μM、100μM、200μM t-BHP组CCND2表达下调(P0.001)；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。
[Abstract]:background
Oxidative damage and reactive oxygen species (also known as Reactive Oxygen, oxygen free radical, Species, ROS) and drug deafness, noise deafness, aging and deafness are closely related to cisplatin, aminoglycosides and continuous noise can make the cochlear hair cells to produce a high concentration of ROS caused by the damage of hair cells. In addition, antioxidants and survival to maintain its normal function in cochlear hair cells. Studies have shown that ROS can regulate the expression of gene transcription, gentamicin and cisplatin can regulate gene transcription in cochlear hair cells by high concentration of ROS, and the signal transduction pathways related to oxidative damage. However, in the post transcriptional regulation of gene expression and cell survival plays an important role, only found in myocardial cells and vascular smooth muscle cells through the regulation of ROS can affect gene expression after transcription, but not related to cochlear hair Therefore, exploring the regulation and regulation mechanism of ROS on cochlear hair cells after gene transcription is important for elucidating the mechanism of oxidative damage in cochlear hair cells.
MicroRNAs (miRNAs) is a kind of endogenous non encoding, small RNA, can be used to target gene mRNAs degradation and translational inhibition and negatively regulate gene expression. A miRNA can regulate the expression of multiple target genes, and several miRNAs can also synergistic effects on a target gene, regulating cell the proliferation / differentiation, growth, migration and apoptosis of.MiRNAs play an important role in the development and maturation of mouse inner ear sensory epithelium, hearing loss is also important regulatory factors. Recent studies have shown that abnormal miRNA may be the cause of hearing loss in humans and mice. However, expression of miRNA damage related cochlear hair cells and no oxidation in the report of gene regulation. Therefore, study on the effect of cochlear hair cell oxidative damage and high concentration of ROS on the expression of miRNAs, and the role of miRNAs in gene regulation mediated by ROS and its biological function in Yes, it will be of great significance in understanding the mechanism of cochlear hair cell damage and hearing loss.
research objective
(1) the use of organic oxidant tert butyl hydroperoxide (tert-Butyl, Hydroperoxide, t-BHP) in the cochlea hair cells (House Ear Institue-Organ of Corti 1, HEI-OC1), through the detection of t-BHP on cell proliferation, cell apoptosis and ROS, establish the model of cochlear hair cell oxidative damage;
(2) the expression of miRNA and mRNA in the oxidative damage of cochlear hair cells was studied by the miRNA expression chip and the whole genome mRNA expression chip.
(3) by bioinformatics analysis of differences in the cochlear hair cell oxidative damage and mRNA expression of miRNA, expression of cochlear hair cell oxidative damage miRNA and regulation of mRNA network and its biological function, to provide more direct scientific clues for gene expression in cochlear hair cell oxidative damage and post transcriptional regulation.
research method
(1) t-BHP cell proliferation was detected by HEI-OC1 cells (0 M, 25 M, 50 M, 100 M, 200 M, 400 M) were 12h, and 100 M t-BHP in 0h, 3h, 6h, 12h, 24h, 48h, Counting and Kit-8 respectively by Cell (CCK-8) detection of different concentrations and different time of exposure to t-BHP after the change of cell proliferation ability.
(2) t-BHP cell apoptosis was detected by HEI-OC1 (0 M, 25 M, 50 M, 100 M, 200 M, 400 M) at 12h, annexin V (Annexin V) and propidium iodide (Propidium Iodide, PI) by double labeling, the effect of flow cytometry in different dose t-BHP on the apoptosis of HEI-OC1 cells.
(3) intracellular ROS detection was set at 0 M, 25 M, 50 M, 100 M M, 200 M, 400 M6 M6 concentration t-BHP cell infected HEI-OC1 cell group, labeled by DCFH-DA probe, the intracellular ROS production was observed by fluorescence inverted microscope, and the level of intracellular HEI-OC1 was quantitatively detected by flow cytometry.
(4) microRNA chip was used to detect HEI-OC1 cells after 0 12h, 50 M, 100 M, 200 Mt-BHP Mt-BHP 12h exposure. Exiqon LNA probe was used to mark the total RNA of cells. After concentration, hybridization and image scanning, the expression profiles of oxidative damage in cochlear hair cells were analyzed.
(5) whole genome expression profiling chip was used to detect HEI-OC1 cells after 0 12h M, 50 M, 100 M, 200 t-BHP M t-BHP 12h exposure. Agilent expression was detected using Agilent Agilent Agilent expression chip.
(6) real time quantitative RT-PCR detection of miRNA and mRNA expression of t-BHP (0 M, 50 M, 100 M, 200 M) in HEI-OC1 cells after 12h, using real time quantitative RT-PCR detection of mmu-miR-29a, the concentration of t-BHP treated cells in mmu-miR-203, CCND2, the expression level of ATF7IP, and using U6 and GAPDH as a reference, the relative quantification of 2- Delta Delta CT method.
(7) the bioinformatics analysis of miRNA gene expression by Targetscan 5.1 prediction of cochlear hair cell oxidative damage and the integration of differences, combined with differential expression analysis of mRNA, 1.2.0 miRNA and mRNA Osprey through the construction of regulatory networks; and the use of DAVID up regulates the expression of miRNA on differentially expressed (and down) analysis of GO and Pathway. The target gene.
(8) statistical analysis showed that all the experimental results were expressed by mean + standard deviation (x + SD). According to the nature of the experimental data, SPSS16.0 software was used for variance analysis.P0.05, with significant difference.
Research results
(1) the effect of t-BHP on cochlear hair cell proliferation of HEI-OC1 cells exposed to different concentration of t-BHP groups after 12h cell growth rate had significant difference (F=79.445, P0.001), and more than 25 M concentration of t-BHP after 12h exposure can inhibit the proliferation of HEI-OC1 cells (P0.05); 100 Mt-BHP exposed HEI-OC1 cells at different time after the cell growth rate had significant difference (F=16.056, P0.001), and more than 100 M t-BHP in HEI-OC1 cells can inhibit the proliferation of 6h (P0.01).
(2) the effect of t-BHP on the cochlear hair cell apoptosis in different concentrations of t-BHP group early apoptosis rate showed no significant difference (F=1.416, P=0.287); the concentration of the apoptosis rate of t-BHP group had statistically significant difference (F=8.372, P=0.001), more than 50 mu M concentration t-BHP induced HEI-OC1 cell apoptosis rate increased (P0.05), and the main late apoptosis (P0.05) increase.
(3) the influence of t-BHP on the level of ROS in cochlear hair cells in cells under the fluorescence microscope ROS on intracellular distribution of different concentration of t-BHP treated group, there is a great difference between intracellular fluorescence and flow cytometry quantitative detection of intracellular ROS results showed that the concentration of t-BHP group of fluorescent cells was statistically significant (F=347.897, P0.001) no, the exposure control group of fluorescent cells was only 3.45%, 25 M t-BHP group of fluorescent cells was 4.26% (P=0.651); 50 M t-BHP group of fluorescent cells was 7.59% (P0.05); 100 M, 200 M, 400 M t-BHP group (P0.001), fluorescent cells were 17.26%, 27.90%, 59.85% the above results show that; more than 50 mu M concentration could induce t-BHP oxidative damage in HEI-OC1 cells, and induced intracellular ROS generation increased significantly.
(4) spectrum based on 0 M t-BHP as control group the expression of microRNA in cochlear hair cell oxidative damage, 50 M, 100 M, 200 M t-BHP treated group were upregulated 40 miRNA, 35 miRNA expression. Compared with non exposed control group, 50 M group t-BHP expression of 21 miRNA, 30 miRNA expression; 100 Mt-BHP group expression of 19 miRNA, 17 miRNA expression; 200 M group t-BHP expression of 21 miRNA, 33 miRNA expression.
(5) expression profiles based on 0 M t-BHP as control group the whole genome mRNA oxidative damaged hair cells in the cochlea, 50 M, 100 M, 200 M t-BHP treated group were upregulated 2076 mRNA, 580 mRNA expression. Compared with non exposed control group, 50 M group t-BHP expression 62 mRNA, 26 mRNA expression; 100 M group t-BHP expression of 1803 mRNA, 298 mRNA expression; 200 M group t-BHP expression of 533 mRNA, 367 mRNA expression.
(6) the validation of real-time RT-PCR chip miRNA and mRNA results by quantitative real-time RT-PCR assay, expression of 200 M t-BHP mmu-miR-29a group (P0.05), 100 M, 200 M in group t-BHP decreased expression of mmu-miR-203 (P0.05); 100 M, 200 M t-BHP group up-regulated expression of ATF7IP (P0.01), 50 M, 100 M, 200 M in group t-BHP decreased expression of CCND2 (P0.001); chip detection results are basically consistent with the miRNA and mRNA expression, indicating that miRNA and mRNA chip experimental results are reliable.
(7) cochlear oxidative damage in miRNA cells and mRNA regulatory network analysis of the concentration of t-BHP group miRNA target gene expression screening showed that 3 up-regulated miRNA 50 M t-BHP HEI-OC1 exposure in groups of cells 5 target gene expression down regulated and 4 down regulated miRNA 4 expression 5 upregulation of miRNA expression of the target gene of.100 M t-BHP in HEI-OC1 cell group in the 20 target gene expression down regulated, downregulation of 8 of the 121 miRNA 8 up-regulated the expression of miRNA target genes.200 M t-BHP in HEI-OC1 cell group 63 target gene expression by 11. Downregulation of miRNA 65 expression of target genes. The integration of differences between the 3 dose t-BHP HEI-OC1 cells expression of miRNA target gene screening results showed that 11 up-regulated miRNA 81 gene expression down regulated and 15 down regulated miRNA 180 A target gene for up-regulated expression.
(8) GO analysis and Pathway GO analysis showed that 180 genes up-regulated by down-regulation of miRNA expression regulated by the 97 GO (Biological Process) classification, the "gene cellular process" classification number, 105 genes; 81 target genes expression is up-regulated by miRNA regulation of the genus 153 GO (Biological Process) classification, including "of biological gene regulation process" and "biological regulation" in the classification of the largest number of all 42 genes.Pathway analysis showed that 180 genes up-regulated by down-regulation of miRNA expression regulated the 6 Pathway classification, 81 target genes expression is up-regulated by miRNA the regulation of 14 Pathway classification.
conclusion
(1) t-BHP in HEI-OC1 cells 12h more than 50 mu M concentration can inhibit cell proliferation, induce cell apoptosis rate increased, and caused the generation of intracellular ROS increased, confirming by 50 M, 100 M, 200 M t-BHP in HEI-OC1 cells, 12h, a light, cochlear hair cell oxidation severe injury model.
(2) compared with the unexposed control group, 50 miRNA M, 100 M and 200 M t-BHP exposure group had 40 miRNA expression up-regulated, 35 miRNA down regulated, 2076 mRNA expression up-regulated, 580 mRNA down regulated, and cochlear hair cell oxidative damage miRNA and mRNA expression profiles were constructed.
(3) bioinformatics integration analysis shows that there are 11 up-regulated miRNA oxidative damaged hair cells in the cochlea of 81 target gene expression is down regulated, 97 GO (Biological Process), 6 Pathway; 15 expression decreased miRNA 180 expression of the target gene is 153 GO (Biological Process) classification, 14 Pathway classification; clear cochlear oxidative damage in miRNA cells and mRNA regulatory network and its biological function.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764

【引证文献】

相关期刊论文 前2条

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2 王艳玲;;噪声与其他职业病危害因素的联合作用[J];职业与健康;2014年07期

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