氡致人支气管上皮细胞线粒体损伤及RNA表达改变
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摘要
目的：流行病学调查和实验研究证实高浓度氡(Rn)暴露和肺癌的发生密切相关。支气管上皮细胞和细胞核DNA是氡及其子体作用的直接靶细胞和重要靶分子。线粒体在α粒子辐射旁效应的调节中起重要作用,而线粒体DNA (mtDNA)是致癌物作用的重要靶点。本课题在建立的线粒体DNA敲除人支气管上皮细胞(p-HBE)模型的基础上,观察氡及其子体照射对p-HBE细胞的损伤效应,同时采用高通量芯片技术,筛选p+HBE与p-HBE细胞间以及氡染毒前后差异表达的mRNA和微小RNA (miRNA),为探讨线粒体在氡致HBE细胞恶性转化中的作用机制提供实验依据。
方法：采用溴化乙锭(EB)诱导法构建p-HBE细胞株,并将其置于特制的氡染毒装置中,氡暴露浓度为20, OOOBq/m3,染毒时间20min,染毒3d后重复染毒1次,连续染毒2次后作为染毒第1代(Rn1),分别染氡10代(Rn10)和30代(Rn30)。以线粒体正常的HBE细胞(p+HBE)为对照细胞,用Real-time PCR测定pHBE细胞氡染毒前后线粒体拷贝数,活细胞探针标记线粒体DNA数目、线粒体形态、线粒体膜电位以及细胞内活性氧(ROS)的水平,用Annexin V和PI染色测定细胞凋亡及细胞周期的改变。选用6种细胞：p+HBE、p+HBE染氡10代细胞(低剂量染氡组)、p+HBE染氡30代细胞(高剂量染氡组)、p-HBE、p-HBE染氡10代(低剂量染氡组p-HBE)以及p-HBE染氡30代细胞(高剂量染氡组p-HBE),采用NimbleGen-135K mRNA表达谱芯片和miRCURYTM芯片筛选差异表达的mRNA和miRNA,并用实时荧光定量PCR进行验证。
结果：用EB诱导30d后,p+HBE细胞中的线粒体拷贝数减少了约77%,细胞浆内mtDNA的数量显著减少,线粒体膜电位降低,同时在无尿嘧啶的培养液中细胞开始大量死亡,表明EB已成功诱导线粒体DNA缺失,建立了p-HBE细胞。经氡染毒后,p+HBE细胞生长速率加快,克隆形成率和存活分数明显下降,而p"HBE细胞的克隆形成率和存活分数却明显升高,提示线粒体DNA敲除后,p-HBE细胞更易受到氡暴露的损伤,可能促进细胞发生恶性转化。p-HBE细胞中ROS水平低于p+HBE细胞,经氡染毒后两种类型细胞中ROS水平都显著升高,但p-HBE细胞内ROS水平仅为p+HBE细胞的48%,提示除线粒体外,细胞内ROS的产生还有其他的来源。p-HBE细胞的线粒体膜电位经氡照射后显著下降,表明线粒体的膜结构和功能受到了严重损伤。p-HBE细胞染氡后的凋亡率有所升高但明显低于p+HBE细胞,提示线粒体缺失细胞增殖能力的增强与其凋亡功能的降低存在密切的相关。p-HBE细胞在高剂量氡染毒后S期细胞增加,G1和G2/M期细胞减少,提示发生了G1期和G2/M期阻滞,延缓细胞进入S期和M期的速度,导致细胞周期的延长。mRNA表达谱的分析结果显示,氡染毒致p+HBE细胞mRNA表达明显上调的有TFF1、TFF2和IGHG1等,表达明显下调的mRNA包括NCOA7,FN1, EGR1, IL-8, RASEF和BNDF等。GO通路分析表明,上调的]mRNA主要是影响信号传导通路,下调的基因70%以上影响蛋白质的结合和酶活性。在p-HBE细胞中,低剂量染氡组p-HBE上调变化最大的mRNA为SPRR家族中的SPRR2E、 SPRR2A、SPRR2D等,下调表达的基因包括HAND2和FCRLM1等。高剂量染氡组p"HBE上调变化最大的mRNA为BICC1、ASNA、KIAA1045等,表达下调的有GNAI3、TSGA2、BAX、PKP2、GNPDA1等。GO通路分析表明,这些上调和下调的mRNA主要功能是影响受体信号、信号传导以及蛋白质结合等通路。microRNA表达谱的分析结果显示,氡暴露致p+HBE细胞miRNA表达上调的有miR-107, miR-3651和miR-3074-3p等,表达下调的有miR-1227以及miR486-5p等。氡暴露致p-HBE细胞miRNA表达上调的有miR-1919-5p、miR-3157-3p和miR-484等,表达下调的有miR-3607-3p,miR-98和miR-1275等。
结论：(1)采用EB诱导法成功构建了mtDNA部分敲除的人支气管上皮细胞株(p-HBE),并通过观察细胞形态学、营养缺陷和活细胞染色及检测线粒体DNA拷贝数、线粒体膜电位的改变,了解了p-HBE细胞的生物学特性,为研究氡致线粒体DNA敲除细胞的损伤效应提供了细胞模型。(2)氡染毒后,p-HBE细胞在生长速度、ROS产生量、线粒体膜电位、细胞凋亡及细胞周期等生物学指标上均出现了明显改变,表明氡染毒后所产生的细胞损伤效应与线粒体的结构和功能障碍密切相关,为进一步从分子水平上探讨氡致肺癌的发生机制提供了基础资料。(3)通过高通量芯片筛选,发现了一批p+HBE与p-HBE细胞间以及氡染毒前后差异表达的mRNA和miRNA,对这些mRNA和miRNA进行深入的生物信息学分析,将有助于阐明在氡致肺癌的过程中,核基因组和线粒体基因组所起的不同作用,以及可能涉及的基因网络和细胞信号通路。
Objective:Both Epidemiological investigation and experimental research confirmed the correlation between exposure of high level of radon and lung cancer. Bronchial epithelial cells, as well as their nuclei, are the targets of radon and its progeny. Mitochondrial has been shown to play an important role in the regulation of bystander effects of a particle, and mitochondrial DNA is one of the targets of carcinogens. This study is to set up a model of human bronchial epithelial cells (HBE) with knockdown of mitochondrial DNA (p") to observe the adverse effects of radon irradiation, and to screen differentially expressed mRNA/miRNA beteen p+HBE and p" HBE cells upon radon exposure, in order to provide experimental data for exploring potential mechanisms underlying malignant transformation induced by radon.
Methods:p" HBE cells were generated by treatment of ethidium bromide (EB), and exposed to radon gas in a special inhalation chamber. The exposure concentration was set to20,000Bq/m3and lasted for20min each time and repeated in3days. Ten generations (Rn10) and30generations (Rn30) were exposed to radon, and the copy numbers of mitochondrial were determined by Real-time PCR, the number, morphology, membrane potential of mitochondrial and intracellular reactive oxygen species (ROS) were labeled by live cell probes. The alternation of apoptosis and cell cycle distribution were evaluated by Annexin V and PI staining. The NimbleGen-135K mRNA chip and miRCURYTM chip were used to reveal changes in mRNA and miRNA expression, and Q-PCR was performed to further confirm the data.
Results:Thirty days after the EB induction, the number of mitochondrial in p-HBE cells decreased77%of that in p+HBE cells. Intracellular mitochondrial DNA was reduced significantly and mitochondrial membrane potential (MMP) was decreased. After removal of uridine for3days, lots of cells could not survive, suggesting that the p"HBE cells were successfully established. Upon exposure to radon, the proliferation of p+HBE cells was accelerated, with decreased clonogenic rate and survival fraction. In p'HBE cells, the clonogenic rate and survival fraction were increased, implicating a more severe damage by radon. The ROS level in both cells were elevated by radon exposure, but the level in p-HBE cells was only48%of that in p+HBE cells. The membrane potential in p"HBE cells was lowered as a result of mitochondrial damage. The apoptotic rate of the p-HBE cells was also found lower than in the p+HBE cells, although both cells showed increased apoptosis when exposed to radon, suggesting a correlation between mitochondrial function and cell proliferation. DNA partial depletion could inhibit apoptosis. Cells in S phase increased and those in G1and G2decreased after radon exposure, indicating a G1and G2/M phase arrest and prolonged cell cycle. The screening of mRNA expression revealed an up-regulation of TFF1, TFF2and IGHG1, and down-regulation of NCOA7, FN1, EGR1, IL-8, RASEF and BNDF. The GO analysis showed that these mRNA mainly affect signal transduction and protein/enzyme activity. In radon exposed p+HBE cells, BICC1, ASNA, KIAA1045were up-regulated, and GNAB, TSGA2, BAX, PKP2and GNPDA1were down-regulated. In radon exposed p-HBE cells, SPRR2E, SPRR2A, and SPRR2D were up-regulated and HAND2and FCRLM1were down-regulated, with functions in receptor signal, signal transduction and protein synthesis. The screening of miRNA expression revealed an up-regulation of miR-107, miR-3651and miR-3074-3p, and a down-regulation of miR-1227and miR486-5p in radon exposed p+HBE cells. In radon exposed p-HBE cells, miR-1919-5p,miR-3157-3p and miR-484were up-regulated, and miR-3607-3p, miR-98and miR-1275were down-regulated.
Conclusions:
1. A p-HBE cell model was successfully established by EB induction. The copy numbers and membrane potential of mitochondria, intracellular reactive oxygen species, apoptosis and cell cycle were determined to characterize the p-HBE cells.
2. Upon radon exposure, indexes reflecting cell growth, ROS production, membrane potential of mitochondria, apoptosis and cell cycle were altered indicating a correlation between mitochondrial disfunction and the cell damage.
3. High-through chip screening revealed a set of differential expressed mRNAs and miRNAs between p+HBE and p-HBE cells and between pre-and after radon exposure. Bioinformatic analysis will help elucidate roles that nuclear DNA and mtDNA may play in signal transduction of radon induced lung cancer.
方法：采用溴化乙锭(EB)诱导法构建p-HBE细胞株,并将其置于特制的氡染毒装置中,氡暴露浓度为20, OOOBq/m3,染毒时间20min,染毒3d后重复染毒1次,连续染毒2次后作为染毒第1代(Rn1),分别染氡10代(Rn10)和30代(Rn30)。以线粒体正常的HBE细胞(p+HBE)为对照细胞,用Real-time PCR测定pHBE细胞氡染毒前后线粒体拷贝数,活细胞探针标记线粒体DNA数目、线粒体形态、线粒体膜电位以及细胞内活性氧(ROS)的水平,用Annexin V和PI染色测定细胞凋亡及细胞周期的改变。选用6种细胞：p+HBE、p+HBE染氡10代细胞(低剂量染氡组)、p+HBE染氡30代细胞(高剂量染氡组)、p-HBE、p-HBE染氡10代(低剂量染氡组p-HBE)以及p-HBE染氡30代细胞(高剂量染氡组p-HBE),采用NimbleGen-135K mRNA表达谱芯片和miRCURYTM芯片筛选差异表达的mRNA和miRNA,并用实时荧光定量PCR进行验证。
结果：用EB诱导30d后,p+HBE细胞中的线粒体拷贝数减少了约77%,细胞浆内mtDNA的数量显著减少,线粒体膜电位降低,同时在无尿嘧啶的培养液中细胞开始大量死亡,表明EB已成功诱导线粒体DNA缺失,建立了p-HBE细胞。经氡染毒后,p+HBE细胞生长速率加快,克隆形成率和存活分数明显下降,而p"HBE细胞的克隆形成率和存活分数却明显升高,提示线粒体DNA敲除后,p-HBE细胞更易受到氡暴露的损伤,可能促进细胞发生恶性转化。p-HBE细胞中ROS水平低于p+HBE细胞,经氡染毒后两种类型细胞中ROS水平都显著升高,但p-HBE细胞内ROS水平仅为p+HBE细胞的48%,提示除线粒体外,细胞内ROS的产生还有其他的来源。p-HBE细胞的线粒体膜电位经氡照射后显著下降,表明线粒体的膜结构和功能受到了严重损伤。p-HBE细胞染氡后的凋亡率有所升高但明显低于p+HBE细胞,提示线粒体缺失细胞增殖能力的增强与其凋亡功能的降低存在密切的相关。p-HBE细胞在高剂量氡染毒后S期细胞增加,G1和G2/M期细胞减少,提示发生了G1期和G2/M期阻滞,延缓细胞进入S期和M期的速度,导致细胞周期的延长。mRNA表达谱的分析结果显示,氡染毒致p+HBE细胞mRNA表达明显上调的有TFF1、TFF2和IGHG1等,表达明显下调的mRNA包括NCOA7,FN1, EGR1, IL-8, RASEF和BNDF等。GO通路分析表明,上调的]mRNA主要是影响信号传导通路,下调的基因70%以上影响蛋白质的结合和酶活性。在p-HBE细胞中,低剂量染氡组p-HBE上调变化最大的mRNA为SPRR家族中的SPRR2E、 SPRR2A、SPRR2D等,下调表达的基因包括HAND2和FCRLM1等。高剂量染氡组p"HBE上调变化最大的mRNA为BICC1、ASNA、KIAA1045等,表达下调的有GNAI3、TSGA2、BAX、PKP2、GNPDA1等。GO通路分析表明,这些上调和下调的mRNA主要功能是影响受体信号、信号传导以及蛋白质结合等通路。microRNA表达谱的分析结果显示,氡暴露致p+HBE细胞miRNA表达上调的有miR-107, miR-3651和miR-3074-3p等,表达下调的有miR-1227以及miR486-5p等。氡暴露致p-HBE细胞miRNA表达上调的有miR-1919-5p、miR-3157-3p和miR-484等,表达下调的有miR-3607-3p,miR-98和miR-1275等。
结论：(1)采用EB诱导法成功构建了mtDNA部分敲除的人支气管上皮细胞株(p-HBE),并通过观察细胞形态学、营养缺陷和活细胞染色及检测线粒体DNA拷贝数、线粒体膜电位的改变,了解了p-HBE细胞的生物学特性,为研究氡致线粒体DNA敲除细胞的损伤效应提供了细胞模型。(2)氡染毒后,p-HBE细胞在生长速度、ROS产生量、线粒体膜电位、细胞凋亡及细胞周期等生物学指标上均出现了明显改变,表明氡染毒后所产生的细胞损伤效应与线粒体的结构和功能障碍密切相关,为进一步从分子水平上探讨氡致肺癌的发生机制提供了基础资料。(3)通过高通量芯片筛选,发现了一批p+HBE与p-HBE细胞间以及氡染毒前后差异表达的mRNA和miRNA,对这些mRNA和miRNA进行深入的生物信息学分析,将有助于阐明在氡致肺癌的过程中,核基因组和线粒体基因组所起的不同作用,以及可能涉及的基因网络和细胞信号通路。
Objective:Both Epidemiological investigation and experimental research confirmed the correlation between exposure of high level of radon and lung cancer. Bronchial epithelial cells, as well as their nuclei, are the targets of radon and its progeny. Mitochondrial has been shown to play an important role in the regulation of bystander effects of a particle, and mitochondrial DNA is one of the targets of carcinogens. This study is to set up a model of human bronchial epithelial cells (HBE) with knockdown of mitochondrial DNA (p") to observe the adverse effects of radon irradiation, and to screen differentially expressed mRNA/miRNA beteen p+HBE and p" HBE cells upon radon exposure, in order to provide experimental data for exploring potential mechanisms underlying malignant transformation induced by radon.
Methods:p" HBE cells were generated by treatment of ethidium bromide (EB), and exposed to radon gas in a special inhalation chamber. The exposure concentration was set to20,000Bq/m3and lasted for20min each time and repeated in3days. Ten generations (Rn10) and30generations (Rn30) were exposed to radon, and the copy numbers of mitochondrial were determined by Real-time PCR, the number, morphology, membrane potential of mitochondrial and intracellular reactive oxygen species (ROS) were labeled by live cell probes. The alternation of apoptosis and cell cycle distribution were evaluated by Annexin V and PI staining. The NimbleGen-135K mRNA chip and miRCURYTM chip were used to reveal changes in mRNA and miRNA expression, and Q-PCR was performed to further confirm the data.
Results:Thirty days after the EB induction, the number of mitochondrial in p-HBE cells decreased77%of that in p+HBE cells. Intracellular mitochondrial DNA was reduced significantly and mitochondrial membrane potential (MMP) was decreased. After removal of uridine for3days, lots of cells could not survive, suggesting that the p"HBE cells were successfully established. Upon exposure to radon, the proliferation of p+HBE cells was accelerated, with decreased clonogenic rate and survival fraction. In p'HBE cells, the clonogenic rate and survival fraction were increased, implicating a more severe damage by radon. The ROS level in both cells were elevated by radon exposure, but the level in p-HBE cells was only48%of that in p+HBE cells. The membrane potential in p"HBE cells was lowered as a result of mitochondrial damage. The apoptotic rate of the p-HBE cells was also found lower than in the p+HBE cells, although both cells showed increased apoptosis when exposed to radon, suggesting a correlation between mitochondrial function and cell proliferation. DNA partial depletion could inhibit apoptosis. Cells in S phase increased and those in G1and G2decreased after radon exposure, indicating a G1and G2/M phase arrest and prolonged cell cycle. The screening of mRNA expression revealed an up-regulation of TFF1, TFF2and IGHG1, and down-regulation of NCOA7, FN1, EGR1, IL-8, RASEF and BNDF. The GO analysis showed that these mRNA mainly affect signal transduction and protein/enzyme activity. In radon exposed p+HBE cells, BICC1, ASNA, KIAA1045were up-regulated, and GNAB, TSGA2, BAX, PKP2and GNPDA1were down-regulated. In radon exposed p-HBE cells, SPRR2E, SPRR2A, and SPRR2D were up-regulated and HAND2and FCRLM1were down-regulated, with functions in receptor signal, signal transduction and protein synthesis. The screening of miRNA expression revealed an up-regulation of miR-107, miR-3651and miR-3074-3p, and a down-regulation of miR-1227and miR486-5p in radon exposed p+HBE cells. In radon exposed p-HBE cells, miR-1919-5p,miR-3157-3p and miR-484were up-regulated, and miR-3607-3p, miR-98and miR-1275were down-regulated.
Conclusions:
1. A p-HBE cell model was successfully established by EB induction. The copy numbers and membrane potential of mitochondria, intracellular reactive oxygen species, apoptosis and cell cycle were determined to characterize the p-HBE cells.
2. Upon radon exposure, indexes reflecting cell growth, ROS production, membrane potential of mitochondria, apoptosis and cell cycle were altered indicating a correlation between mitochondrial disfunction and the cell damage.
3. High-through chip screening revealed a set of differential expressed mRNAs and miRNAs between p+HBE and p-HBE cells and between pre-and after radon exposure. Bioinformatic analysis will help elucidate roles that nuclear DNA and mtDNA may play in signal transduction of radon induced lung cancer.
引文
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