宫颈癌细胞和正常上皮细胞对锌离子胁迫耐受性差异的机制研究

中文摘要

背  景   

锌做为一种可降解医用金属受到越来越广泛的关注。锌属于过渡金属元素，其原子轨道外层的两个电子容易被氧捕获形成活性氧自由基ROS。正常细胞发生恶性转化后，低氧环境和Warburg效应代谢特征使癌细胞具有相对较高的ROS水平基础，以适应调节肿瘤细胞的无限制增生与侵袭转移等恶性特征。ROS同时是一把双刃剑，当其水平超过细胞的耐受极限，可以引起细胞死亡，目前许多化疗药便是利用这一特征来杀伤肿瘤细胞。锌基生物材料植入人体后的最终腐蚀降解产物为锌离子，利用锌离子引发活性氧自由基增高进而杀伤肿瘤细胞，这对于拓展锌基生物材料的临床应用范围具有重要意义。

目  的 

比较研究肿瘤细胞与正常细胞对锌离子胁迫产生耐受性差异的生物学机制，为实现锌基生物材料的潜在临床肿瘤治疗应用提供相应的基础理论。

方  法  

首先，通过CCK8检测比较分析外源性锌离子干预下人体不同肿瘤细胞和正常细胞的活度变化，确立锌离子具有杀伤人体多种肿瘤细胞的表型特征。然后，选择宫颈癌细胞为研究对象，通过锌离子荧光探针FluoZin^TM-3，ROS荧光探针DCFHDA，流式细胞术建立Zn²⁺-ROS-Regulated Cell Death关联分析，然后通过AnnexinV PI FITC检测细胞凋亡情况并利用实时荧光定量（qPCR）检测锌转运蛋白家族、氧化还原系统和细胞死亡相关重要基因mRNA水平分子变化，推测锌引起细胞死亡的可能途径及机制；通过检测氧化应激关键指标如氧化还原调节分子、抗氧化酶、ROS与抑制ROS能力，分析锌离子导致宫颈癌细胞ROS升高的重要机理；最后,通过转录组测序分析,提出Zn²⁺-ROS-Ferroptosis信号通路假设，进而通过多种抑制剂干预、qPCR、蛋白免疫印迹（Western blot）、免疫组化和RNA干扰等方法，找到锌离子诱导铁死亡的关键性分子，进一步探索锌离子引起宫颈癌细胞发生铁死亡的主要信号通路。

结  果

第一部分  锌离子作用下宫颈癌细胞和正常上皮细胞的表型变化

1. 160μM锌离子，可诱导人生殖、消化和呼吸系统源性12种癌细胞发生死亡。

2. 宫颈癌细胞的ROS基础水平较高，外源性锌离子干预下，宫颈癌细胞的ROS水平随其作用时间延长呈现明显升高趋势，而正常细胞则无明显变化。

3. 锌离子促进宫颈癌细胞锌转运蛋白Zip4、ZnT1的mRNA转录上调，ZnT7则转录下调，而宫颈正常上皮细胞中， Zip1、Zip4、Zip6、Zip7、Zip9、Zip10、Zip14、ZnT1、ZnT4和ZnT7的mRNA均转录上调。

4. 宫颈正常上皮细胞发生恶性转化即正常上皮组织向癌前病变与浸润癌进展的过程中，临床宫颈组织标本免疫组化结果显示：恶性转化细胞的Zip4表达不断升高，ZnT1逐渐降低，提示发生恶性转化的宫颈上皮细胞对锌的依赖性增强。

第二部分  锌离子作用下宫颈癌细胞和正常上皮细胞的氧化应激指标改变

1. 锌离子导致宫颈癌细胞的谷胱甘肽GSH不断耗竭，而宫颈正常上皮细胞的GSH含量不断增加，这是锌离子引起宫颈癌细胞ROS水平不断升高的关键原因之一。

2. 宫颈癌细胞锰超氧化物歧化酶（SOD2）、谷胱甘肽过氧化物酶（Gpx）、过氧化氢酶（CAT）和硫氧蛋白还原酶（TrxR）等抗氧化酶的活性均低于宫颈正常上皮细胞；锌离子作用下，宫颈癌细胞的SOD2、CAT与TrxR活性水平不断上升，这表明锌离子引起宫颈癌细胞O2^.-和H₂O₂等活性氧自由基成分不断升高，进而引起抗氧化酶活性不断升高以中和消除其不断产生的ROS。

3. 锌离子作用下，宫颈癌细胞抑制OH^•-能力下降十分显著（P<0.01），其抑制OH^•-能力约是正常上皮细胞的一半，这间接表明OH^•-升高是锌离子导致宫颈癌细胞死亡的重要原因。

4. 临床宫颈组织标本免疫组化结果显示：Keap1表达逐渐升高，Nrf2表达降低，说明发生恶性转化的宫颈上皮细胞抗氧化能力下降。

第三部分  宫颈癌细胞和正常上皮细胞转录组测序分析及铁死亡分子机制研究

1. 在无外源性锌离子胁迫情况下，转录组学测序结果显示：与宫颈正常上皮细胞相比，宫颈癌细胞的铁死亡相关分子COX2、ALOX15、ACSL4的mRNA转录水平均升高2倍以上，Keap1升高近50 倍, 细胞膜铁出胞转运蛋白Ferroportin（FPN1）降低10倍。

2. 锌离子作用下，KEEG富集通路分析显示：铁死亡信号通路关键分子HO-1、SLC7A11、TF、Ferritin、GCL和ACSL4均明显表达上调，其中HO-1在4h和8h分别上调约50和30倍。提示HO-1可能是锌诱导宫颈癌细胞发生铁死亡的关键信号分子。

3. 添加铁离子螯合剂（DFO）、铁死亡抑制剂（Fer-1）和维生素E，均可使细胞死亡受到明显抑制；添加坏死抑制剂（Nec1），部分细胞死亡受到抑制，提示部分细胞通过坏死途径死亡；添加凋亡抑制剂（Z-VAD）和自噬抑制剂（Apocynin），细胞死亡均未受抑制。这提示，锌离子引起的宫颈癌细胞死亡可能主要为铁死亡。

4. qPCR和WB结果显示锌离子导致TF、HO-1、COX2和ALOX15等铁死亡关键分子的转录表达均明显上调，细胞膜铁出胞转运蛋白FPN1的mRNA转录水平则显著下调，这些分子可能是锌离子引起宫颈癌细胞发生铁死亡的重要分子。

5. HO-1siRNA实验结果显示：HO-1被干扰后，锌离子作用下宫颈癌细胞的ROS水平明显降低，这说明HO-1基因在锌离子诱导宫颈癌细胞ROS升高方面起着关键作用。

6. 临床宫颈组织组织标本免疫组化结果显示：在宫颈正常上皮细胞发生恶性转化过程中，ALOX15、ALOXE3和COX2等促进铁死亡的信号分子表达升高；TF表达升高，FPN1表达降低，提示发生恶性转化的宫颈上皮细胞对铁的依赖性升高；SLC7A11和Gpx4表达升高，提示恶性转化细胞的抗氧化能力增强以适应其自身较高的氧化应激水平。

结论  宫颈癌Siha细胞和宫颈正常上皮HcerEpic细胞对锌离子胁迫的耐受性存在差异，锌离子导致宫颈癌细胞内GSH耗竭和ROS生成增多是其杀伤宫颈癌细胞的关键机制，并通过Zn²⁺-Keap1–Nrf2-HO-1通路诱导癌细胞铁死亡。

Study on the mechanism of the difference of tolerance stressed by zinc ions between cervical cancer cells and normal epithelial cells                           Abstract

Background  Zinc could be used as biodegradable medical material, and is gaining more attention now. Zinc is a transition metal element. Two electrons in the outer layer of its atomic orbital are easily captured by oxygen to form ROS.After malignant transformation of normal cells, hypoxia environment and Warburg effect metabolism features enable cancer cells to have relatively high ROS level basis, so as to adapt to malignant characteristics such as unlimited proliferation, invasion and metastasis of tumor cells.However, ROS is also a double-edged sword. When ROS exceeds the tolerance limit, it can cause cell death. At present, many chemotheraputic drugs utilize ROS to eradicate tumor cells. The final degradation products of zinc-based biomaterials implanted into human body are zinc ions, which can cause the increase of active oxygen free radicals and then kill cancer cells, which is of great significance for expanding the clinical application range of zinc-based biomaterials.

Objective  To study the biological mechanism of the difference of tolerance to zinc ions stress between tumor cells and normal cells,providing the basic theory for the potential clinical application of zinc-based biomaterials.

Methods  Firstly, the CCK8 assays were used to compare and analyze the activity changes of different tumor cells and normal cells lines under the intervention of exogenous zinc salts. Zinc ions were established with the phenotype of killing various tumor cells in human body. Secondly, cervical cancer cells was selected as the research object, a correlation analysis of Zn²⁺-ROS-Regulated Cell Death was established through zinc ion fluorescent probe FluoZin^TM-3, ROS fluorescent probe DCFHDA, flow cytometry. Thirdly, AnnexinV PI FITC was adopted to detect the apoptosis of cells and real-time fluorescence quantitative PCR(qPCR) was used to observe the molecular changes of zinc transporter family, redox system as well as mRNA level of candidate genes related to cell death, speculating the possible ways and causes of zinc-induced cell death. Fourthly, the key indicators of oxidative stress such as redox regulation molecule, antioxidant enzymes, ROS and their ability to inhibit ROS were detected to analyze the mechanism for the soaring ROS in cervical cancer cells induced by zinc ions. Lastly, through transcriptome sequencing analysis, the hypothesis of Zn²⁺-ROS-Ferroptosis signaling pathway was proposed. With a variety of inhibitors intervention, qPCR and Western blot (WB), immunohistochemistry (IHC) and RNA interference experiments, key molecules of zinc-induced ferroptosis were identified, furtherly exploring the main signaling pathway of ferroptosis in cervical cancer cells stressed by zinc ions.

Results

Part I  Phenotype changes of cervical cancer cells and normal epithelial cells under the stress of zinc ions

1. Under the stress of zinc ions (160μM), cell death was induced in 12 kinds of cancer cells derived from human reproduction, digestion and respiratory system.

2. The basal level of ROS in cervical cancer cells is high. Under the intervention of exogenous zinc ions, the ROS level of cancer cells increased significantly with the time of zinc ions, compared with no significant change in normal cells.

3. Zinc ions enhanced the mRNA transcription of Zip4 in cervical cancer cells, the mRNA level of ZnT1 was up-regulated, ZnT7 was down-regulated, and in normal cervical epithelial cells, all of the mRNA level of Zip1, Zip4, Zip6, Zip7, Zip9, Zip10, Zip14, ZnT1, ZnT4 and ZnT7 showed a significant increase.

4. In the process of malignant transformation of normal cervical epithelial cells, which refers to the progress of precancerous lesions and invasive cancer, the IHC results of clinical cervical tissue samples suggested that the expression of Zip4 level was diminished and the ZnT1 level was decreased in malignant transformed cells, indicating that the dependence of the malignant transformed cervical epithelial cells on zinc was increased.

Part II  Oxidative stress changes of cervical cancer cells and normal epithelial cells under the stress of zinc ions

1. Zinc ions led to the continuous depletion of GSH in cervical cancer cells, while normal cells showed continuous increase of GSH under the stress of zinc ions, indicating that zinc ions are the trigger to the continuous increase of ROS in cervical cancer cells.

2. The activity of antioxidant enzymes such as manganese superoxide dismutase (SOD2), glutathione peroxidase (Gpx), catalase (CAT) and thioredoxin reductase (TxrR) in cervical cancer cells are significantly lower than normal epithelial cells. Under the stress of zinc ion, the activity levels of SOD2, CAT and TrxR in cervical cancer cells were increased, which indicated that zinc ions may stimulate the increase of reactive oxygen radicals such as O2^.- and H₂O₂ in cervical cancer cells, enhancing activity of antioxidant enzymes to neutralize and eliminate ROS produced by cervical cancer cells.

3. Under the stress of zinc ions, cervical cancer cells showed a significant decrease in the ability to inhibit OH^•-(P<0.01), and such OH^•- inhibiting ability was half of normal epithelial cells, suggesting that the increase in OH^•- induced by zinc ions was the important mechanism of cervical cancer cell death.

4. The IHC results of clinical cervical tissue samples suggested Keap1 expression gradually increased and Nrf2 decreased in cervical cancer cells, indicating that the cervical cancer cells had reduced antioxidant capacity.

Part III  Transcriptome sequencing analysis of cervical cancer cells and normal epithelial cells as well as the molecular mechanism of ferroptosis

1. In the absence of exogenous zinc stress，the results of transcriptomics sequencing showed that the mRNA transcription levels of the related ferroptosis molecules such as COX2, ALOX15, and ACSL4 in cervical cancer cells were 2 times higher than that of normal cervical epithelial cells. Keap1 increased by nearly 50 times, the membrane iron transport protein FPN1 decreased by nearly 10 times, comparing with normal cevical cells.

2. Under the stress of zinc ions, KEEG enrichment pathway analysis showed that the key molecules of ferroptosis signaling pathway such as HO-1, SLC7A11, TF, Ferritin, GCL and ACSL4 were significantly up-regulated, among which HO-1 was at 4, 8h up-regulated by approximately 50 and 30 times respectively. It was suggested that HO-1 is a key signaling molecule for zinc ion to induce ferroptosis in cervical cancer cells.

3. The addition of iron chelating agent (DFO), ferroptois inhibitor (Fer-1), and vitamin E could significantly inhibit cell death. When adding necrosis inhibitor (Nec1), cell death was partially inhibited, suggesting that some cells died through the necrosis pathway under the stress of zinc ions. If apoptosis inhibitor (Z-VAD), or autophagy inhibitor (Apocynin) were added, cell death was not inhibited. This suggests that the death of cervical cancer cells stressed by zinc ions may be mainly through ferroptosis.

4. QPCR and WB results suggested that zinc ions significantly enhance TF, HO-1, NOX1, COX2, ALOX15 and other ferroptosis molecules transcription expression levels, while FPN1 transcription expression was diminished. These markers may be the important molecules triggering ferroptosis in cervical cancer cells.

5. The results of RNA interference assay showed that after HO-1 was interfered, the ROS level under zinc ion stress was significantly reduced, which indicated that the HO-1 gene played a key role in the zinc ion-induced increase of ROS in cancer cells.

6.The IHC results of clinical cervical tissue specimens showed that during the malignant transformation of normal cervical epithelial cells, the expression of the ferroptosis signaling molecules ALOX15, ALOXE3 and COX2 increased; the expression of TF increased, and the expression of FPN1 decreased, suggesting that malignant transformation of cervical epithelial cells have an increased dependence on iron; The increase of SLC7A11 and Gpx4 expression, suggested that malignant transformed cells could enhance antioxidant capacity to adapt to their own higher levels of oxidative stress.

Conclusion  Cervical cancer cells-Siha and normal epithelial cells-HcerEpic had different tolerances stressed by zinc ions. Zinc ions caused GSH depletion and increased ROS production in cervical cancer cells, which was the key mechanism of inducing  ferroptosis through Zn²⁺-Keap-Nrf2-HO-1 pathway.