Potential Mechanism of Thioridazine in Anti-cervical Cancer
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摘要:
目的 运用生物信息学分析, 探讨硫利达嗪抗宫颈癌的潜在作用机制。 方法 通过PharmMapper工具预测能够与硫利达嗪相互作用的靶基因,然后使用STRING在线工具对靶基因进行通路和组织表达富集分析。使用GeneCards和DisGeNET数据库筛选宫颈癌相关基因,与硫利达嗪的靶基因取交集,得到硫利达嗪可能作用于宫颈癌的交互基因。通过STRING构建蛋白互作(PPI)网络,推测核心靶点,评估其重要性。使用clusterProfiler软件进行GO和KEGG通路分析。 结果 通过PharmMapper预测得到47个靶基因,富集到肿瘤相关通路和宫颈癌细胞。与669个宫颈癌基因取交集,获得硫利达嗪和宫颈癌交互基因21个,其中10个关键节点基因为EGFR、PPARG、AR、NOS3、ALB、ESR1、MAPK1、MAPK14、ANXA5和MAPK8,且在宫颈癌PPI网络中处于重要位置。交互基因涉及到的生物进程主要有负离子转运的正调控、丝氨酸肽基磷酸化、类固醇代谢过程、血液凝固、细胞对化学应激的反应等。富集的KEGG通路包括调控癌症通路、松弛素信号通路、内分泌耐药、蛋白聚糖与肿瘤、细胞衰老、GnRH信号通路和VEGF信号通路等。 结论 硫利达嗪具有潜在的抗肿瘤作用,可能通过多靶点和多信号通路的方式在宫颈癌的治疗上发挥作用。 Abstract:Objective To explore the potential mechanism of thioridazine in the treatment of cervical cancer by bioinformatics analysis. Methods The target genes that can interact with thioridazine was predicted by PharmMapper, and then conducted pathway and tissue expression enrichment analysis by STRING online tool. The cervical cancer related genes were screened through GeneCards and DisGeNET databases, and cross the target genes of thioridazine to obtain the interaction genes that thioridazine may act on cervical cancer. The protein-protein interaction(PPI) network was constructed using STRING, and the core targets were speculated and evaluated their importance.The GO and KEGG enrichment analysis were conducted using clusterProfiler package. Results A total of 47 target genes were predicted by PharmMapper, which were enriched in tumor-related pathways and cervical cancer cells. Intersection with 669 cervical cancer genes, 21 common genes were obtained, of which 10 key genes were EGFR, PPARG, AR, NOS3, ALB, ESR1, MAPK1, MAPK14, ANXA5 and MAPK8. These key genes were important in the cervical cancer PPI network. The biological processes involved in interactive genes mainly include positive regulation of anion transport, peptidyl-serine phosphorylation, steroid metabolic process, blood coagulation, and cellular response to chemical stress. Enriched KEGG pathways include pathways in cancer, relaxin signaling pathway, endocrine resistance, proteoglycans in cancer, cellular senescence, GnRH signaling pathway, and VEGF signaling pathway. Conclusion Thioridazine has potential anti-tumor effects and may play a role in the treatment of cervical cancer through multiple targets and multiple signaling pathways. -
Key words:
- Thioridazine /
- Cervical cancer /
- Target /
- Action mechanism
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射频电磁场(radiofrequency electromagnetic field,RF)是指频率在100 kHz~300 GHz的电磁场,主要由手机、基站等通信设备产生[1]。相关研究报道,长期的射频电磁场暴露可能对生物体的神经系统、消化系统、血液系统、生殖系统等方面均有不同程度的影响[2]。机体大脑是对射频电磁场敏感的器官之一[3],脑组织受到射频电磁场后,会出现学习和记忆功能的改变,这是射频电磁场生物学效应的重点研究领域之一。胶原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)是星形胶质细胞中特异性的中间丝蛋白,随着生长发育过程发生变化,并在神经退行性病变过程中发生相应改变[4]。神经细胞黏附分子(neural cell adhesion molecule,NCAM)是成人神经可塑性的主要贡献者,可能参与中枢神经系统损伤修复、神经发育以及与年龄相关的认知功能下降等过程[5-6]。γ-氨基丁酸(gamma-amino butyric acid,GABA)是中枢神经系统内重要的抑制性神经递质,许多研究都已经证实GABA与认知、学习记忆密切相关[7-8]。本研究通过观察SD大鼠暴露后的体重及其海马内的GFAP、NCAM和GABA受体的变化,评估射频电磁场对大鼠的生理发育指标和海马体的影响。
1. 材料与方法
1.1 动物分组
14只7周龄SPF级SD大鼠,雄鼠体重均在250~270 g,雌鼠体重均在200~220 g,由上海斯莱克实验动物公司提供[动物许可证号:SCXK(沪)2017-0005]。适应环境1周后,分为暴露组和对照组,每组7只。暴露组雌雄比例3∶4,对照组雌雄比例4∶3。每天暴露时间固定12 h(20:00~8:00),持续3周。
1.2 暴露装置
射频电磁场暴露装置如之前研究所描述[9],由惠普公司生产的信号发生装置(8614A Signal Generator 0.8~2.4 GHz)连接1个放大器(SCD Amplificateur Lineaire 1.3~2.6 GHz Made France)产生频率
1800 MHz微波,功率密度为0.5 mW/cm2。1.3 主要仪器与试剂
兔抗鼠GFAP多克隆抗体(武汉博士德公司),山羊抗兔SP免疫组化试剂盒和DAB染色剂(福州迈新公司),多聚甲醛、明胶等免疫组化实验常规试剂均为分析纯,8592C频谱分析仪(惠普,美国),Model7620微波辐射测试仪(Narda,美国),8614A信号发生装置、信号放大器(惠普,美国),TX-B8H WiFi
2400 MHz 发射装置(深圳市特信电子有限公司,中国),CM1900冰冻切片机(德国莱卡公司),90i摄影生物显微镜(日本尼康公司),Image Pro Plus 6.0病理图像分析系统(美国 Media Cybernetics 公司)。1.4 大鼠海马组织CA1区、CA3区、DG区GFAP表达的测定
1.4.1 取材
大鼠用戊巴比妥钠溶液腹腔注射麻醉,解剖后经主动脉插管,用4%多聚甲醇溶液(pH = 7.4)灌流固定,断头取脑,浸入装有多聚甲醇溶液的容器中,放在4 ℃冰箱中后固定
1.4.2 组织切片
将固定好的脑组织块应用梯度蔗糖溶液脱水至脑组织沉底,随后将大鼠海马组织从脑组织中分离,再用冰冻切片机将海马组织沿长轴方向行20 μm厚的连续冠状切片,每个海马组织取20~30张切片,将切片放入0.01 ml/L磷酸盐缓冲液中孵育保存并尽快测定。
1.4.3 免疫组化染色
使用免疫组化反应链霉菌抗生物素蛋白-过氧化物酶法(streptavidin-peroxidase method,SP法)进行染色,按说明书操作。用PBS冲洗,加一抗(1∶
4000 )4 ℃过夜。再次用PBS冲洗,二抗37 ℃ 2 h。常规脱水,透明,封片。1.4.4 图像及分折
每张切片分别在海马CA1区、CA3区、DG区随机选取2个视野摄片,每张切片拍摄6次。拍片时均使用研究级显微镜,预实验找到最佳拍摄条件后,所有标本均按此条件进行拍片。拍摄完成后用IPP6.0图像分析软件进行图片分析,测定GFAP、NCAM、GABA阳性染色平均光密度值(mean optical density,MOD),然后求6个视野所测MOD值的平均值,此平均值即为该区域的最终MOD值。
1.5 统计学处理
应用SPSS17.0录入数据进行分析,对数据进行正态性检验。如数据符合正态分布,采用独立样本t检验进行比较,数据采用均数±标准差( $ \bar x \pm s $)表示,P < 0.05差异有统计学意义。
2. 结果
2.1 一般结果
各组未发现老鼠出现死亡和其他异常情况,外观正常。各组体重指标经统计分析,差异无统计学意义(p > 0.05),2组大鼠的体重无明显不同,见 表1。
表 1 大鼠一般情况分析( $ \bar x \pm s $)Table 1. General situation analysis of rat ( $ \bar x \pm s $)分组 动物数(n) 性别(雌/雄) 体重(g) t/P 暴露组 7 3/4 223.50 ± 35.94 t= −0.49 对照组 7 4/3 215.57 ± 34.51 P= 0.633 2.2 GFAP在大鼠海马各区的表达
GAFP在大鼠海马CA1区、CA3区和DG区均有表达,阳性免疫组化染色结果呈褐色或棕褐色,形似蜘蛛,见图1。
2.2.1 GFAP在大鼠海马各区MOD结果比较
海马各区GFAP经统计分析,与对照组相比,CA1区、DG区差异有统计学意义(P < 0.05),CA3区无统计学意义( P > 0.05)。经射频电磁辐射21 d,暴露组大鼠海马CA1区、DG区GFAP的表达下降,见 表2。
表 2 大鼠海马不同区域的GFAP表达MOD值( $ \bar x \pm s $)Table 2. MOD value of GFAP expression in different regions of rat hippocampus ( $ \bar x \pm s $)分组 动物数(n) CA1区 CA3 DG区 暴露组 7 0.07 ± 0.06* 0.11 ± 0.05 0.07 ± 0.06* 对照组 7 1.50 ± 0.52 0.05 ± 0.06 1.50 ± 0.51 t = −3.349 t = −1.894 t = −3.809 P = 0.006 P = 0.083 P = 0.002 与对照组相比,*P < 0.05。 2.3 NCAM在大鼠海马各区的表达
NCAM受体在海马CA1区、CA3区及DG区均有表达,阳性表达免疫组化呈棕色或棕褐色,形状为颗粒状,见图2。
2.3.1 NCAM免疫组织MOD结果比较
NCAM表达的海马各区MOD值经统计分析分析,差异无统计学意义(P > 0.05)。经射频电磁辐射21 d,暴露组大鼠海马各区NCAM的表达无改变,见 表3。
表 3 大鼠海马不同区域的NCAM表达MOD值( $ \bar x \pm s $)Table 3. MOD value of NCAM expression in different regions of rat hippocampus( $ \bar x \pm s $)分组 动物数(n) CA1区 CA3 DG区 暴露组 7 0.05 ± 0.03 0.03 ± 0.02 0.05 ± 0.02 对照组 7 0.04 ± 0.03 1.50 ± 0.52 0.06 ± 0.03 t = 0.591 t = −0.200 t = −1.100 P = 0.565 P = 0.854 P = 0.293 2.4 GABA在海马各区的表达
GABA 受体在海马CA1区、CA3区及DG区均有表达,阳性表达免疫组化呈棕色或棕褐色,形状为颗粒状,见图3。
2.4.1 GABA免疫组织MOD值比较结果
GABA表达的海马各区MOD值经统计分析结果如下,差异无统计学意义(P > 0.05)。经射频电磁辐射21 d,暴露组大鼠海马各区NCAM的表达无改变,见 表4。
表 4 大鼠海马不同区域的GABA表达MOD值( $ \bar x \pm s $)Table 4. MOD value of GABA expression in different regions of rat hippocampus ( $ \bar x \pm s $)分组 动物数(n) CA1区 CA3 DG区 暴露组 7 0.07 ± 0.04 0.07 ± 0.03 0.03 ± 0.02 对照组 7 0.05 ± 0.04 0.04 ± 0.02 0.05 ± 0.05 t = 0.550
t = 1.662 t = −0.870 P = 0.593 P = 0.131 P = 0.402 3. 讨论
本次实验选择的GFAP、NCAM、GABA受体变化可反应RF-EMF对脑部的损伤程度暴露条件,模拟人群所处的
1800 MHz射频电磁场环境,进行全身暴露。经过3周,12 h/d的暴露后,2组大鼠的形态学未发现改变,大鼠海马CA1区、DG区GFAP的表达下降。海马体是哺乳动物中枢神经系统的重要组成部分,是学习记忆功能的结构基础[10-14]。Maskey等[15]将大鼠暴露于835 MHz射频电磁场中,暴露1个月后,海马CA1区神经变性。在Altum等[16]的报告中,在射频电磁场暴露15 d,海马CA1区和CA2区神经元明显减少,而CA3区无明显差异。有报道,每天通过手机产生的900 MHz射频电磁场,动物暴露此环境15 d后,在海马CA1、CA3和齿状回亚区出现神经变性[17]。由此,笔者推测射频电磁场可能海马的CA1、CA3和DG区有所影响。
星形胶质细胞能够调节脑内的离子和神经递质的稳态,具有代谢功能,并在脑损伤时做出反应[18]。GFAP与阿尔茨海默病(alzheimer’s disease,AD)、抑郁症、中风和脑缺血等脑部疾病有关[19-21]。相关研究发现,将大鼠暴露于高强度射频电磁场后发现,脑内GFAP含量增加并对记忆功能有所影响[22]。国内研究发现,将50只雄性大鼠暴露在高功率的射频电磁场后发现,大鼠的学习记忆明显降低,星形胶质细胞中的GFAP含量增加[23]。因此,GFAP含量的增多可能会导致学习和记忆功能下降。NCAM与胶质细胞瘤、一氧化碳中毒后迟发性脑病和痴呆等疾病有关[24-26],同时也参与了学习记忆的过程。研究发现,
1800 MHz电磁波,度值为0.5 m W/cm2的暴露下可致出生后SD大鼠海马NCAM表达下调,并且NCAM的下调可能是低剂量电磁波影响学习记忆的原因之一[27-28]。γ-氨基丁酸是一种天然存在的非蛋白组成氨基酸,具有极重要的功能,与认知功能存在一定的关系。早期便有研究者证明了在一定条件的电磁辐射暴露下,GABA受体表达较对照组增高进而可能影响人的神经行为[29]。另外,在强度为2450 MHz,功率密度为为5.0、10.0 mW/cm2的条件下,研究者发现昆明小鼠的GABA含量增高,学习记忆损伤逐渐加重[30]。在其他领域也证实,低功率密度微波辐射可以通过增加GABA、降低谷氨酸对小鼠的学习记忆产生损害[31]。本研究结果表明,大鼠暴露于射频电磁场21 d后,大鼠海马CA1区、DG区GFAP的表达下降。根据相关的研究,笔者认为,在本实验条件下,
1800 MHz射频电磁场暴露可对海马CA1和DG区的GFAP表达有影响,本实验只进行了初步探索,在同样条件是否对海马CA1区、CA3区以及DG区的GFAP、NCAM和GABA受体表达产生影响,还需要进一步的研究。 -
表 1 排名前10的靶基因通路分析结果
Table 1. Top10 pathways results of target genes
ID号 名称 基因数 FDR hsa04926 松弛素信号通路 9 1.50E-08 hsa05200 肿瘤通路 13 4.88E-08 hsa01522 内分泌耐药 7 6.70E-07 hsa05205 蛋白聚糖与肿瘤 8 3.01E-06 hsa05166 人类T细胞白血病
病毒1感染8 4.18E-06 hsa04218 细胞衰老 7 6.70E-06 hsa04912 促性腺激素释放
激素信号通路6 6.70E-06 hsa04914 孕酮介导的卵
母细胞成熟6 7.36E-06 hsa05161 乙型肝炎 7 7.36E-06 hsa04933 糖尿病并发症中
的AGE-RAGE信号通路6 7.85E-06 表 2 排名前10的靶基因疾病富集分析结果
Table 2. Top10 disease enrichment results of target genes
ID号 名称 基因数 FDR DOID:162 肿瘤 12 0.0022 DOID:14566 细胞增殖疾病 13 0.0022 DOID:4 疾病 31 0.0022 DOID:65 结缔组织疾病 10 0.0064 DOID:7 解剖实体疾病 25 0.0064 DOID:0050636 家族性内脏
淀粉样变性3 0.0183 DOID:28 内分泌系统疾病 7 0.0183 DOID:0080001 骨疾病 8 0.0184 DOID:11801 蛋白质-能量营
养不良2 0.0184 DOID:0060075 雌激素受体
阳性乳腺癌2 0.0244 表 3 排名前10的靶基因组织表达富集分析
Table 3. Top10 tissue expression enrichment results of target genes
ID号 名称 基因数 FDR BTO:0000180 宫颈癌细胞 10 2.03E-06 BTO:0000174 胚胎结构 22 2.03E-06 BTO:0000759 肝 20 2.71E-06 BTO:0000132 血小板 10 4.54E-06 BTO:0001078 胎盘 16 4.54E-06 BTO:0001546 慢性淋巴细胞
白血病细胞8 8.77E-06 BTO:0001489 全身 46 9.93E-06 BTO:0000345 消化腺 22 1.02E-05 BTO:0001491 脏器 30 1.07E-05 BTO:0000522 腺体 34 1.77E-05 表 4 关键靶基因在宫颈癌PPI网络中的排名
Table 4. Ranking of key target genes in the cervical cancer PPI network
靶基因 Degree值 排名 EGFR 329 6 ALB 271 13 ESR1 255 20 ANXA5 200 32 PPARG 182 37 AR 159 53 MAPK1 143 72 MAPK14 141 77 MAPK8 138 79 NOS3 105 127 合计 525 -
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