摘要
目的探讨子宫颈癌发生与肿瘤组织代谢物及代谢途径关键酶表达变化的关系。方法收集子宫颈鳞癌、宫颈上皮内瘤变(CIN)及慢性宫颈炎(正常对照)新鲜及石蜡包埋组织标本,采用超高效液相色谱联合三重四级杆串联质谱(UHPLC-MS/MS)技术检测宫颈组织代谢组分,应用正交偏最小二乘判别分析法(OPLS-DA)对UHPLC-MS/MS数据进行模式识别分析,确定3种宫颈组织差异性代谢物组分,并应用代谢通路分析软件(Metabo Analyst和KEGG)综合分析,预测关键代谢通路。采用定量RT-PCR方法和免疫组织化学SP法检测关键代谢通路关键酶谷氨酸脱羧酶1(GAD1)、糖原合成激酶3(GSK-3)、丙酮酸激酶(PKM2)和肉毒碱棕榈酰基转移酶(CPT 1)的转录及蛋白表达情况。结果 3种宫颈组织之间有34种差异性小分子代谢物;代谢通路分析预测出18条关键代谢通路,其中能量代谢与宫颈癌发生可能最为密切;定量RT-PCR方法结果显示,宫颈癌组织内GAD1及GSK-3β表达水平明显降低(P<0.05),而PKM2及CPT1表达水平明显升高(P<0.05);免疫组织化学结果显示,从正常对照组织到CIN和宫颈癌,GAD1和GSK-3β表达水平明显下降(P<0.05),从强阳性和中等阳性逐渐向弱阳性和阴性变化,而PKM2和CPT1蛋白质表达水平明显上升(P<0.05),进一步验证了定量RT-PCR结果。结论宫颈癌发生伴有肿瘤组织内糖和脂肪酸分解代谢加剧,能量代谢增强,处于物质代谢紊乱的病理状态,其可能与关键代谢途径的限速酶表达调控存在密切关系。
Objective To explore the relationship between the progress of squamous cell carcinoma of the cervix and the regulation of metabolic network.Methods Fresh specimens and paraffin-embedded tissue slices form patients with cervical squamous cell carcinoma（CSCC）,cervical intraepithelial neoplasia（CIN）Ⅱ-Ⅲ and normal controls（NC）were collected.Ultra high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry（UHPLC-MS/MS）was performed to identify metabolites in the three group specimens.Orthogonal partial least-squares discriminant analysis（OPLS-DA）was used to model the systematic variation related to patients with CIN or CSCC with healthy controls.The metabolomics data was additionally analyzed by professional softwares recognizing metabolic pathways（KEGG;MetaboAnalyst 2.0）and online databases,to identify certain metabolic pathways and key enzymes associated with the metabolic regulation.Quantitative RT-PCR was used to reveal the association of cervical carcinoma with the mRNA expression level of candidate key enzymes.Meanwhile,the protein expression level of candidate key enzymes in paraffin-embedded tissue specimens were provided by the pathological tissue specimen bank.Results 34 small molecular metabolites were identified that were different in cervical carcinoma.This profile was comprehensively analyzed by professional softwares for recognizing metabolic pathways（MetaboAnalyst and KEGG）and predicted 18 key metabolic pathways,among which cervical carcinogenesis was the most probably associated with 4 key metabolic pathways,the energy metabolism may be the most closely related to the occurrence of cervical cancer.By analysis of transcriptions for candidate genes coding these key enzymes by quantitative RT-PCR,the results showed that the expression of PKM2 and CPT1 was significantly upregulated,and GAD1 and GSK-3βdownregulated,respectively,in tissue specimens of cervical carcinoma compared with normal controls（P〈0.05）.In immunohistochemical staining analysis further confirmed the result of quantitative RT-PCR,the protein expression of GAD1 and GSK-3βwas gradually decreased,and PKM2 and CPT1 increased,respectively,from normal controls to precancerous lesions（CIN Ⅱ-Ⅲ）and cervical carcinoma with significant differences（P〈0.05）.Conclusion The occurrence of cervical cancer is accompanied by increased glucose and fatty acid catabolism and increased energy metabolism in the tumor tissue.It is in a pathological state of substance metabolism disorder.It may be closely related to the expression of speed limiting enzymes in key metabolic pathways.
作者
阿仙姑.哈斯木
努尔满古力.肉孜
徐丽秀
哈提拉.吐尔逊
马俊旗
Axiangu Haimu;Nuermangu Rouz;XU Lixiu;Hatila Tuerxun;MA Junqi(Department of Pathology, School of Pre clinical Medicine, Xingjiang Medical University Urumqi 830011, China;Department of Gynaecology of the People＇s Hospital of Xinjiang Autonomous Region, Ururnqi 830000, China;Department of Gynecology, the First Affiliated Hospital of Xinjiang Medical University, Ururnqi 830054, China)
出处
《新疆医科大学学报》
CAS
2018年第5期521-527,533,共8页
Journal of Xinjiang Medical University
基金
国家自然科学基金(81360332)
关键词
宫颈癌
能量代谢
组织代谢组分
关键酶
cervical cancer
energy metabolism
tissue metabolism component
key enzymes
