HPV感染及宫颈鳞癌的共性代谢调控网络及分子标志物研究

Cervical cancer is a kind of gynecologic malignancy, in which HPV infection is an acknowledged risk factor. In spite of the fact that a tumor tissue is restricted to a certain organ, cancer is believed to be a disease that the whole body of patient is switched to a pathological state, in which the body is functionally unable to defeat or remove tumor cells. This suggest a dynamic change of metabolic regulatory network of whole body or tumor tissue, which can be our base of studying the mechanism of tumorigenesis with metabolomics approach. Our previous study shown that there are differences in serum metabolomic profile among cervical cancer patients, precancerous disease patients and normal control subjects, but the molecular mechanism of how tumorigenesis is associated with certain metobolic pathways remains unclear.In this project, we will analyze the association of carcinogenesis and progression of cervical cancer with the change in metabolic regulatory networks by analyzing tissue-specific differential metabolomic profile of various groups and evaluating candidate metabolic marker, with the aid of high-resolution magic angle spinning nuclear magnetic resonance and high performance liquid chromatography. And we will analyze the interaction between metabolic marker and HPV infection by selective inhibition of HPV16 gene expression by RNAi in cervical cancer cells. With these results, we will have a breakthrough understanding of cervical cancer tumorigenesis and HPV-associated initiation of cancer on the metabolic regulatory level. This will aid to improve the early diagnosis index system of cervical cancer, which is of great significance in prevention and treatment of cervical cancer in Xinjiang.

宫颈癌是妇科恶性肿瘤，HPV是公认的危险因素。宫颈癌发生不仅是局部组织病变，也是患者整体上进入病理状态的结果，可反映整体或局部代谢调控网络的动态变化，此正是代谢组学研究肿瘤发病机制的切入点。前期研究中，我们通过血浆代谢谱研究，发现维吾尔族宫颈癌共性代谢组分，但只能间接认识肿瘤发生与特定代谢通路的关系。为深入了解宫颈癌组织代谢调控机制，本项目拟依托磁共振及高效液相色谱技术平台，利用宫颈癌及癌旁组织标本，通过宫颈癌组织特异代谢组谱分析、代谢标记物发现及关键酶活性测定，探讨宫颈癌发生与细胞代谢调控网络改变关系。以RNAi选择性抑制宫颈癌细胞HPV16基因表达及代谢组学分析，研究HPV感染特异性改变的细胞代谢调控网络。以此，发现HPV感染宫颈癌特异的代谢标记物群、关键酶及代谢途径，从代谢调控水平阐释宫颈癌发病机制，寻找HPV致癌生物学规律，丰富宫颈癌诊断指标体系，对提高新疆宫颈癌防治水平有重要意义

宫颈癌是妇科恶性肿瘤，HPV是公认的危险因素。宫颈癌发生不仅是局部组织病变，也是患者整体上进入病理状态的结果，可反映整体或局部代谢调控网络的动态变化，此正是代谢组学研究肿瘤发病机制的切入点。前期研究中，我们通过血浆代谢谱研究，发现维吾尔族宫颈癌共性代谢组分，但只能间接认识肿瘤发生与特定代谢通路的关系。为深入了解宫颈癌组织代谢调控机制，本项目提取组织DNA和HPV分型后依托高分辨魔角旋转核磁共振技术（High-resolution magic angle spinning nuclear magnetic resonance，HRMAS-1HNMR）和高效液相色谱联合三重四级杆串联质谱（UHPLC-MS/MS）技术平台，利用宫颈癌及癌旁组织标本，通过宫颈癌组织特异代谢组谱分析、代谢标记物发现及关键酶活性测定，探讨宫颈癌发生与细胞代谢调控网络改变关系。鉴别出17种宫颈癌及HPV16感染共性差异表达的小分子代谢物， 18条关键代谢通路，其中牛磺酸和亚牛磺酸代谢，丙氨酸、天冬氨酸和谷氨酸代谢，D-谷氨酰胺和D-谷氨酸代谢及硫胺素代谢等4条代谢可能与宫颈癌发生密切相关。谷氨酸脱羧酶 1（GAD1）、糖原合成激酶 3（GSK-3）GSK-3β 、肌肉丙酮酸激酶同工酶 2（PKM2）和肉毒碱棕榈酰基转移酶（CPT A1）等代谢限速酶在宫颈病变组织中活性发生变化，其中 GAD1 调节氨基酸分解代谢，GSK-3 调节糖分解代谢、糖异生和糖原合成，PKM2 调节糖氧化分解，CPT A1调节脂肪酸分解代谢。构建了 HPV16 编码基因 E6 和 E7 的 RNAi 表达载体，以此转染 SiHa 宫颈癌细胞（HPV16 阳性），同步表达 RNAi 片段和绿色荧光蛋白质（GFP），通过激光共聚焦显微成像分析，评价转染效率，建立了 RNAi 细胞模型；通过基于 RNAi 细胞模型的代谢谱和定量 RT-PCR 分析，RNAi 细胞模型系列与正常对照细胞相比较，共有13 种差异表达的小分子代谢物，GAD1 和 GSK-3β 表达水平上调，而 PKM2 和 CPT1 表达水平下调，其变化趋势与上述基于宫颈癌组织内表达水平变化趋势正好相反，提示 HPV16 编码基因表达可能影响上述限速酶介导代谢调节过程，为从代谢调控水平上揭示 HPV16 致病和致癌生物学本质提供了重要依据。