H2AFX基因多态性与非霍奇金淋巴瘤的相关性及机制研究

Non-Hodgkin lymphoma (NHL) is the most common hematological malignancy，the cause is not clear. May be associated with the genetic mutations, viruses, radiation, chemical drugs and autoimmune diseases.The DNA repair system is crucial in maintaining genomic integrity and preventing carcinogenesis. Several studies suggested that altered DNA repair function may play an important role in the pathogenesis and development of NHL. The single nucleotide polymorphism (SNP)s of H2AFX associated with various cancers.   Our Previous study showed that H2AFX gene polymorphism and Hodgkin lymphoma (NHL) have a relationship. In this study we investigate association of H2AFX gene polymorphisms with the risk of NHL in 100NHL and 300 controls subjects, through Real-time PCR and sequencing. Establishment of relevant data. One important form of DNA damage is double-strand breaks (DSBs).DSBs induce phosphorylation of the H2A histone family member X (H2AFX), which is a core histone involved in the cellular response to DSBs, Phosphorylated H2AFX（γ-H2AX） induces the recruitment of repair factors to damaged DNA or tethered DNA ends. γ-H2AX affect the sensitivity of chemotherapy, This project research the content of γ-H2AX in lymphoma cells in Medication before and after,clarify relationship between the H2AFX gene and significance in therapy, explore the possible mechanism of H2AFX in the pathogenesis of NHL, through Immunofluorescence(IF),Flow cytometry(FCM),Western Blot..the possible mechanism of H2AFX in the pathogenesis of NHL, through Immunofluorescence(IF),Flow cytometry(FCM),Western Blot.

DNA修复系统在维持基因组稳定性和防止肿瘤发生中起关键作用，H2AFX(H2AX)是最早被磷酸化的底物，是细胞中感应DNA损伤最敏感的分子。研究发现H2AFX在维持细胞遗传信息的完整性上发挥重要作用，其单一核苷酸多态性（SNP）与各种肿瘤的发生有关。前期工作发现H2AFX基因多态性与非霍奇金淋巴瘤（NHL）发生有一定关系，本研究拟采用病例-对照研究的方法，本研究对300例正常人和100例NHL进行基因多态性研究，通过采用Real-time PCR、测序等方法，搜索延边地区朝鲜族人群中H2AFX基因的SNP，建立相关数据资料。磷酸化H2AFX（γ-H2AX）参与DNA损伤修复，影响化疗敏感性，本项目通过免疫印迹、流式细胞等技术研究药物处理前后淋巴瘤细胞中γ-H2AX的量。探讨H2AFX基因在NHL发病机理中的可能作用。研究成果有助于进一步揭示NHL发病机理，为该病临床治疗提供依据。

DNA修复系统在维持基因组稳定性和防止肿瘤发生中起关键作用，H2AFX(H2AX)是最早被磷酸化的底物，是细胞中感应DNA损伤最敏感的分子。鉴于磷酸化H2AFX（γ-H2AX）参与DNA损伤修复，影响化疗敏感性，通过免疫印迹、流式细胞等技术研究药物处理前后肿瘤细胞中γ-H2AX的变化。本课题利用免疫组织化学染色方法在蛋白质水平上确认了γ-H2AX在非霍奇金淋巴瘤（NHL）以及多种上皮性恶性肿瘤（包括宫颈癌、前列腺癌、甲状腺乳头状癌、肝细胞肝癌、乳腺癌等）中的表达情况，肿瘤组织中的表达均高于正常组织；通过慢病毒介导的RNA干扰技术在淋巴瘤细胞（SU-DHL-2和SU-DHL-4）、前列腺癌细胞株（PC3）和宫颈鳞状细胞癌细胞株（SiHa）中成功完成YAP慢病毒载体的构建。利用集落形成实验和细胞计数（Cell counting kit-8，CCK-8）、划痕实验、transwell侵袭小室实验等方法明确γ-H2AX与肿瘤细胞的增殖、侵袭和迁移相关，（P<0.05）；利用免疫荧光（Immunofluorescence,IF）及蛋白印迹（Western Blot）等方法检测组蛋白去乙酰化酶（HDAC）抑制剂处理前后肿瘤细胞中γ-H2AX的变化，结果HDAC处理后γ-H2AX和E-Cadherin表达增多，提示γ-H2AX可能会抑制肿瘤细胞的上皮膜转化（EMT），目前的研究结果提示H2AX在肿瘤发生、发展和EMT中起到重要作用。而且可能与化疗敏感性相关，为临床治疗提供依据。