【作者】 刘强；

【导师】 谭跃球；

【作者基本信息】 中南大学 ， 遗传学， 2023， 博士

【摘要】 研究背景:同源重组(Homologous recombination HR)是减数分裂的关键过程,是指来自父本和母本的同源染色体通过联会,交换染色体DNA的遗传物质。HR是形成正常配子的前提,也是遗传多样性和基因组稳定性的基础。减数分裂中,同源重组修复(homologous recombination repair,HRR)是DNA双链断裂(double strand break,DSB)的首选修复方式。HR及HRR既发生于减数分裂中,也发生于有丝分裂中。HRR功能障碍可以导致同源重组修复缺陷(homologous recombination deficiency,HRD),通常所指的是细胞水平上的HRR功能障碍状态,可由相关基因的胚系或体细胞发生突变,以及表观遗传失活等多种因素引起细胞水平的异常。HRD是导致精子发生障碍的重要原因,是男性不育特别是非梗阻性无精子症的重要分子标志物。近年来的研究表明,HRD也常存在于多种恶性肿瘤中,如卵巢癌、乳腺癌、胰腺导管癌、前列腺癌等恶性肿瘤。HRR是一条涉及多个步骤的复杂DNA损伤修复过程,HRD的存在会使肿瘤细胞对诱发DNA交联的铂类药物高度敏感,目前HRD正逐步发展成为新型生物标志物并用于肿瘤精准治疗。肺癌是全球发病率第二高、死亡率最高的恶性肿瘤,已成为严重危害人类健康的全球性医学问题。根据病理学特征肺癌分为小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC),其中NSCLC约占80-90%,5年生存率仅约10%。遗传和环境因素引起的DNA损伤是导致NSCLC发生的重要机制之一。近年来的研究表明,HRD相关分子同样参与了NSCLC的发生发展进程,但具体作用机制尚不清楚。研究目的:在非梗阻性无精子症中筛选减数分裂同源重组修复相关基因,分析其中关键HRR基因XRCC2在肺癌等9种肿瘤中的表达情况。明确XRCC2在NSCLC中高表达基础上,探讨XRCC2在NSCLC发展中的作用与分子机制,探究靶向XRCC2在NSCLC治疗中的潜在临床价值,为NSCLC诊疗提供新的靶点和理论基础。研究方法:在279例行全外显子组测序的非梗阻性无精子症患者中对26个减数分裂HRR基因进行筛选,聚焦到减数分裂HRR基因XRCC2。通过生物信息学分析、临床组织样本检测,明确XRCC2在NSCLC中的表达及其与预后和临床特征的相关性;通过CCK8、流式细胞术、Transwell及划痕实验,检测XRCC2对NSCLC细胞株增殖、凋亡、迁移和侵袭的影响;通过IP-质谱、co-IP等技术,分析XRCC2与Vimentin的互作关系;通过CHX半衰期实验,探讨XRCC2调控Vimentin的稳定性;通过RNA测序、Ch IP、荧光素酶报告基因、Rescue等体外实验,探究XRCC2在NSCLC中的上、下游调控机制;裸鼠移植瘤实验检测XRCC2及上、下游基因在体内对NSCLC的作用。结果:279例NOA患者行全外显子组测序,筛选到7个减数分裂HRR基因。经进一步的生物信息学分析及临床样本检测发现,在NSCLC患者中XRCC2表达水平较高,与预后呈现负相关;XRCC2显著促进NSCLC细胞增殖、迁移与侵袭、抑制细胞凋亡。RNA测序结果发现敲低XRCC2增殖通路中FOS基因的表达显著上调,过表达XRCC2引起c-Fos的m RNA和蛋白水平降低,促进细胞增殖,并通过Rescue实验表明XRCC2通过c-Fos调控NSCLC细胞增殖;IP-质谱、co-IP、CHX半衰期实验等发现XRCC2与Vimentin蛋白直接相互作用,并影响Vimentin半衰期与蛋白表达。同时,生物信息学分析结合Ch IP、荧光素酶报告基因证明c-Myc转录调控XRCC2的表达,联合应用c-Myc抑制剂10058-F4有效增强DOX对NSCLC的抑制作用。结论:建立了26个减数分裂同源重组修复的基因集,发现XRCC2可能是与NSCLC相关的新DNA损伤修复相关基因,其作用与机制很可能是:XRCC2通过下调c-Fos诱导NSCLC细胞增殖、减少细胞凋亡,并促进小鼠移植瘤的生长;XRCC2通过结合并稳定Vimentin蛋白增强NSCLC细胞体内外转移能力;c-Myc是XRCC2的上游转录因子,c-Myc抑制剂10058-F4可增强DOX对NSCLC体内、外增殖与转移的抑制作用。图30幅,表16个,参考文献101篇更多还原

【Abstract】 Background:Homologous recombination(HR)is an essential cellular process required for producing normal gametes,generating genetic diversity,ensuring proper chromosome segregation,and repairing certain types of DNA damage.In meiosis,homologous recombination repair(HRR)is the preferred repair pathway for DNA double strand break(DSB).HR and HRR occur in both meiosis and mitosis.HRR dysfunction can lead to homologous recombination defect(HRD),usually due to various factors such as germline mutation or somatic mutation of HRR related genes,and epigenetic abnormalities leading to cellular abnormalities.HRD is an important cause of spermatogenesis disorder and an important molecular marker of male infertility,especially non-obstructive azoospermia(NOA).Recent studies have shown that HRD also exists in a variety of malignant tumors,such as ovarian cancer,breast cancer,pancreatic duct cancer,prostate cancer and other malignant tumors.HRR is a complex DNA damage repaire process that involves multiple steps.HRD can makes tumor cells highly sensitive to platinum drugs that induce DNA cross-linking.Currently,HRD is gradually developing into a new biomarker and used for precise tumor treatment.Lung cancer is the second most common malignant tumor and the highest mortality in the world,which has become a global medical problem seriously endangering human health.Lung cancer is classified based on its pathological features into two main types:small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC comprises approximately 80-90%of all cases,making it the predominant form,and the 5-year survival rate is only about 10%.DNA damage caused by genetic and environmental factors is one of the important mechanisms leading to the development of NSCLC.Recent research indicates the involvement of HDR-associated molecules in the initiation and progression of NSCLC,although the precise underlying mechanism remains elusive.Purpose:To screen meiotic recombination repair-related genes in NOA,and analyze the expression of key HRR gene X-ray repair cross-complementing gene 2(XRCC2)in 9 kinds of tumors,including lung cancer.On the basis of clarifying the high expression of XRCC2 in NSCLC,explore the role and molecu Lar mechanism of XRCC2 in the occurrence and development of NSCLC,explore the potential clinical value of targeting XRCC2 in the treatment of NSCLC,and provide a new target and theoretical basis for the diagnosis and treatment of NSCLC.Methods:Twenty six meiotic HR-related genes were screened from279 cases of non-obstructive azoospermia with whole-exome sequencing(WES),and the key gene XRCC2 was focused.Bioinformatics analysis and clinical tissue sample testing to clarify the expression of XRCC2 in NSCLC and its correlation with prognosis and clinical characteristics.The effects of XRCC2 on the proliferation,apoptosis,migration and invasion of NSCLC cell lines were detected by CCK8,flow cytometry,Transwell and scratch test.Ip-mass spectrometry and co-IP were used to analyze the interaction relationship between XRCC2 and Vimentin;CHX half-life assay to explore the stability of Vimentin regulated by XRCC2.RNA sequencing,Ch IP,luciferase reporter gene,Rescue and other experiments were used to explore the upstream and downstream regulatory mechanism of XRCC2 in NSCLC.Nude mice xenograft experiments were used to detect the effect of XRCC2 and its upstream and downstream genes on NSCLC in vivo.Results:In 279 patients with NOA,seven meiotic HR-related genes were screened by WES.Additional bioinformatics scrutiny and examination of clinical samples revealed heightened expression of XRCC2in individuals afflicted with NSCLC,displaying an adverse association with prognosis.XRCC2 can significantly promote the proliferation,migration and invasion of NSCLC cells,and inhibit cell apoptosis.RNA sequencing results showed that knockdown of XRCC2 significantly up-regulated the expression of FOS gene in the proliferation pathway,and overexpression of XRCC2 decreased the m RNA and protein levels of c-Fos and promoted cell proliferation.Rescue experiments showed that XRCC2regulated NSCLC cell proliferation through c-Fos.Ip-mass spectrometry,co-IP,and CHX half-life assay showed that XRCC2 directly interacted with Vimentin protein and affected Vimentin half-life and protein expression.At the same time,bioinformatics analysis combined with Ch IP and luciferase reporter gene showed that c-Myc transcription regulated the expression of XRCC2,and c-Myc inhibitor 10058-F4effectively enhanced the inhibitory effect of DOX on NSCLC.Conclusion:The meiotic related gene XRCC2 is an HRR gene involved in the development of NSCLC.Its mechanism is to induce cell proliferation and reduce apoptosis by down-regulating c-Fos,and promote the migration and invasion of NSCLC cells by binding and stabilizing Vimentin protein,thus promoting the occurrence and development of NSCLC.c-Myc is an upstream transcription factor of XRCC2.c-Myc inhibitor 10058-F4 inhibits the proliferation and metastasis of NSCLC in vitro and in vivo by down-regulating XRCC2.更多还原