【作者】 陈睿；

【导师】 魏军民；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2023， 硕士

【摘要】 研究背景:根据2022年的癌症统计数据,乳腺癌是女性群体常见的恶性肿瘤之一,死亡率仅次于肺癌,位居第二位。约1 0%-20%的乳腺癌患者人表皮生长因子受体2(HER2)、雌激素受体(ER)和孕激素受体(PR)表达呈阴性,即三阴乳腺癌(triple-negative breast cancer,TNBC)。近年来TNBC的发病率和死亡率均在逐渐上升,且该病患者逐渐倾向于年轻化、低龄化。TNBC具有高度异质性和强侵袭性,与其他乳腺癌亚型相比,具有容易早期复发、早期转移、生存时间短和预后差的特点。目前为止,手术和化疗仍然是TNBC的主要治疗方法,由于TNBC的具体发病机制及其相关影响因素尚未完全阐明,针对TNBC的大多数治疗措施均效果不佳,只有少数个体化治疗手段已被证明可以改善TNBC患者的预后。因此,需要深入探讨TNBC发生发展的原理及机制,进一步发现用于TNBC患者诊断和预后的生物标志物。研究目的:1.本研究应用生物信息学技术,在TCGA数据库筛选出TNBC的相关基因G 蛋白偶联受体 34(G protein-coupled receptor 34,GPR34),分析 TNBC 组织和配对的癌旁组织中GPR34的表达情况。并利用免疫组织化学染色测定GPR34在TNBC的表达水平,进一步分析GPR34的表达水平与TNBC患者临床病理参数的相关性,探讨其与预后的关系。2.探究GPR34是否可作为TNBC的治疗靶点及具有评估疾病预后作用的生物标志物,为TNBC的机制研究及寻求潜在治疗靶点奠定基础。研究方法:1.本研究在TCGA数据库中获取126例TNBC患者的mRNA测序数据和116例TNBC患者的临床数据。2.应用R语言,根据肿瘤微环境相关生存分析、基质细胞及免疫细胞差异分析结果,筛选出TNBC差异表达基因(differentially expressed genes,DEGs)。首先使用STRING网站对筛选出的差异表达基因进行构建蛋白质-蛋白质相互作用网络(protein-protein interaction,PPI),以置信度≥0.400为标准筛选出TNBC的核心基因;其次对差异表达基因同时进行生存分析及临床相关性分析,同时满足p<0.05即确定为TNBC的预后相关基因。通过Venn分析对TNBC的核心基因及预后相关基因取交集后最终筛选出GPR34为TNBC的关键基因。3.验证GPR34与TCGA数据库中TNBC患者生存的关系、使用GSEA分析推测GPR34的作用通路,探讨GPR34的表达与免疫细胞浸润水平的关系。4.收集自2011年6月1日至2017年10月31日于山东大学齐鲁医院乳腺外科行手术治疗的95例TNBC患者。利用免疫组织化学染色(immunohistochemical staining,IHC)检测TNBC肿瘤组织及配对癌旁正常组织石蜡切片标本中GPR34蛋白的表达情况,并用SPSS 26.0分析GPR34蛋白表达水平与TNBC患者临床资料、病理特征的关系,最后根据随访资料,采用Kaplan-Meier法绘制生存曲线。研究结果:1.根据错误发现率 FDR<0.05 和|logfold-change|>1.0 的标准在 TNBC中筛选出135个肿瘤微环境相关差异表达基因,其中133个上调基因,2个下调基因,GPR34属于上调表达基因。2.通过对筛选出的差异表达基因构建蛋白质-蛋白质相互作用网络。首先筛选出72个TNBC的核心基因;其次对差异表达基因进行生存分析及临床相关性分析后筛选出2个TNBC的预后相关基因,并最终确定GPR34为TNBC的关键基因。3.在TCGA数据库中,我们发现GPR34mRNA在TNBC患者中高表达,并且GPR34 mRNA高表达的患者具有更短的总生存时间(overall survival,OS)(p<0.05)。4.GSEA(Gene set enrichment analysis)分析显示 GPR34 主要富集 T细胞受体信号通路、B细胞受体信号通路、趋化因子信号通路和JAK-STAT信号通路等多种信号通路。免疫细胞浸润分析显示,记忆B细胞、巨噬细胞M2、静息肥大细胞和中性粒细胞的浸润与GPR34的表达有关。5.免疫组织化学染色结果显示GPR34蛋白在TNBC组织中高表达(p<0.05)。GPR34蛋白表达高的患者其总生存期较短(p<0.05)。6.GPR34的表达在T1、T2期的患者和T3、T4期的患者间有显著差异(p<0.001),而与TNBC患者的年龄、组织学分级、有无淋巴结转移、TNM分期等无明显相关性(p>0.05)。研究结论:1.在TNBC中GPR34蛋白的表达水平高于周围正常乳腺组织,差异具有统计学意义,提示其可能与TNBC的发病及进展相关。2.在TNBC中,GPR34高表达的患者生存率降低、预后差,推测其可作为评估预后的生物标志物,未来随研究的深入有可能成为TNBC的潜在治疗靶点。更多还原

【Abstract】 Background:Breast cancer is one of the most common malignancies in the female population,with the second highest mortality rate after lung cancer,according to cancer statistics for 2022.10%-20%of breast cancers are negative for human epidermal growth factor receptor 2(HER2),estrogen receptor(ER)and progesterone receptor(PR)expression,known as triple-negative breast cancer(TNBC).In recent years,both the incidence and mortality of TNBC are increasing gradually,and the patients with TNBC tend to be younger and younger.TNBC is highly invasive and heterogeneous.Patients with TNBC have a tendency for earlier recurrence,earlier metastasis,more reduced overall survival(OS)and poorer prognosis compared with other breast cancer patients.Surgery and chemotherapy are still the main treatment methods for TNBC.As the specific pathogenesis of TNBC and its related influencing factors have not been fully clarified,most therapeutic measures for TNBC are not effective.Only a few individualized treatments have been shown to improve the prognosis and outcome of TNBC patients.Therefore,we need to learn more about the mechanisms of the development of the TNBC,as well as to identify new biomarkers for the diagnosis and prognosis of TNBC patients.Objectives:1.In this study,we screened G protein-coupled receptor 34(GPR34),a key gene of TNBC in TCGA database by bioinformatics techniques and analyzed the expression of GPR34 in TNBC tissues and paired paracancerous tissues.To investigate the expression level of GPR34 in TNBC based on immunohistochemistry staining,and to explore the relationship between GPR34 expression levels,clinicopathological parameters and prognosis of TNBC patients.2.To explore whether GPR34 could be used as a therapeutic target of TNBC and a biomarker to evaluate the prognosis of the disease,laying a foundation for the mechanism research of TNBC and seeking potential targeted therapy.Methods:1.In this study,RNA sequencing data of 126 TNBC patients and clinical data of 116 TNBC patients were obtained in TCGA.2.Using R software,TNBC differentially expressed genes(DEGs)were screened based on the results of relevant survival analysis of tumor microenvironment,stromal cell and immune cell differential analysis.First,we used STRING to construct a protein-protein interaction network(PPI)for DEGs and screened the core genes for TNBC with a confidence level higher than 0.400;then we performed both survival analysis and clinical correlation analysis on DEGs to identify prognosis-related genes in TNBC(p<0.05).GPR34 was finally screened as a key gene in TNBC after the intersection of the above two methods by Venn analysis.3.To verify the relationship between GPR34 and survival of TNBC patients in TCGA,to speculate the pathway of GPR34 using GSEA analysis and explore the relationship between GPR34 expression and the level of immune cell infiltration.4.95 TNBC patients who received surgical treatment in the Department of breast surgery,Qilu Hospital of Shandong university from June 1st,2011 to October 31st,2017 were collected.The expression of GPR34 protein was detected by immunohistochemical staining(IHC)in the tumor tissues of TNBC and paraffin sections of TNBC specimens.The relationship between GPR34 protein expression level and the clinicopathological characteristics of TNBC patients were analyzed by SPSS 26.0.Finally,according to the follow-up data,we used Kaplan-Meier method to plot the OS curve.Results:1.Among the 135 tumor microenvironment-related DEGs,133 up-regulated genes and 2 down-regulated genes were screened in TNBC based on the criteria of false discovery rate FDR<0.05 and |log fold-change|>1.0,and GPR34 belonged to upregulated expressed genes.2.We screened 72 core genes for TNBC by constructing protein interaction networks for DEGs and screened 2 prognosis-related genes by performing the survival analysis and clinical correlation analysis.Finally,GPR34 was identified as a key gene for TNBC.3.In TCGA,we found that GPR34 mRNA was highly expressed in TNBC patients,and patients with high GPR34 mRNA expression had relatively shorter OS(p<0.05).4.GSEA analysis showed that GPR34 was enriched in various signaling pathways such as T-cell receptor signaling pathway,B-cell receptor signaling pathway,chemokine signaling pathway and JAK-STAT signaling pathway.Immune cells infiltration analysis showed that memory B cells,macrophage M2,resting mast cells and neutrophils were related to the expression of GPR34.5.Immunohistochemical staining showed that GPR34 protein was highly expressed in TNBC tissues(p<0.05).Patients with high GPR34 protein expression had a shorter OS(p<0.05).6.GPR34 expression was significantly different between TNBC patients in T1,T2 stages and those in T3,T4 stages(p<0.001).There was no significant correlation between GPR34 expression and age,histological grade,presence of lymph node metastasis and TNM stage of TNBC patients(p>0.05)Conclusion:1.The expression level of GPR34 protein in TNBC was significantly higher than that in peripheral normal breast tissue,suggesting that it may be associated with the development of TNBC.2.In TNBC,patients with high GPR34 expression have reduced survival and poor prognosis,and we speculated that it could be used as a biomarker to assess prognosis,and may become a potential future therapeutic target for the treatment of TNBC.更多还原