【作者】 谭爽；

【导师】 韩冰；

【作者基本信息】 吉林大学 ， 口腔医学硕士（专业学位）， 2023， 硕士

【摘要】 背景:口腔鳞状细胞癌(Oral Squamous Cell Carcinoma,OSCC)是口腔颌面部常见的恶性肿瘤,多数患者就诊时即为中晚期病变,对患者的咀嚼、吞咽、呼吸及外形产生较大影响。手术治疗常导致患者术后畸形率高、生存率低,而放化疗也难以得到令人满意的治疗结果。因此对于OSCC的早发现、早诊断、早治疗具有重要的意义。近年来,生物标志物在肿瘤的诊断、治疗和预后的预测中发挥着重要的意义,然而OSCC仍缺乏有效的生物标志物。因此,寻找特异性高的生物标志物以提高对OSCC患者的早期诊断率以及预后的判断成了迫切的要求。丝氨酸蛋白酶(Serine Protease,SP)大约有180个成员,是人类蛋白酶中最古老、第二大的家族,它广泛存在各种生命体中,如病毒、人类等。SP在消化、凝血受精、血管生成、纤溶、细胞凋亡与分化等过程中均发挥着作用,尤其是近年来,大量的证据证实SP在肿瘤的进展以及炎症中具有重要作用。在肿瘤的发展、肿瘤的侵袭以及肿瘤的转移过程中,肿瘤与基质之间的密切相互作用塑造了肿瘤微环境(Tumor Microenvironment,TME),决定了肿瘤细胞的命运。基质细胞还可以影响抗肿瘤免疫和对免疫治疗的反应。因此,了解控制这种复杂而动态相互作用的分子机制对于癌症诊断和治疗非常重要。由癌症和基质细胞表达和分泌的蛋白水解酶在调节肿瘤-基质相互作用中起重要作用,几种SP已经被证实在TME中发挥作用,而且SP中的多个成员被发现与肿瘤的发生、发展及预后有密切关系。作为丝氨酸蛋白酶胰蛋白酶家族的保守成员,丝氨酸蛋白酶23(Serine Protease 23,PRSS23)在癌症中的生物学功能仍然很大程度上未知。目前的研究表明PRSS23在甲状腺癌、胃癌、胰腺癌及结肠癌等均发挥着重要作用,但是目前关于PRSS23与癌症关系的研究还很有限,大多数研究只关注基因表达谱的结果,并显示在各种类型的癌症中都观察到PRSS23表达的增强,但PRSS23在肿瘤发生和癌症进展中的作用尚不清楚。针对OSCC,目前尚未查阅到PRSS23与其相关的研究结果。生物信息学是分子生物学及信息技术的结合体,它利用计算机科学、应用数学、信息学还有统计学等方法来研究生物学的问题,它目前主要用于解决医学问题,包括疾病的诊断以及疾病防治等方面。目的:通过分析PRSS23与OSCC之间的相互关系及检测PRSS23在OSCC中的表达情况,探究PRSS23蛋白在OSCC中的表达意义,分析PRSS23在OSCC发生及发展过程中的作用,并确定PRSS23是否可以成为OSCC的诊断及预后标志物。首先利用生物信息学分析PRSS23与OSCC的关系,然后通过免疫组织化学(Immunohistochemistry,IHC)进一步检测PRSS23蛋白在OSCC组织及癌旁组织中的表达情况,根据检测结果,探究PRSS23蛋白在OSCC中的表达意义,分析PRSS23在OSCC发生及发展过程中的作用,并确定PRSS23是否可以成为OSCC的诊断及预后标志物。方法:通过在中外数据库(包括中国知网、万方、Pubmed、Web of Science)检索文献,结合目前的研究情况,发现PRSS23与多种肿瘤的发生发展有着密切的关系,确定目的基因为PRSS23。首先通过生物信息学方法验证PRSS23在OSCC组织和正常组织间的表达差异,并对其进行生存分析,然后对PRSS23进行受试者工作特征(Rreceiver Operating Characteristic,ROC)曲线分析、共表达基因分析以及免疫浸润分析,对PRSS23作为OSCC的诊断及预后的标志物的可信性及其在OSCC诊断以及预后方面的意义进行分析。然后选取术前未经放化疗、均为首次手术的OSCC患者的标本,其中包括OSCC组织标本20例及OSCC患者的癌旁组织标本14例,使用免疫组织化学(Immunohistochemistry,IHC)检测在OSCC组织及癌旁组织中PRSS23的表达水平是否存在差异,通过t检验和非参数秩和检验来测定PRSS23蛋白在不同临床病理参数的组织间的表达量是否有差异,结果以p<0.05来体现差异具有统计学意义。结果:1.基因差异表达分析结果显示:PRSS23在OSCC组织中呈显著高表达。2.PRSS23预后分析结果显示:PRSS23的高表达与患者的OS(Overall Survival)之间的关系无统计学意义。3.ROC曲线的分析结果:凭借PRSS23的表达结果来诊断OSCC较为可信(AUC=0.810,p<0.05)4.共表达基因分析结果:在OSCC中,PRSS23与NAT1(Arylamine N-acetyltransferase 1)、ADH1B(Alcohol Dehydrogenase 1B)、BIRC5(Baculoviral IAP Repeat Containing 5)、AQP5(Aquaporin 5)、BCL2A1(B-cell lymphoma-2 A1)基因均有较低的相关性,提示在OSCC中,PRSS23可能独立发挥作用。5.免疫浸润:对PRSS23与24种免疫细胞的免疫情况进行分析,发现PRSS23的表达与OSCC的免疫细胞的免疫浸润水平均有较低的相关性,提示在OSCC的免疫浸润过程中,PRSS23可能不发挥作用。6.通过IHC对PRSS23蛋白在OSCC组织和癌旁组织中的表达量进行检测,对数据进行统计学分析,结果显示表达量无明显差异,二者无统计学意义(p>0.05)。7.PRSS23蛋白的表达与OSCC临床分期相关,差异具有统计学意义(p<0.05);PRSS23蛋白的表达水平与OSCC患者的年龄、患者的性别、肿瘤的分化程度、肿瘤的浸润深度、有无淋巴结转移均无明显相关性,差异不具统计学意义(p>0.05)。结论:1.PRSS23在OSCC组织和癌旁组织中的表达差异不具有统计学差异(p>0.05),PRSS23可能不参与OSCC的发生发展过程。2.PRSS23的表达与患者的预后无关,这可能是由于TCGA数据库中OSCC的样本量不足以及部分预后信息没有跟进等。3.PRSS23可能不参与调控OSCC的免疫浸润过程。4.PRSS23蛋白表达与OSCC患者临床分期相关。5.PRSS23不能作为OSCC的诊断标志物,为临床治疗提供理论基础。更多还原

【Abstract】 Background:Oral squamous cell carcinoma(OSCC)has a low survival rate and a high deformity rate due to its high metastasis rate and ability to destroy the upper gastrointestinal and respiratory functions.The lack of clinical diagnostic indicators for OSCC often leads to late diagnosis.Chemoradiotherapy combined with surgery is crucial for patients with advanced disease(stage III and IV)or with metastases.However,chemotherapeutic drugs such as cisplatin and 5-fluorouracil failed to produce satisfactory results due to the drug resistance of OSCC cells.In addition,clinically,the prognosis of OSCC is mainly predicted by TNM staging,and supportive testing at the molecular level is also needed.Therefore,new biomarkers are urgently needed to improve the diagnosis,treatment and prognosis prediction of OSCC patients.In recent years,there are many biomarkers related to OSCC being discovered.For the diagnosis and treatment of OSCC,the role of biomarkers has also been paid more and more attention.It is hoped that OSCC can be diagnosed early through biomarkers,so that improve the survival rate of OSCC patients.With approximately 180 members,serine proteases are the oldest and second largest family of human proteases,and they exist widely,from viruses to humans.Serine proteases play an important role in digestion,coagulation and fertilization,fibrinolysis,apoptosis and differentiation,angiogenesis and other processes.Especially in recent years,a large amount of evidence shows that serine proteases play an important role in tumor progression and inflammation.During tumor development,invasion and metastasis,the close interaction between tumor and stroma shapes the tumor microenvironment(the tumor microenvironment,TME)and determines the fate of tumor cells.Stromal cells can also influence antitumor immunity and response to immunotherapy.Therefore,understanding the molecular mechanisms governing this complex and dynamic interaction is important for cancer diagnosis and therapy.Proteolytic enzymes expressed and secreted by cancer and stromal cells play an important role in regulating tumor-stromal interactions,several serine proteases have been shown to function in the tumor microenvironment,and multiple members of the serine proteases were found to be associated with tumor occurrence,development and prognosis are closely related.As a conserved member of the trypsin family of serine proteases,the biological function of the serine protease PRSS23(Serine Protease 23)in cancer remains largely unknown.Current studies have shown that PRSS23 plays an important role in thyroid cancer,gastric cancer,pancreatic cancer and gastric cancer,but the current research on the relationship between PRSS23 and cancer is still very limited.Most studies only focus on the results of gene expression profiling and show that enhanced expression of PRSS23 is observed in various types of cancer,but the role of PRSS23 in tumorigenesis and cancer progression remains unclear.Especially for OSCC,there is no relevant research literature on PRSS23 and OSCC.Bioinformatics is a combination of molecular biology and information technology.It uses methods such as applied mathematics,informatics,statistics and computer science to study biological problems.It can solve complex biomedical problems and it plays an important role in the diagnosis,treatment and prevention of diseases.Objective:The first part of this study finds out the target gene by reading a large number of literatures,and uses bioinformatics methods to analyze the target gene.Using bioinformatics to analyze the relationship between PRSS23 and OSCC,but the real situation of PRSS23 protein expression in OSCC tissue has not been verified,so the second part further verifies the relationship between PRSS23 and OSCC by performing immunohistochemistry(IHC),By detecting the expression of PRSS23 protein in OSCC tissues and paracancerous tissues,we can explore the expression and significance of PRSS23 protein in OSCC,discuss the role of PRSS23 in the occurrence and development of OSCC,and determine whether PRSS23 can be the key to OSCC.Diagnostic and prognostic markers.Method:Firstly,to select the appropriate gene for research by searching literature in databases such as Pubmed and Web of Science,and synthesizing the research situation at home and abroad.ROC curve analysis,co-expression gene analysis and immune infiltration analysis were performed on the gene to analyze the credibility of the gene as a marker for the diagnosis and prognosis of OSCC and its biological significance in OSCC.Then selects the specimens of OSCC patients who have not undergone radiotherapy and chemotherapy before operation and are the first operation,including 20 cases of OSCC specimens and 14 cases of paracancerous tissue specimens of OSCC patients.IHC is used to detect Specimens were tested to detect whether there was a difference in the expression level of PRSS23 between OSCC tissues and adjacent tissues.The t test and non-parametric rank sum test were used to determine whether the expression level of PRSS23 protein was different among tissues with different clinicopathological parameters.All results P<0.05 means the difference is statistically significant.Result:1.Analysis of the results in the TCGA database showed that PRSS23 was significantly highly expressed in oral squamous cell carcinoma tissues.2.Prognostic analysis results of PRSS23: The high expression of PRSS23 was not correlated with the decrease of OS in patients.3.ROC curve analysis results: the diagnosis of oral squamous cell carcinoma based on the detection of PRSS23 expression is more reliable(AUC=0.810,p<0.05).4.The results of co-expression gene analysis showed that PRSS23 was not correlated with other gene expression in OSCC,and PRSS23 may play a role independently in OSCC.5.The results of immune infiltration showed that there was no significant correlation between the expression of PRSS23 and the level of immune infiltration of immune cells in oral squamous cell carcinoma,PRSS23 may not play a role in the immune infiltration process of oral squamous cell carcinoma.6.Statistical analysis was performed on the expression of PRSS23 protein in OSCC tissues and adjacent tissues,and the results showed that there was no significant difference in expression,and there was no statistical significance between the two(p>0.05).7.The expression of PRSS23 protein was correlated with the clinical stage of the tumor,and the difference was statistically significant(p<0.05);the expression level of PRSS23 protein was not correlated with the gender,age,degree of tumor differentiation,depth of tumor invasion,and lymph node metastasis.No statistical significance(p>0.05).In conclusion:1.There was no significant difference in the expression of PRSS23 in OSCC and paracancer tissues(p > 0.05),and PRSS23 may not be involved in the occurrence and development of OSCC.2.The expression of PRSS23 was not correlated with the prognosis of patients,which may be due to the insufficient sample size of OSCC in the TCGA database and the lack of follow-up of some prognostic information.3.PRSS23 may not be involved in regulating the immune infiltration process of oral squamous cell carcinoma.4.PRSS23 protein expression is correlated with the clinical stage of OSCC patients.5.PRSS23 can not be used as a diagnostic marker for oral squamous cell carcinoma,providing a theoretical basis for clinical targeted molecular therapy.更多还原