【作者】 陈浩；

【导师】 朱克祥；

【作者基本信息】 兰州大学 ， 临床医学·外科学（专业学位）， 2023， 硕士

【摘要】 背景:胰腺癌(Pancreatic Cancer,PC)作为预后最差的消化系统肿瘤之一,起病隐匿、疗效不佳、转移迅速最终导致其预后不良。过半患者在确诊时已无法行根治性切除,因此,当前胰腺癌诊治的重点在于早诊断、早治疗。鉴于胰腺癌具体发病机制仍不完全清楚,探讨胰腺癌发生、发展机制成为当下的研究热点之一。本文旨在探究内质网蛋白27(Endoplasmic Reticulum Protein 27,ERP27)对胰腺癌细胞增殖及迁移影响,为寻找胰腺癌新型治疗靶点提供参考。蛋白质二硫键异构酶家族(Protein Disulfide Isomerase,PDI)通过催化底物两个半胱氨酸残基二硫键的合成、异构化或充当分子伴侣来协助蛋白底物进行折叠,在内质网相关降解及功能蛋白合成上发挥重要作用,对维持内质网蛋白稳态、避免异常蛋白质折叠引起的细胞应激和疾病至关重要。ERP27是PDI家族内质网驻留蛋白之一,其本身不具有催化活性,但能结合并转运未折叠的蛋白底物,在维持内质网稳态中发挥重要作用。胰腺癌细胞本身更重的代谢负荷及缺乏血供的微环境导致其内质网稳态容易失衡,因此,ERP27作为一种内质网应激效应蛋白,可能对胰腺癌细胞的增殖及迁移有着重要影响。目的:通过生信学方法探讨ERP27在胰腺癌组织和正常组织中表达差异性,利用体外实验研究ERP27在人胰腺癌细胞和正常胰腺导管上皮细胞中的表达量差异及过表达ERP27对胰腺癌细胞增殖和迁移能力的影响。方法:1.生信学方法:在公共数据库(GEPIA数据库、CCLE数据库及TCGA数据库等)中检索选取胰腺癌相关数据集,通过生物信息学方法分析ERP27在人胰腺组织和和癌旁组织中的差异表达情况,通过GO、KEGG富集分析和相关性分析研究胰腺癌中ERP27正相关基因集,探讨ERP27对胰腺癌进展抑制作用的机制。2.体外实验:(1)使用q PCR及Western Blot检测并比较人正常胰腺导管上皮细胞HPNE和4株胰腺癌细胞(As PC-1、Bx PC-3、PANC-1、Pa Ca2)中ERP27在m RNA及蛋白水平上差异性表达。(2)构建过表达靶向ERP27 sh RNA慢病毒,空白对照组为未转染病毒的Pa Ca2细胞(Pa Ca2-BC),阴性对照组为转染空载体慢病毒的Pa Ca2细胞株(Pa Ca2-NC),实验组为转染ERP27过表达慢病毒Pa Ca2细胞株(Pa Ca2-VC),使用细胞增殖实验(CCK-8法)和细胞划痕实验检测并对比三株细胞间增殖及迁移能力的差异。结果:1.生物信息学分析发现ERP27在胰腺癌组织中表达显著低于正常胰腺组织(P<0.001),且与预后相关,高表达组与低表达组中位生存期分别为23.03月和17.23月,HR=0.55,P=0.0061差异具有统计学意义(P<0.05)。对TCGA胰腺癌数据库中ERP27正相关基因集进行GO富集分析,结果显示:生理功能(bioligocal Process,BP)富集表明此群基因主要参与细胞修饰氨基酸分解代谢、抗菌体液反应、细胞对铜离子的反应、水溶性维生素代谢、脂质消化、四吡咯代谢及钴胺素代谢过程,胞内定位(cellular Component,CC)富集结果显示此群基因功能定位于以肌动蛋白为基础的细胞突起、酶原颗粒、基质侧质膜、顶端质膜、锚定成分膜、细胞突起膜等位置,分子功能(Molecular Function,MF)富集提示此基因群主要发挥配体门控通道、离子门控通道、外肽酶、甘油三酯脂肪酶、内肽酶、肽聚糖结合、寡糖结合、丝氨酸型水解酶及丝氨酸型内肽酶活性等功能,KEGG富集分析结果显示,此群基因主要参与代谢通路为:胰腺分泌、胃酸分泌、淀粉和蔗糖代谢、神经活性配体-受体相互作用、甘油脂质代谢、矿物质吸收、近端小管碳酸氢盐重吸收、脂肪消化吸收和蛋白质消化吸收等代谢过程。2.q PCR及WB结果显示:ERP27 m RNA及蛋白在人胰腺癌细胞株As PC-1、Bx PC-3、PANC-1及Pa Ca2中均低表达,在正常胰腺导管上皮细胞HPNE高表达,差异具有统计学意义(P<0.05)。3.细胞增殖实验(CCK-8法)及细胞划痕实验结果表明:与空白对照组及阴性对照组相比,过表达ERP27后Pa Ca2细胞增殖和迁移能力明显下降,且差异具有统计学意义(P<0.05),空白对照组与阴性对照组之间差异无统计学意义(P>0.05)。结论:1.与正常胰腺导管上皮细胞及胰腺组织相比,ERP27在胰腺癌细胞及胰腺癌组织中表达量均显著降低。2.ERP27对胰腺癌细胞增殖及迁移能力具有抑制作用,ERP27可能抑制胰腺癌进展。更多还原

【Abstract】 Background Pancreatic cancer is one of the worst prognostic tumors in the digestive system,with insidious onset,poor efficacy and rapid metastasis ultimately leading to a poor prognosis.More than half of the patients can’t undergo radical resection at the time of diagnosis,therefore,early diagnosis and early treatment are particularly important.Given that the specific pathogenesis of pancreatic cancer remains unclear,it has become one of the research hotspots to explore the occurrence and progression of pancreatic cancer.The aim of this paper is to interrogate the effect of ERP27 on the proliferation and migration of pancreatic cancer cells,and to provide references for finding new therapeutic targets for pancreatic cancer.The family of protein disulfide isomerase(PDI)catalyzing the formation and isomerization of disulfide bonds by linking two cysteine residues or act as chaperons and assist in the degradation and functional protein synthesis,which is essential for maintaining endoplasmic reticulum homeostasis and avoiding abnormal protein folding induced cellular stress and disease.ERP27 is one of the endoplasmic reticulum-resident proteins of the PDI family,which is not catalytically active it self,but can distinguish and bind unfolded protein substrates and play an important role in maintaining endoplasmic reticulum homeostasis.The heavier metabolic load and lower blood supply microenvironment predispose pancreatic cancer cells to imbalance in endoplasmic reticulum homeostasis.As an endoplasmic reticulum stress effector protein,ERP27 may have an important impact on the proliferation and migration of pancreatic cancer cells.Objective Bioinformatics methods were used to investigate the differential expression of ERP27 in pancreatic cancer tissues and normal tissues.Then study the differential expression of ERP27 in human pancreatic cancer cells between normal pancreatic ductal epithelial cells and finding the effect of overexpression of ERP27 on the proliferation and migration capacity of pancreatic cancer cells by in vitro experiments.Methods 1.Bioinformatics method:The relevant data sets of pancreatic cancer were retrieved from public databases(GEPIA database,CCLE database,TCGA database,etc)analyzed the differential expression of ERP27 in human pancreatic tissues and paracancerous tissues by bioinformatics methods.Investigating the positively related gene set of ERP27 in pancreatic cancer by GO,KEGG enrichment analysis and correlation analysis in order to find the possible pathways which the ERP27 inhibits pancreatic cancer progression.2.In vitro experiments(1)Using q PCR and Western Blot to detect and compare the differential expression of ERP27 at m RNA and protein levels in human normal pancreatic ductal epithelial cells HPNE with four pancreatic cancer cells(As PC-1,Bx PC-3,PANC-1,Pa Ca2).(2)After constructing overexpression targeting ERP27 sh RNA lentivirus,blank control group was Pa Ca2 cells without virus transfection(Paca2-BC),negative control group was Pa Ca2 cell line transfected with empty vector virus(Pa Ca2-NC),and experimental group was Pa Ca2 cell line transfected with ERP27 overexpression lentivirus(Pa Ca2-VC).The differences in proliferation and migration capacity between three cell lines were detected and compared by CCK-8 method and Wound healing.Results 1.Bioinformatics analysis revealed that ERP27 expression was significantly lower in pancreatic cancer tissues than in normal pancreatic tissues(P<0.001)and correlated with prognosis.The median survival time of high expression group and low expression group was 23.03 months and 17.23 months,HR=0.55,P=0.0061.And the difference was statistically significant(P<0.05).GO enrichment analysis of the ERP27 positively related genes in the TCGA pancreatic cancer database showed that the enrichment of Bioligocal Process(BP)indicated this group of genes was mainly involved in cellular modification of amino acid catabolism,antibacterial humoral response,cellular response to copper ions,water-soluble vitamin metabolism,lipid digestion,tetrapyrrole metabolism and cobalamin metabolism process.The Cellular Component(CC)enrichment results showed that this gene cluster is functionally located in actin-based cellular protrusions,zymogen particles,matrix lateral plasma membranes,apical plasma membranes,anchoring component membranes,cellular protrusion membranes,etc.The Molecular Function(MF)enrichment suggests that this gene cluster mainly plays the role of ligand-gated channels,ion gated channels,ectopeptide membranes,triglyceride lipase,endopeptidase,peptidoglycan binding,oligosaccharide binding,serine hydrolase and serine endopeptidase activities.The KEGG enrichment analysis showed that this gene cluster is mainly involved in the following metabolic pathways: pancreatic secretion,gastric acid secretion,starch and sucrose metabolism,neuroactive ligand-receptor interactions,glycerolipid metabolism,mineral uptake,proximal tubule bicarbonate reabsorption,fat digestion and protein digestion and absorption.2.q PCR and WB results showed low expression of ERP27 m RNA and protein in human pancreatic cancer cell lines As PC-1,Bx PC-3,PANC-1 and Pa Ca2,but high expression in normal pancreatic cells HPNE,and the difference was statistically significant(P < 0.05).3.The results of CCK-8 method and Wound healing showed that the proliferation and migration capacity of Pa Ca2 cells were significantly decreased after overexpression of ERP27 compared with blank control and negative control groups,and the difference was statistically significant(P<0.05).conclusion 1.The expression of ERP27 was significantly reduced in pancreatic cancer cells and pancreatic cancer tissues compared with normal pancreatic ductal epithelial cells and pancreatic tissues.2.ERP27 has an inhibitory effect on the proliferation and migration capacity of pancreatic cancer cells,ERP27 may inhibit pancreatic cancer progression.更多还原