【作者】 张帆；

【导师】 严永敏；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2025， 博士

【摘要】 目的:胃癌是我国高发的消化道肿瘤,早期诊断与治疗是胃癌临床诊疗的关键,但目前尚缺乏理想的血清诊断标志物。本研究旨在筛选胃癌特异的血浆外泌体源hsa＿circ＿0006718(简称外泌体circ6718)标志物,分析外泌体circ6718的临床诊断价值。探讨肿瘤微环境中胃癌细胞外泌体circ6718调控胃癌间充质干细胞(gastric cancer mesenchymal stem cells,GC-MSCs)向癌症相关成纤维细胞(cancer-associated fibroblasts,CAF)转化,促进胃癌进展的作用和机制,为胃癌的诊断和治疗提供新的潜在靶点。方法:(1)采用circRNA芯片技术,筛选胃癌患者血浆外泌体中差异性表达的circ6718,使用实时荧光定量PCR技术(real-time quantitative reverse transcription polymerase chain reaction,q RT-PCR)检测其在健康人群、慢性萎缩性胃炎患者以及胃癌患者的血浆外泌体中的相对表达量,根据病理资料分析circ6718的临床相关性,通过受试者工作(receiver operating characteristic curve,ROC)曲线分析其诊断效能,利用生存曲线评估其预后价值。(2)使用circ6718的小干扰RNA(small interference RNA,siRNA)和过表达质粒对胃癌细胞转染后进行体外功能学实验,采用CCK-8实验动态评估circ6718对胃癌细胞增殖活性的调控作用,采用克隆形成实验检测circ6718对胃癌细胞集落形成能力的影响,构建Transwell跨膜迁移实验和Matrigel基质胶侵袭实验评估circ6718对胃癌迁移和侵袭功能的影响,采用Western blot方法检测胃癌细胞增殖、凋亡和上皮间质转化(epithelial-mesenchymal transition,EMT)指标表达量。构建裸鼠皮下荷瘤和腹腔转移瘤模型探究circ6718在体内对胃癌进展的生物学作用,通过免疫组化和Western blot技术检测增殖和转移相关蛋白表达量。(3)应用RNA荧光原位杂交(fluorescence in situ hybridization,FISH)和核质分离实验确定circ6718在胃癌细胞中的定位。RNA结合蛋白免疫沉淀(RNA-binding protein immunoprecipitation RNA,RIP)实验检测circ6718与真核翻译起始因子2(argonaute 2,AGO2)蛋白的结合,联合Starbase、Circ Interactome等生物信息学数据库预测circ6718可能结合的靶微小RNA(micro RNA,miRNA),在转染circ6718 siRNA和过表达质粒后通过q RT-PCR检测miRNA的表达量。构建circ6718双荧光素酶报告基因质粒和miRNA mimics共转染至胃癌细胞,确定circ6718与miRNA的具体结合位点。将circ6718过表达质粒与miRNA mimics共转染至胃癌细胞验证其对胃癌进展的作用。构建miRNA inhibitor并与circ6718 siRNA共转染至胃癌细胞验证其对胃癌进展的作用。联合Target Scan、miRDB等生物信息学数据库预测miRNA可能结合的靶基因,在转染了miRNA mimics和circ6718过表达质粒和siRNA的细胞中通过q RT-PCR检测靶基因的表达量,并在胃癌组织标本中进行验证。采用双荧光素酶报告基因实验证实miRNA和靶基因的结合。将circ6718的过表达质粒和siRNA与靶基因的siRNA和过表达质粒共转染至胃癌细胞并通过功能学实验和Western blot技术确定其对胃癌进展的作用。查阅相关文献寻找靶基因下游发挥促癌作用的信号通路,采用生物信息学预测、免疫共沉淀(co-immunoprecipitation,Co-IP)、免疫荧光实验确定其调控信号通路的具体作用机制。(4)通过慢病毒转染技术筛选出稳定过表达和敲减circ6718的胃癌细胞并收集其外泌体处理胃癌细胞和GC-MSCs,应用细胞功能学实验、q RT-PCR和Western blot技术确定其对胃癌细胞和GC-MSCs的作用并阐明具体作用机制。(5)基于circ6718促进胃癌进展的效应,将其siRNA与HEK-293T细胞外泌体联合构建工程化外泌体,通过电穿孔技术将circ6718 siRNA导入HEK-293T细胞外泌体并验证装载效率,随后进行体内外实验验证其对胃癌增殖和转移的抑制效果,通过生物安全性实验评估工程化外泌体对裸鼠肝肾等主要脏器的功能有无影响。结果:(1)Circ6718在胃癌患者血浆外泌体中的表达量较健康人群和慢性萎缩性胃炎患者高,临床资料分析显示,其高表达与胃癌的远端转移密切相关,其ROC曲线下面积为0.68,将其与传统诊断标志物癌胚抗原(carcino-embryonic antigen,CEA)和糖链抗原-50(carbohydrate antigen-50,CA-50)联合时诊断效能为最佳,ROC曲线下面积为0.86,且其在胃癌患者术后血浆外泌体中的表达较术前下调,Kaplan-Meier生存分析显示高表达circ6718的患者预后较差。(2)Circ6718由10号染色体类似细胞周期控制因子1(CWF19 like cell cycle control factor 1,CWF19L1)基因上2-4号外显子经过反向剪切形成。q RT-PCR结果显示,其在多种胃癌细胞和胃癌细胞外泌体中高表达,敲减circ6718可以在体内外抑制胃癌细胞的增殖、迁移、侵袭和上皮间质转化(EMT)进程,过表达则相反。(3)核质分离和RNA FISH实验显示,circ6718在多种胃癌细胞核和胞质中均有表达,且大多分布于细胞质中。RIP实验证明circ6718可以与AGO2蛋白结合,联合多个数据库预测和q RT-PCR实验找寻下游靶分子。采用双荧光素酶报告基因实验证实hsa-miR-561-3p与circ6718的结合。体外细胞功能学实验结果显示,过表达hsa-miR-561-3p会部分削弱circ6718的促癌效应。联合多个数据库预测和q RT-PCR实验找寻下游靶基因。双荧光素酶报告基因实验证实SAAL1与hsa-miR-561-3p的结合,敲减SAAL1可以逆转circ6718的促癌作用。Western blot结果显示其高表达可以激活TGFβ1/Smad2/3信号通路。Co-IP和免疫荧光实验显示,SAAL1可以结合转录因子PRRX1,染色质免疫共沉淀技术(chromatin immunoprecipitation,Ch IP)和双荧光素酶报告基因实验证明PRRX1可以与TGFβ1的启动子区域结合进而促进TGFβ1的转录激活,促进TGFβ1/Smad2/3信号通路的激活。(4)过表达circ6718的胃癌细胞外泌体明显促进了胃癌细胞的增殖、迁移、侵袭和EMT,上调了GC-MSCs的CAF标志物的表达,敲减circ6718的外泌体则起到相反的作用,在GC-MSCs中,circ6718可通过靶向hsa-miR-561-3p/SAAL1/PRRX1激活TGFβ1/Smad2/3信号通路,从而促进GC-MSCs的活化。(5)成功构建装载circ6718 siRNA的工程化HEK-293T细胞外泌体,体内外实验证实工程化外泌体具有抑制胃癌增殖和转移的生物学效应,生物安全性实验显示工程化外泌体对裸鼠肝肾等主要脏器的功能没有影响。结论:血浆外泌体circ6718与胃癌患者预后不良及远端转移正相关,具备诊断及预后评估潜力。胃癌细胞外泌体circ6718可通过靶向hsa-miR-561-3p/SAAL1/PRRX1轴激活TGFβ1/Smad2/3通路,促进胃癌细胞增殖、迁移、侵袭及EMT进程,诱导GC-MSCs分化为CAF,形成促转移微环境。此外,circ6718 siRNA工程化外泌体在体内外均能有效抑制肿瘤进展。更多还原

【Abstract】 Objective:Gastric cancer is a highly prevalent gastrointestinal cancer in China,early diagnosis and treatment is crucial for the clinical diagnosis and treatment of gastric cancer,but currently lacking ideal serum diagnostic markers.In this study,we aimed to screen plasma exosome-derived hsa＿circ＿0006718(exosomal circ6718)biomarker specific for gastric cancer and analyze the clinical diagnostic value of exosomal circ6718.Additionally,we explored the role and mechanism of gastric cancer cell exosomal circ6718 in the tumor microenvironment to regulate the transformation of gastric cancer mesenchymal stem cells(GC-MSCs)to cancer-associated fibroblasts(CAFs)and to promote the progression of gastric cancer,providing a new potential target for the diagnosis and treatment of gastric cancer.Methods:(1)We identified circ6718,a circular RNA differentially expressed in the plasma exosomes of gastric cancer patients,using circRNA microarray technology.Its relative expression was then measured in the plasma exosomes of healthy individuals,patients with chronic atrophic gastritis,and gastric cancer patients through real-time fluorescence quantitative PCR(q RT-PCR).The clinical relevance of circ6718 was analyzed in relation to pathological data,and its prognostic value using receiver operating characteristic(ROC)curves.Additionally,survival analysis was conducted to evaluate its diagnostic efficacy and prognostic value.(2)The small interfering RNA(siRNA)and overexpression plasmid of circ6718 were used to perform in vitro functional experiments on gastric cancer cells after transfection.CCK-8 assay was used to dynamically assess the regulatory effect of circ6718 on the proliferative activity of gastric cancer cells.Clone formation assay was used to detect the effect of circ6718on the colony formation ability of gastric cancer cells.Transwell transmembrane migration assay and Matrigel matrix gel invasion assay were conducted to evaluate the effect of circ6718on the migration and invasion function of gastric cancer.The expression of proliferation,apoptosis,and epithelial-mesenchymal transition(EMT)were detected by Western blot.The biological role of circ6718 on gastric cancer progression in vivo was explored using constructing subcutaneous loaded tumor and intraperitoneal metastasis models in nude mice.The expression of proliferation and metastasis-related proteins was detected using immunohistochemistry and Western blot assay.(3)RNA fluorescence in situ hybridization(FISH)and nucleoplasm separation experiments were employed to identify the localization of circ6718 in gastric cancer cells.RNA-binding protein immunoprecipitation RNA(RIP)experiments were conducted to detect the binding of circ6718 to argonaute 2(AGO2)protein.By Starbase,Circ Interactome and other bioinformatics databases,the potential target micro RNAs(miRNAs)that circ6718 might bind were predicted.After transfecting circ6718 siRNA and overexpression plasmids,q RT-PCR was used to detect the expression of these miRNAs.A circ6718 dual luciferase reporter gene plasmid and miRNA mimics were constructed and co-transfected into gastric cancer cells to identify the specific binding sites between circ6718 and miRNA.The circ6718overexpression plasmid and miRNA mimics were co-transfected into gastric cancer cells to verify their effects on gastric cancer progression.A miRNA inhibitor was constructed and co-transfected with circ6718 siRNA into gastric cancer cells to verify its impact on gastric cancer progression.Target Scan,miRDB and other bioinformatics databases were combined to predict the target genes that miRNA might bind.q RT-PCR was performed to detect the expression of target genes in cells transfected with miRNA mimics,circ6718 overexpression plasmid,and siRNA.These findings were further verified in gastric cancer tissue specimens.The dual-luciferase reporter gene assay was used to confirm the binding between miRNAs and target genes.The overexpression plasmid and siRNA of circ6718 were co-transfected with the siRNA and overexpression plasmid of the target gene into gastric cancer cells.The effects on the progression of gastric cancer were determined using functional assay and Western blot technique.The relevant literature was reviewed to explore the signaling pathways downstream of the target gene.The specific regulatory mechanism was determined using bioinformatics prediction,co-immunoprecipitation(Co-IP),and immunofluorescence experiments.(4)Gastric cancer cells with stable overexpression and knockdown of circ6718 were selected by lentiviral transfection.Exosomes derived from these cells were collected and treated with GC cells and GC-MSCs.The effects on GC cells and GC-MSCs were evaluated using cellular functional assays,q RT-PCR,and Western blot techniques,and the underlying mechanisms were explored.(5)Given the role of circ6718 in promoting gastric cancer progression,its siRNA was combined with the advantage of easy modification and good biosafety of HEK-293T cell derived exosomes to construct engineered exosomes.The circ6718 siRNA was introduced into HEK-293T cell exosomes by electroporation,and the loading efficiency was subsequently verified.In vitro and in vivo experiments were performed to assess the inhibitory effect on the proliferation and metastasis of gastric cancer.Additionally,biosafety experiments were performed to assess whether the engineered exosomes affected the function of major organs,including liver and kidney,in nude mice.Results:(1)Circ6718 exhibited higher expression levels in the plasma exosomes of gastric cancer patients compared to healthy individuals and patients with chronic atrophic gastritis.Analysis of clinical data showed that elevated circ6718 expression was closely related to the distal metastasis in gastric cancer.The area under the ROC curve for circ6718 was found to be 0.68,however,its diagnostic efficacy improved significantly when combined with traditional diagnostic markers such as carcino-embryonic antigen(CEA)and carbohydrate antigen-50(CA-50),resulting in an AUC of 0.86.Furthermore,the expression of circ6718 in the plasma exosomes of gastric cancer patients decreased after surgery compared to preoperative levels.Kaplan-Meier survival analysis showed that patients with high circ6718 expression had a poorer prognosis.(2)Circ6718 is formed by reverse shearing of exons 2-4 on the CWF19 like cell cycle control factor 1(CWF19L1)gene on chromosome 10.q RT-PCR results showed that circ6718was highly expressed in various gastric cancer cells and their exosomes.Both in vitro and in vivo studies showed that knocking down circ6718 inhibited the proliferation,migration,invasion,and epithelial mesenchymal transition(EMT)of gastric cancer cells,whereas overexpression of circ6718 produced the opposite effect.(3)Nucleoplasm isolation and RNA FISH experiments revealed that circ6718 was expressed both in the nucleus and cytoplasm of various gastric cancer cells,with a predominant distribution in the cytoplasm.RIP experiments demonstrated that circ6718bonded to the AGO2 protein.Combined with prediction from multiple databases and q RT-PCR experiments,downstream target molecules were identified.A dual luciferase reporter assay confirmed the binding of hsa-miR-561-3p to circ6718.Results from in vitro cellular functional assay showed that overexpression of hsa-miR-561-3p partially attenuated the pro-carcinogenic effects of circ6718.Further combined multiple database predictions and q RT-PCR experiments were performed to identify downstream target genes.A dual luciferase reporter gene assay confirmed that SAAL1 bonded to hsa-miR-561-3p,and knockdown of SAAL1 reversed the pro-carcinogenic effects of circ6718.SAAL1,as a serum amyloid analogue,played a pro-inflammatory and pro-cancer role.Co-IP and immunofluorescence experiments showed that SAAL1 interacted with the transcription factor PRRX1.Chromatin immunoprecipitation(Ch IP)and dual luciferase reporter gene experiments revealed that PRRX1 bonded to the promoter region of TGFβ1,thereby promoting its transcriptional activation and facilitating the activation of the TGFβ1/Smad2/3 signaling pathway.(4)Gastric cancer cell exosomes overexpressing circ6718 significantly promoted gastric cancer proliferation,migration,invasion,and EMT,additionally upregulated the expression of CAF markers in GC-MSCs.In contrast,knocking down circ6718 in exosomes yielded opposite effects.Moreover,in GC-MSCs,circ6718 was able to target the hsa-miR-561-3p/SAAL1/PRRX1axis,thereby activating the TGFβ1/Smad2/3 signaling pathway to promote the activation of GC-MSCs.(5)Successfully constructed exosomes of engineered HEK-293T cells loaded with circ6718 siRNA.Both in vitro and in vivo experiments confirmed that the engineered exosomes effectively inhibited the proliferation and metastasis of gastric cancer.Furthermore,biosafety assessments demonstrated that these engineered exosomes did not significantly affect the function of the liver,kidney,and other major organs of nude mice.Conclusions:Plasma exosomal circ6718 positively associated with poor prognosis and distal metastasis in gastric cancer patients,with potential for diagnostic and prognostic assessment.Gastric cancer cell exosomal circ6718 activates the TGFβ1/Smad2/3 pathway by targeting the hsa-miR-561-3p/SAAL1/PRRX1 axis,promoting gastric cancer cell proliferation,migration,invasion,and EMT process,and inducing GC-MSCs to differentiate into CAFs,creating a pro-metastatic microenvironment.In addition,circ6718 siRNA-engineered exosomes effectively inhibited tumor progression both in vivo and in vitro.更多还原