【作者】 赵坤；

【导师】 高春正；

【作者基本信息】 山东大学 ， 外科学（专业学位）， 2025， 博士

【摘要】 研究目的随着科学研究的深入,微小RNA(microRNA,miRNA)作为重要生物分子,因其在癌症患者体内的显著变化而受到广泛关注。这些miRNA分子不仅参与复杂的生物过程调控,还可能成为预测癌症患者预后、转移及病情发展的关键生物标志物。尽管这一领域进展迅速,关于肺癌骨转移(bone metastasis,BM)与外泌体来源miRNAs(exosomal miRNAs,exo-miRNAs)之间关系的研究仍较为匮乏。本研究旨在筛选与肺癌骨转移密切相关的exo-miRNAs,并初步评估其在预测肺癌骨转移中的潜在价值。通过高通量测序、生物信息学分析、细胞生物学实验和动物实验等方法,系统探索exo-miRNAs在肺癌骨转移中的表达特征、功能作用和具体分子机制,为肺癌的早期诊断、治疗及预后评估提供新的思路和方法。研究方法本次研究综合运用生物信息学、细胞生物学、分子生物学实验和动物实验等方法,深入探讨了肺癌骨转移相关的exo-miRNAs及其调控机制,并建立了一个有助于肺癌骨转移早期诊断和预后评价的miRNA标志物模型。具体而言,采用差速离心结合超速离心的高精度分离技术,从患者血清样本和细胞培养上清中提取外泌体。采用高通量测序技术全面检测exo-miRNAs,并结合生物信息学分析及ROC曲线分析,筛选出与肺癌骨转移诊断和治疗相关的候选exo-miRNAs。为进一步验证这些候选 miRNAs 的表达模式与功能作用,我们在肺癌患者样本及具有不同侵袭能力的肺癌细胞中开展表达检测,并通过实时定量PCR、细胞功能实验、动物模型和Western blotting(WB)等手段,深入分析其在肺癌骨转移中的具体调控机制。通过上述方法,构建了基于exo-miRNAs的预测模型,评估其与肺癌骨转移发生风险的相关性及诊断效能。进一步结合其来源的肿瘤特异性和分子稳定性,探讨其作为肺癌骨转移早期诊断标志物的潜在应用价值。研究结果为筛选肺癌骨转移相关的exo-miRNAs,使用具备不同生物学特性的肺癌细胞系A549和95D细胞培养上清中的外泌体,旨在识别普适性肺癌骨转移相关的exo-miRNAs。通过高通量测序与差异分析,研究鉴定出29个与肺癌骨转移紧密相关的exo-miRNAs,并预测它们调控101个骨转移相关靶基因,这些基因主要涉及癌细胞转录调控相关通路。进一步构建了由7个核心miRNA和10个关键枢纽基因构成的外泌体调控网络,揭示了 miRNA与靶基因之间的复杂相互作用。结合ROC曲线分析,结果显示miR-151a-3p和miR-877-5p在区分肺癌骨转移患者方面具有较高的诊断准确性,可能成为重要的生物标志物。分子机制和功能学实验选择来源于同一细胞株的95C和95D,它们在侵袭性上存在显著差异,为探索miRNA的作用机制提供了理想的研究模型。通过细胞迁移、侵袭、划痕愈合和上皮-间质转化等实验,研究发现这两个exo-miRNAs显著影响细胞的迁移和侵袭能力。进一步的检测结果表明,外泌体来源的miR-151a-3p抑制了 95C和95D肺癌细胞的迁移和侵袭能力,而miR-877-5p则显著促进了这两种细胞的迁移和侵袭能力。通过调节95D和95C细胞外泌体中miRNA的表达,反向验证了 miRNA在外泌体介导的细胞间通讯中的作用。最后,结合生物信息学分析、荧光素酶报告实验和WB实验等方法,筛选并验证了 miR-877-5p抑制靶基因NR4A3表达。转录组测序和WB实验发现,miR-877-5p通过抑制NR4A3表达,进而激活MAPK/ERK信号通路,促进95C和95D肺癌细胞的迁移和侵袭。动物实验进一步证实了 miR-877-5p在体内亦具有显著的促骨转移效应,进一步增强了研究的临床相关性与转化潜力。研究结论本研究成功鉴定了与肺癌骨转移密切相关的差异表达外泌体miRNA表达谱,并据此构建了外泌体miRNA-mRNA枢纽靶基因调控网络。结合ROC曲线分析,我们发现miR-151a-3p和miR-877-5p展现出良好的敏感度和肺癌骨转移特异性,提示它们具备成为新一代诊断生物标志物的巨大潜力。分子生物学、细胞生物学和动物实验进一步证实,miR-877-5p显著抑制了肺癌细胞的迁移、侵袭和EMT。机制上,miR-877-5p通过抑制NR4A3的表达,从而激活MAPK/ERK信号通路,促进肺癌细胞的迁移和侵袭。这些发现不仅加深了我们对肺癌骨转移分子机制的理解,还为该疾病的早期诊断、个性化治疗方案的制定及疗效评价提供了理论基础和应用前景。特别是在早期诊断方面,miR-877-5p的高敏感性和特异性使其有望成为临床上区分肺癌骨转移患者与非骨转移患者的重要工具,帮助医生更早地识别并干预疾病进程。本研究通过多层次手段(包括临床样本、体外实验和动物模型)系统验证了其在肺癌骨转移中的功能作用,这些发现为肺癌骨转移的早期诊断和精准治疗提供了理论基础与实践前景。更多还原

【Abstract】 Research ObjectiveWith the advancement of scientific research,microRNAs(miRNAs)have gained significant attention as important biological molecules due to their notable changes in cancer patients.These miRNA molecules are involved in regulating complex biological processes and may serve as key biomarkers for predicting prognosis,metastasis,and disease progression in cancer patients.Despite rapid progress in this field,investigations into the relationship between bone metastasis(BM)in lung cancer and exosome-derived miRNAs(exo-miRNAs)remain sparse.This study aims to identify exo-miRNAs closely associated with bone metastasis in lung cancer and to preliminarily assess their potential value in predicting such metastasis.Methods such as high-throughput sequencing,bioinformatics analysis,cellular biology experiments,and animal studies will be employed to explore the role and specific mechanisms of exo-miRNAs in lung cancer bone metastasis,providing new ideas and approaches for early diagnosis,treatment,and prognosis evaluation of lung cancer.MethodsThis study employs a combination of bioinformatics,cell biology,molecular biology experiments,and animal studies to thoroughly investigate exo-miRNAs associated with bone metastasis in lung cancer and their regulatory mechanisms.Additionally,we aim to establish a miRNA biomarker model that aids in the early diagnosis and prognosis evaluation of lung cancer bone metastasis.Specifically,highprecision separation techniques combining differential centrifugation with ultracentrifugation will be used to extract exosomes from patient serum samples and cell culture supernatants.High-throughput sequencing will comprehensively analyze exo-miRNAs,while bioinformatics and ROC curve analyses will be utilized to screen candidate exo-miRNAs related to the diagnosis and treatment of lung cancer bone metastasis.To further validate the specificity and related molecular mechanisms of these candidate exo-miRNAs in regulating lung cancer bone metastasis,we will use real-time quantitative PCR to assess the expression of candidate exo-miRNAs in a patient cohort.Through cellular,animal,and molecular biology experimental techniques,we will construct a prediction model based on exo-miRNAs,evaluating their role and specific mechanisms in regulating bone metastasis in lung cancer patients.The analysis will target the correlation between candidate miRNAs and the risk of lung cancer bone metastasis,evaluating their effectiveness in the diagnosis of such metastasis.By verifying the tumor specificity and high stability of exo-miRNAs,we will explore their potential as early diagnostic markers for lung cancer bone metastasis.ResultsTo identify exo-miRNAs related to lung cancer bone metastasis,exosomes from the culture supernatants of two lung cancer cell lines,A549 and 95D,with different biological characteristics were utilized to identify universally relevant exo-miRNAs linked to lung cancer bone metastasis.The study identified 29 exo-miRNAs closely associated with lung cancer bone metastasis and predicted their regulation of 101 bone metastasis-related target genes,which mainly involve pathways related to cancer cell transcriptional regulation.A regulatory network comprising 7 core miRNAs and 10 key hub genes was constructed,revealing the complex interactions between miRNAs and target genes.ROC curve analysis indicated that miR-151a-3p and miR877-5p exhibited high diagnostic accuracy in distinguishing lung cancer patients with bone metastasis,suggesting their potential as important biomarkers.For exploring the molecular mechanisms and functional experiments,the 95C and 95D cell lines,derived from the same parent cell line,were chosen due to their significant differences in invasiveness,providing an ideal model for studying the action mechanisms of miRNAs.Experiments,including cell migration,invasion,scratch healing,and epithelial-mesenchymal transition,revealed that these two exomiRNAs significantly affected the migration and invasion capabilities of the cells.Further results showed that exosomal miR-151a-3p inhibited the migration and invasion abilities of both 95C and 95D lung cancer cells,while miR-877-5p significantly promoted the migration and invasion of these two cell lines.By modulating the expression of miRNAs in exosomes from 95D and 95C cells,the role of miRNAs in exosome-mediated intercellular communication was validated.Ultimately,through bioinformatics analysis,luciferase assays,and Western blotting(WB)experiments,miR-877-5p was identified and validated as a suppressor of the target gene NR4A3 expression.Transcriptomic sequencing and WB results indicated that miR-877-5p activates the MAPK/ERK/signaling pathway by inhibiting NR4A3 expression,thereby promoting the migration and invasion of 95C and 95D lung cancer cells.Animal experiments further confirmed the role of miR-877-5p in lung cancer bone metastasis,enhancing the proliferation,migration,and invasion abilities of lung cancer cells.ConclusionThis study successfully identified a differentially expressed exosomal miRNA expression profile closely related to lung cancer bone metastasis and constructed an exosomal miRNA-mRNA hub target gene regulatory network based on these findings.Based on ROC curve analysis,we found that miR-151a-3p and miR-877-5p exhibit extremely high sensitivity and specificity for lung cancer bone metastasis,suggesting their significant potential as next-generation diagnostic biomarkers.Further molecular biology,cell biology,and animal experiments revealed that miR-877-5p markedly inhibited the migration,invasion,and EMT abilities of lung cancer cells.Mechanistically,miR-877-5p promotes the migration and invasion of lung cancer cells by inhibiting NR4A3 expression and activating the MAPK/ERK signaling pathway.These findings not only deepen our understanding of the molecular mechanisms underlying lung cancer bone metastasis but also provide a theoretical foundation and potential for the early diagnosis and development of personalized treatment strategies for this disease.Notably,in the context of early diagnosis,the high sensitivity and specificity of miR-877-5p position make it a promising tool for clinically distinguishing lung cancer patients with bone metastasis from those without,enabling physicians to identify and intervene in disease progression at an earlier stage.更多还原