List of DEmiRNAs identified from GSE119481.

One of the critical challenges in managing colorectal cancer (CRC) is the development of oxaliplatin (OXP) resistance. Long non-coding RNAs (lncRNAs) have a crucial role in CRC progression and chemotherapy resistance, with exosomal lncRNAs emerging as potential biomarkers. This study aimed to predict key lncRNAs involved in OXP-resistance using in-silico methods and validate them using RT-qPCR methods in CRC cells and their isolated exosomes. Two public datasets, GSE42387 and GSE119481, were downloaded from the GEO database to identify differentially expressed genes (DEGs) and miRNAs (DEmiRNAs) associated with OXP-resistance in the HCT116 cell line. The analysis of GSE42387 revealed 210 DEGs, and GSE119481 identified 73 DEmiRNAs. A protein-protein interaction (PPI) network analysis of the DEGs identified 133 interconnected genes, from which the top ten genes with the highest degree scores were selected. By intersecting predicted miRNAs targeting these genes with the DEmiRNAs, 38 common miRNAs were found. Subsequently, 224 lncRNAs targeting these common miRNAs were predicted. LncRNA-miRNA-mRNA network were constructed and the top five lncRNAs with the highest degree scores were identified. Analysis using the Kaplan-Meier plotter database revealed that the key lncRNAs NEAT1, OIP5-AS1, and MALAT1 are significantly associated with the overall survival of CRC patients. To validate these lncRNAs, OXP-resistant HCT116 sub-cell line (HCT116/OXR) was developed by exposing parental HCT116 cells to gradually increasing concentrations of OXP. Exosomes derived from both HCT116 and HCT116/OXR cells were isolated and characterized utilizing dynamic light scattering (DLS), transmission electron microscopy (TEM), and Western blotting. RT-qPCR confirmed elevated levels of NEAT1, OIP5-AS1, and MALAT1 in HCT116/OXR cells and their exosomes compared to parental HCT116 cells and their exosomes. This study concludes that NEAT1, OIP5-AS1, and MALAT1 are associated with the OXP-resistance in CRC. The high levels of these lncRNAs in exosomes of resistant cells suggest their involvement in intercellular communication and resistance propagation. This positioning makes them promising biomarkers for OXP-resistance in CRC.

结直肠癌（colorectal cancer, CRC）诊疗面临的关键挑战之一，是奥沙利铂（oxaliplatin, OXP）耐药的产生。长链非编码RNA（long non-coding RNAs, lncRNAs）在结直肠癌进展与化疗耐药中发挥关键作用，而外泌体长链非编码RNA正成为潜在的生物标志物。本研究旨在通过计算机模拟方法筛选参与奥沙利铂耐药的关键长链非编码RNA，并在结直肠癌细胞及其分离的外泌体中通过实时定量聚合酶链反应（RT-qPCR）进行验证。本研究从GEO数据库下载了两个公共数据集GSE42387与GSE119481，用于鉴定HCT116细胞系中与奥沙利铂耐药相关的差异表达基因（differentially expressed genes, DEGs）与差异表达微小RNA（differentially expressed miRNAs, DEmiRNAs）。对GSE42387的分析筛选出210个差异表达基因，对GSE119481的分析则鉴定出73个差异表达微小RNA。通过对差异表达基因进行蛋白质相互作用（protein-protein interaction, PPI）网络分析，得到133个相互关联的基因，并从中选取连接度得分最高的10个核心基因。将靶向这些核心基因的预测微小RNA与差异表达微小RNA取交集，共得到38个共有微小RNA。随后预测得到224个靶向这些共有微小RNA的长链非编码RNA。构建长链非编码RNA-微小RNA-信使RNA（lncRNA-miRNA-mRNA）调控网络，并筛选出连接度得分最高的5个关键长链非编码RNA。通过Kaplan-Meier绘图仪数据库分析发现，关键长链非编码RNA NEAT1、OIP5-AS1及MALAT1与结直肠癌患者的总生存期显著相关。为验证上述长链非编码RNA，本研究通过逐步递增奥沙利铂浓度的方式，诱导亲本HCT116细胞构建奥沙利铂耐药的HCT116亚细胞系（HCT116/OXR）。分离并鉴定了亲本HCT116细胞与HCT116/OXR细胞分泌的外泌体，所用方法包括动态光散射（dynamic light scattering, DLS）、透射电子显微镜（transmission electron microscopy, TEM）及蛋白质印迹（Western blotting）。实时定量聚合酶链反应结果证实，相较于亲本HCT116细胞及其外泌体，HCT116/OXR细胞及其外泌体中NEAT1、OIP5-AS1与MALAT1的表达水平显著升高。本研究最终得出结论：NEAT1、OIP5-AS1及MALAT1与结直肠癌奥沙利铂耐药相关。耐药细胞外泌体中这些长链非编码RNA的高表达水平提示其参与了细胞间通讯与耐药性传播，有望成为结直肠癌奥沙利铂耐药的潜在生物标志物。