Abstract：
BACKGROUND: PDAC, also known as pancreatic ductal adenocarcinoma, is often diagnosed at a late stage due to nonspecific symptoms and a distinct lack of reliable biomarkers for timely diagnosis. Ferroptosis, a novel non-apoptotic cell death mode discovered in recent years, is strongly linked to the progression of PDAC and the evasion of the immune system. The objective of this study is to discover a novel ceRNA biomarker associated with ferroptosis and investigate its possible molecular mechanisms and therapeutic potential in PDAC.
METHODS: Based on the FerrDb and TCGA databases, the R survival package was used to screen for ferroptosis-related mRNAs associated with PDAC prognosis. The ferroptosis-related ceRNA network was identified by miRTarBase, miRNet, and starBase and visualized using Cytoscape. The LASSO regression analysis was used to build a risk model associated with ceRNA. Additionally, we investigated the correlation between the ceRNA axis and the infiltration of immune cells in PDAC by employing the ssGSEA algorithm. Spearman correlation analysis was used to investigate the association between the ceRNA network and the expression levels of immune checkpoint genes in PDAC. The prediction of potential medications for PAAD patients with high risk scores was conducted using the R package oncoPredict and the Genomics of Drug Sensitivity in Cancer (GDSC) repository. Expression levels of LINC02535 in clinical specimens and PDAC cell lines were determined using qRT-PCR. CCK-8, colony formation, EdU, wound healing, and transwell assays were performed to assess the impact of reducing LINC02535 on the growth, migration, and invasion of PDAC cell lines BxPC3 and PANC1.
RESULTS: We first discovered a new LINC02535/miR-30c-5p/EIF2S1 axis associated with ferroptosis and created a prognostic nomogram for predicting overall survival. Meanwhile, the risk scores of the LINC02535/miR-30c-5p/EIF2S1 axis associated with ferroptosis were linked to immune subtypes in PDAC. The high immune infiltration subtype exhibited elevated ceRNA risk scores and EIF2S1 expression. The correlation analysis revealed a positive correlation between ceRNA risk scores and four immune cells, namely Activated CD4 T cell, Memory B cell, Neutrophil, and Type 2 T helper cell, as well as four immune checkpoint genes, namely CD274, HAVCR2, PDCD1LG2, and TIGIT. The analysis of drug sensitivity indicated that individuals with a high-risk score may exhibit greater sensitivity to inhibitors targeting MEK1/2 compared to those with a low-risk score. In our validation experiments, it was observed that the expression of LINC02535 was increased in both PDAC tissues and cell lines. Additionally, the inhibition of LINC02535 resulted in decreased proliferation, migration, and invasion of PDAC cells. Rescue experiments demonstrated that LINC02535 promoted PDAC cell growth and metastasis by upregulating EIF2S1 expression.
CONCLUSIONS: To summarize, a novel ferroptosis-associated LINC02535/miR-30c-5p/EIF2S1 ceRNA network for PDAC patients was established. The analysis of this network\'s functionality offers potential insights for clinical decision-making and the advancement of precision medicine.
METHODS: Based on the FerrDb and TCGA databases, the R survival package was used to screen for ferroptosis-related mRNAs associated with PDAC prognosis. The ferroptosis-related ceRNA network was identified by miRTarBase, miRNet, and starBase and visualized using Cytoscape. The LASSO regression analysis was used to build a risk model associated with ceRNA. Additionally, we investigated the correlation between the ceRNA axis and the infiltration of immune cells in PDAC by employing the ssGSEA algorithm. Spearman correlation analysis was used to investigate the association between the ceRNA network and the expression levels of immune checkpoint genes in PDAC. The prediction of potential medications for PAAD patients with high risk scores was conducted using the R package oncoPredict and the Genomics of Drug Sensitivity in Cancer (GDSC) repository. Expression levels of LINC02535 in clinical specimens and PDAC cell lines were determined using qRT-PCR. CCK-8, colony formation, EdU, wound healing, and transwell assays were performed to assess the impact of reducing LINC02535 on the growth, migration, and invasion of PDAC cell lines BxPC3 and PANC1.
RESULTS: We first discovered a new LINC02535/miR-30c-5p/EIF2S1 axis associated with ferroptosis and created a prognostic nomogram for predicting overall survival. Meanwhile, the risk scores of the LINC02535/miR-30c-5p/EIF2S1 axis associated with ferroptosis were linked to immune subtypes in PDAC. The high immune infiltration subtype exhibited elevated ceRNA risk scores and EIF2S1 expression. The correlation analysis revealed a positive correlation between ceRNA risk scores and four immune cells, namely Activated CD4 T cell, Memory B cell, Neutrophil, and Type 2 T helper cell, as well as four immune checkpoint genes, namely CD274, HAVCR2, PDCD1LG2, and TIGIT. The analysis of drug sensitivity indicated that individuals with a high-risk score may exhibit greater sensitivity to inhibitors targeting MEK1/2 compared to those with a low-risk score. In our validation experiments, it was observed that the expression of LINC02535 was increased in both PDAC tissues and cell lines. Additionally, the inhibition of LINC02535 resulted in decreased proliferation, migration, and invasion of PDAC cells. Rescue experiments demonstrated that LINC02535 promoted PDAC cell growth and metastasis by upregulating EIF2S1 expression.
CONCLUSIONS: To summarize, a novel ferroptosis-associated LINC02535/miR-30c-5p/EIF2S1 ceRNA network for PDAC patients was established. The analysis of this network\'s functionality offers potential insights for clinical decision-making and the advancement of precision medicine.
摘要：
背景：PDAC，也被称为胰腺导管腺癌，由于非特异性症状和明显缺乏及时诊断的可靠生物标志物，通常在晚期诊断。Ferroptosis,近年来发现的一种新的非凋亡细胞死亡模式，与PDAC的进展和免疫系统的逃避密切相关。本研究的目的是发现一种与铁凋亡相关的新型ceRNA生物标志物，并研究其在PDAC中的可能分子机制和治疗潜力。
方法：基于FerrDb和TCGA数据库，使用R生存包筛选与PDAC预后相关的铁死亡相关mRNA.通过miRTarBase鉴定铁凋亡相关的ceRNA网络，miRNet,和starBase，并使用Cytoscape可视化。LASSO回归分析用于建立与ceRNA相关的风险模型。此外,我们采用ssGSEA算法研究了ceRNA轴与PDAC中免疫细胞浸润之间的相关性。使用Spearman相关性分析来研究ceRNA网络与PDAC中免疫检查点基因表达水平之间的关联。使用R包oncoPredict和癌症药物敏感性基因组学（GDSC）资料库对具有高风险评分的PAAD患者的潜在药物进行预测。使用qRT-PCR测定临床样本和PDAC细胞系中LINC02535的表达水平。CCK-8,集落形成,EdU,伤口愈合，和transwell测定进行评估减少LINC02535对生长的影响，迁移,以及PDAC细胞系BxPC3和PANC1的侵袭。
结果：我们首次发现了一个新的LINC02535/miR-30c-5p/EIF2S1轴与铁性凋亡相关，并创建了预测总生存期的预后列线图。同时,与铁凋亡相关的LINC02535/miR-30c-5p/EIF2S1轴的风险评分与PDAC中的免疫亚型相关.高免疫浸润亚型表现出升高的ceRNA风险评分和EIF2S1表达。相关性分析显示，ceRNA风险评分与四种免疫细胞呈正相关，即活化的CD4T细胞，记忆B细胞，中性粒细胞,和2型辅助T细胞，以及四个免疫检查点基因，即CD274、HAVCR2、PDCD1LG2和TIGIT。药物敏感性分析表明，与具有低风险评分的个体相比，具有高风险评分的个体可能对靶向MEK1/2的抑制剂表现出更高的敏感性。在我们的验证实验中，观察到LINC02535的表达在PDAC组织和细胞系中均增加。此外,LINC02535的抑制导致增殖减少，迁移,和PDAC细胞的侵袭。挽救实验表明，LINC02535通过上调EIF2S1表达促进PDAC细胞生长和转移。
结论：总结一下，我们为PDAC患者建立了一个新的铁凋亡相关LINC02535/miR-30c-5p/EIF2S1ceRNA网络.对该网络功能的分析为临床决策和精准医学的发展提供了潜在的见解。
方法：基于FerrDb和TCGA数据库，使用R生存包筛选与PDAC预后相关的铁死亡相关mRNA.通过miRTarBase鉴定铁凋亡相关的ceRNA网络，miRNet,和starBase，并使用Cytoscape可视化。LASSO回归分析用于建立与ceRNA相关的风险模型。此外,我们采用ssGSEA算法研究了ceRNA轴与PDAC中免疫细胞浸润之间的相关性。使用Spearman相关性分析来研究ceRNA网络与PDAC中免疫检查点基因表达水平之间的关联。使用R包oncoPredict和癌症药物敏感性基因组学（GDSC）资料库对具有高风险评分的PAAD患者的潜在药物进行预测。使用qRT-PCR测定临床样本和PDAC细胞系中LINC02535的表达水平。CCK-8,集落形成,EdU,伤口愈合，和transwell测定进行评估减少LINC02535对生长的影响，迁移,以及PDAC细胞系BxPC3和PANC1的侵袭。
结果：我们首次发现了一个新的LINC02535/miR-30c-5p/EIF2S1轴与铁性凋亡相关，并创建了预测总生存期的预后列线图。同时,与铁凋亡相关的LINC02535/miR-30c-5p/EIF2S1轴的风险评分与PDAC中的免疫亚型相关.高免疫浸润亚型表现出升高的ceRNA风险评分和EIF2S1表达。相关性分析显示，ceRNA风险评分与四种免疫细胞呈正相关，即活化的CD4T细胞，记忆B细胞，中性粒细胞,和2型辅助T细胞，以及四个免疫检查点基因，即CD274、HAVCR2、PDCD1LG2和TIGIT。药物敏感性分析表明，与具有低风险评分的个体相比，具有高风险评分的个体可能对靶向MEK1/2的抑制剂表现出更高的敏感性。在我们的验证实验中，观察到LINC02535的表达在PDAC组织和细胞系中均增加。此外,LINC02535的抑制导致增殖减少，迁移,和PDAC细胞的侵袭。挽救实验表明，LINC02535通过上调EIF2S1表达促进PDAC细胞生长和转移。
结论：总结一下，我们为PDAC患者建立了一个新的铁凋亡相关LINC02535/miR-30c-5p/EIF2S1ceRNA网络.对该网络功能的分析为临床决策和精准医学的发展提供了潜在的见解。
