Pancreatic adenocarcinoma (PAAD) is the eighth leading cause of cancer-related mortality that causes serious physical and mental burden to human. Reactive oxygen species accumulation and iron overload might enable ferroptosis-mediated cancer therapies. This study was to elusive novel ferroptosis regulator and its association with immune microenvironment and PD-L1 in PAAD. RNA-seq data and relevant information were obtained from The Cancer Genome Atlas and Genotype-Tissue Expression. The R packages “ggplot2” and “pheatmap” were used to the expression of 20 ferroptosis regulators between PAAD and normal tissues. The R package “ConsensusClusterPlus”, “survival”, “survminer”, “immunedeconv”, and TIDE algorithm performed consensus clustering, overall survival, progression-free survival, disease free survival, immune infiltration level, and immunotherapy responses between cluster 1 and cluster 2. The prognostic value was confirmed by the Kaplan–Meier curves, receiver operating characteristic curve, univariate and multivariate cox regression, and nomogram. Moreover, the relationship of FANCD2 and immunity, drug sensitivity was investigated by R package “ggstatsplot”, “immunedeconv”, “ggalluvial” and “pRRophetic”. Besides, the qRT-PCR, immunohistochemistry and western blotting detected the expression of FANCD2 in PAAD cell lines. Most ferroptosis regulators were up-regulated in PAAD, while the expression of LPCAT3, MT1G, and GLS2 was down-regulated in PAAD (P < 0.05), indicting there was a positively correlation among ferroptosis regulators. Based on clustering parameter, we identified cluster 1 and cluster 2, and cluster 2 had a better prognosis for patients with PAAD. The immune infiltration level of cluster 1 was higher in macrophage M1, myeloid dendritic cell, T cell CD4 + Th2, B cell, T cell CD8 + central memory, immune score, and microenvironment score than cluster 2 in PAAD. Moreover, FANCD2 was up-regulated in PAAD by public databases, immunohistochemistry, qRT-PCR and Western blotting, which had closely related to overall survival, immune microenvironment, and drug sensitivity. A novel crosstalk of ferroptosis exhibits a favourable prognostic performance and builds a robust theoretical foundation for mRNA vaccine and personalized immunotherapy. FANCD2 could be an effective for prognostic recognition, immune efficacy evaluation, and mRNA vaccine for patients with PAAD, providing a vital guidance for further study of regulating tumor immunity and vaccine development.

BackgroundIncreasing evidence elucidated N6-methyladenosine (m6A) dysregulation participated in regulating RNA maturation, stability, and translation. This study aimed to demystify the crosstalk between m6A regulators and the immune microenvironment, providing a potential therapeutic target for patients with hepatocellular carcinoma (HCC).MethodsTotals of 371 HCC and 50 normal patients were included in this study. GSE121248 and GSE40367 datasets were used to validate the expression of HNRNPC. The R package “ConsensusClusterPlus” was performed to screen consensus clustering types based on the expression of m6A regulators in HCC. The R package “pheatmap”, “immunedeconv”, “survival”, “survminer” and “RMS” were applied to investigate the expression, immunity, overall survival, and clinical application in different clusters and expression groups. Comprehensive analysis of HNRNPC in pan-cancer was conducted by TIMER2 database. Besides, HNRNPC mRNA and protein expression were verified by qRT-PCR and immunohistochemistry analysis.ResultsMost of m6A regulators were over-expressed excerpt for ZC3H13 in HCC. Three independent clusters were screened based on m6A regulators expression, and the cluster 2 had a favorable prognosis in HCC. Then, the cluster 2 was positively expression in macrophage, hematopoietic stem cell, endothelial cell, and stroma score, while negatively in T cell CD4+ memory and mast cell. We identified HNRNPC was an independent prognostic factor in HCC, and nomogram performed superior application value for clinical decision making. Moreover, PD-L1 was significantly up-regulated in HCC tissues, cluster 1, and cluster 3, and we found PD-L1 expression was positively correlated with HNRNPC. Patients with HCC in high-expression groups was associated with tumor-promoting cells. Besides, HNRNPC was correlated with prognosis, TMB, and immune checkpoints in cancers. Particularly, the experiments confirmed that HNRNPC was positively expression in HCC cells and tissues.ConclusionThe m6A regulators play irreplaceable roles in prognosis and immune infiltration in HCC, and the relationship of HNRNPC and PD-L1 possesses a promising direction for therapeutic targets of immunotherapy response. Exploration of m6A regulators pattern could be build the prognostic stratification of individual patients and move toward to personalized treatment.

CLSPN, an essential molecule of the S-phase checkpoint in DNA replication stress, have not been elucidated in hepatocellular carcinoma (HCC). In this study, we firstly discovered and systematically verified CLSPN expression using RT-qPCR and western blotting assay, and its high expression was an independent prognostic factor in HCC. Patients with CLSPN low-expression had higher infiltration levels of T cell CD4 + memory resting, monocyte, mast cell activated, dryness index and lower immune response in HCC. Then, CLSPN silencing inhibited the proliferation, migration, invasion and cell cycle progression of HCC cells proved by CCK-8, transwell and cell cycle assay. We established a key lncRNA PSMA3-AS1/hsa-miR-101-3p/CLSPN regulator axis in HCC. Furthermore, CLSPN-mediated ubiquitination or deubiquitination may regulate post-transcriptional modifications in HCC. The emerging CLSPN potentialities might be mediated by the β-catenin-mediated Wnt signaling pathway. In addition, we detected CLSPN interaction protein profile, which further confirmed CLSPN involved in posttranscriptional modification, protein turnover, and biogenesis locating in cytoplasm, secreted, and mitochondrion. Therefore, it was the first time to discover and verify expression, prognosis, immunotherapy, RNAs regulator, posttranscriptional modification, and molecular mechanisms of CLSPN in HCC. These novel insights might accelerate the process of individualized diagnosis and precision therapeutics for patients with HCC.

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer deaths worldwide, seriously affecting human community health and care. Emerging evidence has shown that aberrant glycosylation is associated with tumor progression and metastasis. However, the role of glycosylation-related genes in HCC has notbeen reported.MethodsWeighted gene coexpression network analysis and non-negative matrix factorization analysis were applied to identify functional modules and molecularm subtypes in HCC. The least absolute shrinkage and selection operator Cox regression was used to construct the glycosylation-related signature. The independent prognostic value of the risk model was confirmed and validated by systematic techniques, including principal component analysis, T-distributed random neighbor embedding analysis, Kaplan–Meier survival analysis, the ROC curve, multivariate Cox regression, the nomogram, and the calibration curve. The single-sample gene set enrichment analysis, gene set variation analysis, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were evaluated by the immune microenvironment and potential biological processes. The quantitative real-time polymerase chain reaction and immunohistochemistry analysis were used to verify the expression of five genes.ResultsWe identified the glycosylation-related genes with bioinformatics analysis to construct and validate a five-gene signature for the prognosis of HCC patients. Patients with HCC in the high-risk group had a worse prognosis. The risk score could be an independent factor and was associated with clinical features, such as the grade and stage. The nomogram exhibited an accurate score that included the risk score and clinical parameters. The infiltration levels of antitumor cells were upregulated in the low-risk group, including B_cells, Mast_cells, neutrophils, NK_cells, and T_helper_cells. Moreover, glycosylation was more sensitive to immunotherapy, and may play a critical role in the metabolic processes of HCC, such as bile acid metabolism and fatty acid metabolism. In addition, the five-gene messenger RNA (mRNA) and protein expression were overexpressed in HCC cells and tissues.ConclusionsThe glycosylation-related signature is effective for prognostic recognition, immune efficacy evaluation, and substance metabolism in HCC, providing a novel insight for therapeutic target prediction and clinical decision-making.