DNA Replication Pathways Research Articles

RFC3 serves as a novel prognostic biomarker and target for head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer that originates from the squamous cells. The role of the replication factor C subunit 3 (RFC3) in HNSCC progression remains elusive. The aim of this study was to uncover RFC3 significance in HNSCC. The Cancer Genome Atlas (TCGA-HNSCC) dataset was initially used to assess RFC3 expression and its association with HNSCC clinical features. Subsequently, quantitative reverse transcription PCR (RT-qPCR) confirmed RFC3 mRNA expression in oral squamous cell carcinoma (OSCC), a primary HNSCC type. Survival rates were evaluated using the Kaplan-Meier plot, while the Tumor Immune Estimation Resource (TIMER) database probed RFC3-immune cell interaction. Additionally, in silico tools were used to examine the RFC3 protein network and engagement in HNSCC pathways. RFC3 expression is significantly upregulated in HNSCC, including OSCC. Upregulated RFC3 expression was significantly correlated with the clinicopathological features of HNSCC, including tumor stage, grade, metastasis, and patient survival. RFC3 is also associated with immune cell infiltration. Functional analysis has highlighted its involvement in DNA replication, mismatch repair, and cell cycle pathways. Interestingly, RFC3 high expression is linked to well-known oncogenic signaling pathways, such as MYC/MYCN, HIPPO, and mTOR. In conclusion, RFC3 can be considered a novel prognostic biomarker for HNSCC, and further studies on its functional mechanisms may help to use RFC3 as a therapeutic target for HNSCC. The clinical relevance of this study lies in identifying RFC3 as a novel biomarker and prognostic indicator for HNSCC, offering insights that could impact future clinical approaches.

MiR-3200 accelerates the growth of liver cancer cells by enhancing Rab7A

Researches indicate miR-3200 is closely related to tumorigenesis, However, the role of miR-3200 in human hepatocarcinogenesis is still unclear. In this study, we clearly demonstrate that miR-3200 accelerates the growth of liver cancer cells in vivo and in vitro. Obviously, these findings are noteworthy that miR-3200 affects the transcriptional regulation for several genes, including DSP，BABAM2, Rab7A,SQSTM1,PRKAG2,CDK1,ABCE1,BECN1,PTEN,UPRT. And miR-3200 affects the transcriptional ability of several genes, such as, upregulating CADPS, DSP,FBXO32, PPCA,SGK1, PATXN7L1, PLK2,ITGB5,FZD3,HOXC8,HSPA1A,C-Myc,CyclnD1,CyclinE,PCNA and down –regulating SUV39H1, MYO1G, OLFML3, CBX5, PPDE2A, HOXA7, RAD54L, CDC45,SHMT7,MAD2L1,P27,IQGAP3,PTEN,P57,SCAMP3,etc...On the other hand, it is obvious that miR-3200 affects the translational ability of several genes, such as, upregulating GNS,UPRT,EIFAD,YOS1,SGK1,K-Ras,PKM2,C-myc,Pim1,CyclinD1,mTOR,erbB-2,CyclinE,PCNA,RRAS,ARAF,RAPH1,etc.. and down-regulating KDM2A, AATF, TMM17B, RAB8B, MYO1G,P21WAF1/Cip1,GADD45,PTEN,P27,P18,P57,SERBP1,RPL34,UFD1,Bax,ANXA6,GSK3β. Strikingly, miR-3200 affects some signaling pathway in liver cancer, including carbon metabolism signaling pathway, DNA replication pathway, FoxO signaling pathway, Hippo signaling pathway, serine and threonine metabolism signaling pathway, mTOR signaling pathway, Fatty acid biosynthesis signaling pathway, carcinogenesis-receptor activation signaling pathway, autophagy signaling pathway. Furthermore, our results suggest that miR-3200 enhances expression of RAB7A, and then Rab7A regulates the carcinogenic function of miR-3200 by increasing telomere remodeling in human liver cancer. These results are of great significance for the prevention and treatment of human liver cancer.

Mutational Landscape of ER Receptor Negative Endometrial Cancer Patients Categorized as No Specific Molecular Profile (NSMP)

The endometrial cancer (EC) molecular classification introduced by The Cancer Genome Atlas (TCGA) has initiated molecular-based classification with clear prognostic value. Four major tumor subtypes exist: 1) TP53 mutation (p53abn); 2) POLE mutation (POLEmut); 3) mismatch repair protein deficient (MMRd); and 4) no specific molecular profile (NSMP). Of these subtypes, p53abn patients exhibited the worst prognosis. Recently, a prognostic refinement of NSMP EC using estrogen receptor (ER) status, from PORTEC-III trial data, showed that (ER-) NSMP patients had a strikingly poor prognosis, similar to p53abn patients. Currently, drivers of NSMP (ER-) disease are unknown and require genomic characterization. In this study, we perform an integrated molecular analysis to characterize this aggressive and poorly understood cohort. RNAseq and mutational data was downloaded from the TCGA, PanCancer Atlas, Uterine Endometrial Carcinoma dataset via the genomic data commons (GDC). All patients in this cohort demonstrated endometrioid histology. P53abn, and POLE mutated patients were filtered using mutational data. MMRd and NSMP ESR1 gene low expressors (ER Low) were filtered using Z-score cutoffs of RNAseq expression data. Survival analysis was performed using the Survival R-package. Gene expression testing was performed using the EdgeR R-package. Pathway Enrichment analysis was conducted using EnrichR software. Forty-two patients out of 529 samples met our GDC NSMP (ER low) filtering criteria (7%), similar to the 5% of PORTEC-III patients classified as NSMP (ER -). Strikingly 83% percent of GDC NSMP (ER low) patients harbored a mutation in the PI3K-AKT-mTOR signaling pathway, with 48% of GDC NSMP (ER low) patients carrying a mutation in the p110 alpha (PIK3CA) gene. Interestingly, of the patients with PIK3CA mutant tumors, 2/20 patients died in 5 years (10%), compared to 9/22 patients whose tumors were PIK3CA WT (41%) (p = 0.02). Among GDC NSMP (ER low) patients with PIK3CA WT tumors gene expression signatures were enriched for MYC target genes (p = 1.1e-51), DNA replication (p = 4.3e-30), cell cycle (p = 9.32e-20), and cell cycle checkpoints (p = 6.4e-20) pathways. Additionally, strongly in PIK3CA WT tumors are mitochondrial membrane proteins (p = 1.63e-37) and ribosomal proteins (2.9e-24). In this analysis, we show that GDC NSMP (ER low) tumors nearly all harbor a mutation in the PI3K-AKT-mTOR signaling pathway. Further we demonstrate that the most common gene mutation in the cohort, PIK3CA, is counterintuitively a marker of improved survival. Additionally, we show that within that this subpopulation, PIK3CA WT patients exhibit robustly upregulated gene expression programs dedicated to energy production, cell cycling, and division.

The difference of intestinal microbiota composition between Lantang and Landrace newborn piglets

BackgroundThe early development of intestinal microbiota plays a fundamental role in host health and development. To investigate the difference in the intestinal microbial composition between Lantang and Landrace newborn piglets, we amplified and sequenced the V3-V4 region of 16 S rRNA gene in jejunal microbiota of Lantang and landrace newborn.ResultsThe findings revealed that the dominant phyla in the jejunum of Lantang piglets were Firmicutes, Actinobacteria and Bacteroidetes, while the dominant phyla of Landrace is Proteobacteria and Fusobacteria. Specifically, Corynebacterium_1, Lactobacillus, Rothia, Granulicatella, Corynebacteriales_unclassified, Corynebacterium, Globicatella and Actinomycetales_unclassified were found to be the dominant genera of Lantang group, while Clostridium_sensu_stricto_1, Escherichia-Shigella, Actinobacillus and Bifidobacterium were the dominant genera of Landrace. Based on the functional prediction of bacteria, we found that bacterial communities from Lantang samples had a significantly greater abundance pathways of fatty acid synthesis, protein synthesis, DNA replication, recombination, repair and material transport across membranes, while the carrier protein of pathogenic bacteria was more abundant in Landrace samples.ConclusionsOverall, there was a tremendous difference in the early intestinal flora composition between Landang and Landrace piglets, which was related to the breed characteristics and may be one of the reasons affecting the growth characteristics. However, more further extensive studies should be included to reveal the underlying relationship between early intestinal flora composition in different breeds and pig growth characteristics.

Comprehensive analysis and validation of SNX7 as a novel biomarker for the diagnosis, prognosis, and prediction of chemotherapy and immunotherapy response in hepatocellular carcinoma

BackgroundStudies have demonstrated that Sorting nexin 7 (SNX7) functions as an anti-apoptotic protein in liver tissue and plays a crucial role in the survival of hepatocytes during early embryonic development. However, its diagnostic and prognostic value as well as the predictive value of chemotherapy and immunotherapy have not been reported in hepatocellular carcinoma (HCC).MethodsSNX7 mRNA expression and its diagnostic efficacy were examined in GEO datasets, and the findings were further confirmed in TCGA, ICGC cohorts, and cell lines. The protein level of SNX7 was determined using CPTAC and HPA databases, and the results were validated through immunohistochemistry (IHC). Survival analyses were performed in TCGA and ICGC cohorts, and the results were subsequently validated via Kaplan–Meier Plotter. The response to chemotherapy and immunotherapy was predicted via GDSC dataset and TIDE algorithm, respectively. R packages were employed to explore the relationship between SNX7 expression and immune infiltration, m6A modification, as well as the functional enrichment of differentially expressed genes (DEGs).ResultsThe expression of SNX7 at both mRNA and protein levels was significantly upregulated in HCC tissues. SNX7 exhibited superior diagnostic efficacy compared to AFP alone for HCC detection, and combining it with AFP improved the diagnostic accuracy for HCC. High SNX7 was associated with unfavorable outcomes, including poor overall survival, disease-specific survival, progression-free survival, and advanced pathological stage, in patients with HCC, and SNX7 was identified as an independent risk factor for HCC. Moreover, elevated SNX7 expression was positively correlated with increased sensitivity to various chemotherapy drugs, including sorafenib, while it was associated with resistance to immunotherapy in HCC patients. Correlation analysis revealed a relationship between SNX7 and multiple m6A-related genes and various immune cells. Finally, enrichment analysis demonstrated strong associations of SNX7 with critical biological processes, such as cell cycle regulation, cellular senescence, cell adhesion, DNA replication, and mismatch repair pathway in HCC.ConclusionsOur study highlights the association of SNX7 with the immune microenvironment and its potential influence on HCC progression. SNX7 emerges as a promising novel biomarker for the diagnosis, prognosis, and prediction of response to chemotherapy and immunotherapy in patients with HCC.

Overexpression of Nei endonuclease VIII-like protein 3 in hepatocellular carcinoma indicates increased levels of immune infiltration and an unfavorable prognosis

Objective: To evaluate the role and molecular mechanism of Nei endonuclease VIII-like protein 3 (NEIL3) in hepatocellular carcinoma (HCC) through The Cancer Genome Atlas database. Methods: RNA sequencing of HCC samples was the first step in determining the level of gene NEIL3 expression in normal tissues and tumors. Then, NEIL3 was used for the Gene Ontology, the Kyoto Encyclopedia of Genes and Genomes, gene enrichment analysis, immune cell infiltration analysis. The samples were divided into high and low expression groups according to the median expression level of NEIL3 in liver cancer tissues. Logistic regression analysis, Kaplan-Meier analysis, univariate and multivariate Cox regression analysis, and a nomogram prognostic model were used to explore the clinical and prognostic significance of NEIL3 in HCC. Results: Compared with normal samples, NEIL3 was highly expressed in most malignant tumors, including HCC (P < 0.05). High expression of NEIL3 was related to cell cycle, DNA replication, and cell receptor pathways. In addition, the high expression of NEIL3 was significantly positively correlated with T-helper 2 lymphocytes and infiltration levels (R = 0.670, P < 0.001). Compared with the NEIL3 low expression group, the NEIL3 high expression group had a higher level of Th2 cell infiltration in tumor tissues (P < 0.001). Logistic regression analysis showed that NEIL3 overexpression was associated with high T stage, high pathological stage, high tissue grade, AFP > 400 μg/L and vascular invasion of HCC. The Kaplan-Meier analysis results showed that overall survival [hazard ratio (HR) = 2.53, P < 0.001)], disease-specific survival (HR = 2.52, P < 0.001), and progression-free interval (HR = 1.82, P < 0.001) in patients with HCC with high NEIL3 expression were unfavorable. Cox regression analysis results showed that high NEIL3 expression was an independent risk factor for an unfavorable prognosis in HCC patients (P = 0.002). The nomogram and calibration chart further demonstrated that high NEIL3 expression was one of the risk factors for an unfavorable prognosis in HCC patients. Conclusion: Elevated expression of NEIL3 is associated with an unfavorable prognosis and an increased proportion of immune cells in HCC, and it is likely to be used as a potential biomarker for evaluating the prognosis and immune infiltration level.

Comprehensive analysis of Cuproplasia and immune microenvironment in lung adenocarcinoma.

Background: Trace elements such as copper are essential for human health. Recently the journal Nat Rev Cancer has put forward the concept of Cuproplasia, a way of promoting tumor growth through reliance on copper. We attempted to conduct a comprehensive analysis of Cuproplasia-related genes in lung adenocarcinoma (LUAD) to explore the mechanism of action of Cuproplasia-related genes in LUAD. Method: Transcriptome data and clinical information of LUAD were obtained from TCGA-LUAD and GSE31210, and prognostic models of Cuproplasia-related genes were constructed and verified by regression analysis of GSVA, WGCNA, univariate COX and lasso. The signal pathways affected by Cuproplasia-related genes were analyzed by GO, KEGG and hallmarK pathway enrichment methods. Five immunocell infiltration algorithms and IMVIGOR210 data were used to analyze immune cell content and immunotherapy outcomes in the high-low risk group. Results: In the results of WGCNA, BROWN and TURQUOISE were identified as modules closely related to Cuproplasia score. In the end, lasso regression analysis established a Cuproplasia-related signature (CRS) based on 24 genes, and the prognosis of high-risk populations was worse in TCGA-LUAD and GSE31210 datasets. The enrichment analysis showed that copper proliferation was mainly through chromosome, cell cycle, dna replication, g2m checkpoint and other pathways. Immunoinfiltration analysis showed that there were differences in the content of macrophages among the four algorithms. And IMVIGOR210 found that the lower the score, the more effective the immunotherapy was. Conclusion: The Cuproplasia related gene can be used to predict the prognosis and immunotherapy outcome of LUAD patients, and may exert its effect by affecting chromosome-related pathways and macrophages.

Vitamin D3 regulates NSUN2 expression and inhibits melanoma cell proliferation and migration.

The activated form of vitamin D3 [1,25-dihydroxyvitamin D3; 1,25(OH)2D3] is important for various physiological processes, such as bone mineralization and calcium metabolism, and plays an anticancer role in numerous cancers as well. Its role in melanoma cells has yet to be proven. NOP2/Sun RNA methyltransferase 2 (NSUN2) is a typical RNA methyltransferase and is highly expressed in a variety of cancer cells. However, the molecular mechanisms underlying the role of 1,25(OH)2D3 and NSUN2 in melanoma cells remain largely unknown. The current study showed that 1,25(OH)2D3 could significantly and specifically inhibit the proliferation and migration of melanoma B16 cells. 1,25(OH)2D3 enhances vitamin D receptor expression while simultaneously reducing NSUN2 expression in melanoma cells. Subsequently, knockdown of NSUN2 suppressed B16 cell proliferation and migration. RNA-Seq results illuminated that DNA replication, cell proliferation and cell cycle pathways were enriched, and genes promoting these pathways were reduced after knocking down Nsun2. Dual-luciferase reporter assays showed that 1,25(OH)2D3 downregulated reporter gene expression was controlled by the Nsun2 promoter. The results suggest that 1,25(OH)2D3 binds to the vitamin D response element located upstream of the Nsun2 promoter to downregulate Nsun2 transcription activity and then affects the gene expression pattern related to cell proliferation and the cell cycle, thereby restraining B16 cell proliferation and migration.

Oncogenic and immunological roles of RACGAP1 in pan-cancer and its potential value in nasopharyngeal carcinoma.

A particular GTPase-activating protein called RACGAP1 is involved in apoptosis, proliferation, invasion, metastasis, and drug resistance in a variety of malignancies. Nevertheless, the role of RACGAP1 in pan-cancer was less studied, and its value of the expression and prognostic of nasopharyngeal carcinoma (NPC) has not been explored. Hence, the goal of this study was to investigate the oncogenic and immunological roles of RACGAP1 in various cancers and its potential value in NPC. We comprehensively analyzed RACGAP1 expression, prognostic value, function, methylation levels, relationship with immune cells, immune infiltration, and immunotherapy response in pan-cancer utilizing multiple databases. The results discovered that RACGAP1 expression was elevated in most cancers and suggested poor prognosis, which could be related to the involvement of RACGAP1 in various cancer-related pathways such as the cell cycle and correlated with RACGAP1 methylation levels, immune cell infiltration and reaction to immunotherapy, and chemoresistance. RACGAP1 could inhibit anti-tumor immunity and immunotherapy responses by fostering immune cell infiltration and cytotoxic T lymphocyte dysfunction. Significantly, we validated that RACGAP1 mRNA and protein were highly expressed in NPC. The Gene Expression Omnibus database revealed that elevated RACGAP1 expression was associated with shorter PFS in patients with NPC, and RACGAP1 potentially influenced cell cycle progression, DNA replication, metabolism, and immune-related pathways, resulting in the recurrence and metastasis of NPC. This study indicated that RACGAP1 could be a potential biomarker in pan-cancer and NPC.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

A cuproptosis-related lncRNA signature predicts the prognosis and immune cell status in head and neck squamous cell carcinoma.

The incidence of head and neck squamous cell carcinoma (HNSCC), one of the most prevalent tumors, is increasing rapidly worldwide. Cuproptosis, as a new copper-dependent cell death form, was proposed recently. However, the prognosis value and immune effects of cuproptosis-related lncRNAs (CRLs) have not yet been elucidated in HNSCC. In the current study, the expression pattern, differential profile, clinical correlation, DNA methylation, functional enrichment, univariate prognosis factor, and the immune effects of CRLs were analyzed. A four-CRL signature was constructed using the least absolute shrinkage and selection operator (LASSO) algorithm. Results showed that 20 CRLs had significant effects on the stage progression of HNSCC. Sixteen CRLs were tightly correlated with the overall survival (OS) of HNSCC patients. Particularly, lnc-FGF3-4 as a single risk factor was upregulated in HNSCC tissues and negatively impacted the prognosis of HNSCC. DNA methylation probes of cg02278768 (MIR9-3HG), cg07312099 (ASAH1-AS1), and cg16867777 (TIAM1-AS1) were also correlated with the prognosis of HNSCC. The four-CRL signature that included MAP4K3-DT, lnc-TCEA3-1, MIR9-3HG, and CDKN2A-DT had a significantly negative effect on the activation of T cells follicular helper and OS probability of HNSCC. Functional analysis revealed that cell cycle, DNA replication, and p53 signal pathways were enriched. A novel CRL-related signature has the potential of prognosis prediction in HNSCC. Targeting CRLs may be a promising therapeutic strategy for HNSCC.

Dissecting the Methylomes of EGFR-Amplified Glioblastoma Reveals Altered DNA Replication and Packaging, and Chromatin and Gene Silencing Pathways.

Glioblastoma IDH wildtype is the most frequent brain tumor in adults. It shows a highly malignant behavior and devastating outcomes. To date, there is still no targeted therapy available; thus, patients' median survival is limited to 12-15 months. Epithelial growth factor receptor (EGFR) is an interesting targetable candidate in advanced precision medicine for brain tumor patients. In this study, we performed integrated epigenome-wide DNA-methylation profiling of 866,895 methylation specific sites in 50 glioblastoma IDH wildtype samples, comparing EGFR amplified and non-amplified glioblastomas. We found 9849 significantly differentially methylated CpGs (DMCGs) with Δβ ≥ 0.1 and p-value < 0.05 in EGFR amplified, compared to EGFR non-amplified glioblastomas. Of these DMCGs, 2380 were annotated with tiling (2090), promoter (117), gene (69) and CpG islands (104); 7460 are located at other loci. Interestingly, the list of differentially methylated genes allocated eleven functionally relevant RNAs: five miRNAs (miR1180, miR1255B1, miR126, miR128-2, miR3125), two long non-coding RNAs (LINC00474, LINC01091), and four antisense RNAs (EPN2-AS1, MNX1-AS2, NKX2-2-AS1, WWTR1-AS1). Gene ontology (GO) analysis showed enrichment of "DNA replication-dependent nucleosome assembly", "chromatin silencing at rDNA", "regulation of gene silencing by miRNA", "DNA packaging", "posttranscriptional gene silencing", "gene silencing by RNA", "negative regulation of gene expression, epigenetic", "regulation of gene silencing", "protein-DNA complex subunit organization", and "DNA replication-independent nucleosome organization" pathways being hypomethylated in EGFR amplified glioblastomas. In summary, dissecting the methylomes of EGFR amplified and non-amplified glioblastomas revealed altered DNA replication, DNA packaging, chromatin silencing and gene silencing pathways, opening potential novel targets for future precision medicine.

Elucidating the obligate nature and biological capacity of an invasive fungal corn pathogen.

Tar spot is a devasting corn disease caused by the obligate fungal pathogen Phyllachora maydis. Since its initial identification in the United States in 2015, P. maydis has become an increasing threat to corn production. Despite this, P. maydis has remained largely understudied at the molecular level due to difficulties surrounding its obligate lifestyle. Here, we generated a significantly improved P. maydis nuclear and mitochondrial genome using a combination of long- and short-read technologies and also provide the first transcriptomic analysis of primary tar spot lesions. Our results show that P. maydis is deficient in inorganic nitrogen utilization, is likely heterothallic, and encodes for significantly more protein coding genes, including secreted enzymes and effectors, than previous determined. Furthermore, our expression analysis suggests that following primary tar spot lesion formation, P. maydis might reroute carbon flux away from DNA replication and cell division pathways and towards pathways previously implicated in having significant roles in pathogenicity, such as autophagy and secretion. Together, our results identified several highly expressed unique secreted factors that likely contribute to host recognition and subsequent infection, greatly increasing our knowledge of the biological capacity of P. maydis, which have much broader implications for mitigating tar spot of corn.

Integrated lncRNA transcriptomics, proteomics, and metabolomics to identify early cellular response variation in deoxynivalenol-treated IPEC-J2 cells.

Mycotoxins, especially deoxynivalenol (DON), are common contaminants of food and feed, which also has serious threaten to human health and livestock production. Moreover, DON severely impair intestinal epithelial barrier function. Therefore, it is necessary to investigate the mechanism of intestinal epithelial cell injury induced by DON. Here, intestinal porcine enterocyte cell (IPEC-J2) was incubated with 200ng/ml or 2000ng/ml DON for 6h, then lncRNA sequencing, metabolomics and proteomics were applied. Combined with long coding transcriptomics, and proteomics, 200ng/ml DON treatment (LDON group) significantly upregulated ribosome biogenesis in eukaryotes, spliceosome, and ubiquitin mediated proteolysis, RNA transport, and downregulated metabolic pathways in IPEC-J2, 2000ng/ml of DON treatment (HDON group) significantly upregulated ribosome biogenesis in eukaryotes, and spliceosome, and downregulated base excision repair, cell cycle, DNA replication, homologous recombination, and mismatch repair in IPEC-J2. Combined with long coding transcriptomics, and proteomics, as compared with LDON group, HDON group significantly upregulated adherens junction, hippo signaling pathway, and pathways in cancer, and downregulated DNA replication pathways in IPEC-J2. In metabolomics, LDON group and HDON group was mainly downregulated biosynthesis of unsaturated fatty acids, and fatty acid metabolism. These results provide a new insight to prevent and treat DON induced intestinal epithelial cell injury.

Molecular Characterization of Esophageal Squamous Cell Carcinoma Using Quantitative Proteomics

Esophageal squamous cell carcinoma (ESCC) is a heterogeneous cancer associated with a poor prognosis in advanced stages. In India, it is the sixth most common cause of cancer-related mortality. In this study, we employed high-resolution mass spectrometry-based quantitative proteomics to characterize the differential protein expression pattern associated with ESCC. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. In addition, we identified downregulation of esophagus tissue-enriched proteins such as SLURP1, PADI1, CSTA, small proline-rich proteins such as SPRR3, SPRR2A, SPRR1A, KRT4, and KRT13, involved in squamous cell differentiation. We identified several overexpressed proteins mapped to the 3q24-29 chromosomal region, aligning with CNV alterations in this region reported in several published studies. Among these, we identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by genes in the 3q26 region. Functional enrichment analysis revealed proteins involved in cell cycle pathways, DNA replication, spliceosome, and DNA repair pathways. We identified the overexpression of multiple proteins that play a major role in alleviating ER stress, including SYVN1 and SEL1L. The SYVN1/SEL1L complex is an essential part of the ER quality control machinery clearing misfolded proteins from the ER. SYVN1 is an E3 ubiquitin ligase that ubiquitinates ER-resident proteins. Interestingly, there are also other non-canonical substrates of SYVN1 which are known to play a crucial role in tumor progression. Thus, SYVN1 could be a potential therapeutic target in ESCC.

Integrative transcriptome and proteome analyses of clear cell renal cell carcinoma develop a prognostic classifier associated with thrombus

Clear cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) is associated with poor prognosis. Our integrative analyses of transcriptome and proteome reveal distinctive molecular features of ccRCC with VTT, and yield the development of a prognostic classifier to facilitate ccRCC molecular subtyping and treatment. The RNA sequencing and mass spectrometry were performed in normal-tumor-thrombus tissue triples of five ccRCC patients. Statistical analysis, GO and KEGG enrichment analysis, and protein–protein interaction network construction were used to interpret the transcriptomic and proteomic data. A six-gene-based classifier was developed to predict patients’ survival using Cox regression, which was validated in an independent cohort. Transcriptomic analysis identified 1131 tumorigenesis-associated differentially expressed genes (DEGs) and 856 invasion-associated DEGs. Overexpression of transcription factor EGR2 in VTT indicated its important role in tumor invasion. Furthermore, proteomic analysis showed 597 tumorigenesis-associated differentially expressed proteins (DEPs) and 452 invasion-associated DEPs. The invasion-associated DEPs showed unique enrichment in DNA replication, lysine degradation, and PPAR signaling pathway. Integration of transcriptome and proteome reveals 142 tumorigenesis-associated proteins and 84 invasion-associated proteins displaying changes consistent with corresponding genes in transcriptomic profiling. Based on their different expression patterns among normal-tumor-thrombus triples, RAB25 and GGT5 were supposed to play a consistent role in both tumorigenesis and invasion processes, while SHMT2 and CADM4 might play the opposite roles in tumorigenesis and thrombus invasion. A prognostic classifier consisting of six DEGs (DEPTOR, DPEP1, NAT8, PLOD2, SLC7A5, SUSD2) performed satisfactorily in predicting survival of ccRCC patients (HR = 4.41, P < 0.001), which was further validated in an independent cohort of 40 cases (HR = 5.52, P = 0.026). Our study revealed the transcriptomic and proteomic profiles of ccRCC patients with VTT, and identified the distinctive molecular features associated with VTT. The six-gene-based prognostic classifier developed by integrative analyses may facilitate ccRCC molecular subtyping and treatment.

Discovering pathway biomarkers of hepatocellular carcinoma occurrence and development by dynamic network entropy analysis

ObjectiveGene expression profiling techniques measure the transcription of thousands of genes in a parallel manner. With more and more hepatocellular carcinoma (HCC) transcriptomic data becoming available, the high-throughput data provides an unprecedented opportunity to discover HCC diagnostic biomarkers. In this work, we propose a bioinformatics method based on dynamic network entropy analysis, called DNEA, to identify potential pathway biomarkers for HCC occurrence and development by integrating transcriptome and interactome. MethodsWe firstly collect the pathways documented in different knowledge-bases and then impose the genome-wide human transcriptomic data of multistage cancerous tissues during the development and progression of HCC. After linking the gene sets of pathways into individual connected networks, we map the corresponding gene expression information onto these pathways. The dynamic network entropy of individual pathways is calculated to evaluate its activities and dysfunctionalities during the disease occurrence and development. We use the overall significant difference in the entropic dynamics during the time course to prioritize distinctive pathways during disease progression. Then machine learning classification methods are employed to screen out pathway biomarkers with the classification ability to distinguish different-stage samples of HCC progression. ResultsPathway biomarkers discovered based on DNEA demonstrate good classification performance in measuring HCC progression. The classification accuracy is as follows: DNA replication pathway (mean AUC = 0.82, 20 genes) from KEGG, FMLP pathway (mean AUC = 0.84, 14 genes) from BioCarta, and downstream signaling of activated FGFR pathway (mean AUC = 0.80, 15 genes) from Reactome. At the same time, previous studies have shown that these genes and pathways screened are closely related to the occurrence and development of HCC in terms of oncogenesis dysfunctions. ConclusionsOur method for cancer biomarker discovery based on dynamic network entropy analysis is effective and efficient in identifying pathway biomarkers related to the progression of complex diseases.

Proper RPA acetylation promotes accurate DNA replication and repair.

The single-stranded DNA (ssDNA) binding protein complex RPA plays a critical role in promoting DNA replication and multiple DNA repair pathways. However, how RPA is regulated to achieve its functions precisely in these processes remains elusive. Here, we found that proper acetylation and deacetylation of RPA are required to regulate RPA function in promoting high-fidelity DNA replication and repair. We show that yeast RPA is acetylated on multiple conserved lysines by the acetyltransferase NuA4 upon DNA damage. Mimicking constitutive RPA acetylation or blocking its acetylation causes spontaneous mutations with the signature of micro-homology-mediated large deletions or insertions. In parallel, improper RPA acetylation/deacetylation impairs DNA double-strand break (DSB) repair by the accurate gene conversion or break-induced replication while increasing the error-prone repair by single-strand annealing or alternative end joining. Mechanistically, we show that proper acetylation and deacetylation of RPA ensure its normal nuclear localization and ssDNA binding ability. Importantly, mutation of the equivalent residues in human RPA1 also impairs RPA binding on ssDNA, leading to attenuated RAD51 loading and homologous recombination repair. Thus, timely RPA acetylation and deacetylation likely represent a conserved mechanism promoting high-fidelity replication and repair while discriminating the error-prone repair mechanisms in eukaryotes.

Comprehensive analyses of cuproptosis-related gene CDKN2A on prognosis and immunologic therapy in human tumors

Recent studies have identified a novel programmed cell death based on copper, named cuproptosis. However, as an anti-cuproptosis gene, the functional roles, definite mechanisms and prognostic value of CDKN2A in pan-cancer are largely unclear. The GEPIA2, cancer genome atlas (TCGA), the tumor immune estimation resource 2.0 and CPTAC databases were performed to validate the differential expression of CDKN2A in 33 tumors. The clinical features and survival prognosis analysis were conducted by GEPIA2 and UALCAN web tool. Genetic alteration analysis of CDKN2A in pan-cancer was also evaluated. Furthermore, the functional roles of CDKN2A were explored via DNA methylation analysis, tumor microenvironment, infiltration of immune cells, enrichment analysis and gene co-expression associated with cuproptosis and immune regulation. The CDKN2A expression, both at the transcriptional and translational level, was obviously upregulated in most cancer patients, which might lead to poor survival in certain cancer types. CDKN2A expression was significantly associated with tumor pathological stages in some cancer types. In adrenocortical carcinoma (ACC) and kidney renal clear cell carcinoma (KIRC), DNA methylation of CDKN2A was explored to induce poor clinical outcomes. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis indicated that CDKN2A expression was closely related to several cancer-associated signaling pathways, such as the p53 signaling pathway, Cellular senescence, DNA replication and Cell cycle signaling pathways. Gene set enrichment analysis (GSEA) analysis suggested that aberrantly expressed CDKN2A took part in the cell cycle regulation, immune regulation and mitochondrial signaling pathways in certain cancer patients. In addition, aberrant CDKN2A expression was closely correlated to immune infiltration and the levels of immune-regulatory genes. The study deeply defined the concrete roles of cuproptosis-related gene CDKN2A in tumorigenesis. The results provided new insights and pieces of evidence for treatment.

Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy.

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.