Cell Cycle-related Processes Research Articles

Comparative Analysis of Digestion Methods for Bile Proteomics: The Key to Unlocking Biliary Biomarker Potential.

Bile's potential to reflect the health of the biliary system has led to increased attention, with proteomic analysis offering deeper understanding of biliary diseases and potential biomarkers. With the emergence of normothermic machine perfusion (NMP), bile can be easily collected and analyzed. However, the composition of bile can make the application of proteomics challenging. This study systematically evaluated various trypsin digestion methods to optimize proteomics of bile from human NMP livers. Bile was collected from 12 human donor livers that were accepted for transplantation after the NMP viability assessment. We performed tryptic digestion using six different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without additional precipitation before digestion. Proteins were analyzed using untargeted proteomics. Methods were assessed for total protein IDs, variation, and protein characteristics to determine the most optimal method. Methods involving precipitation surpassed crude methods in protein identifications (4500 vs 3815) except for in-gel digestion. Filtered data (40%) resulted in 3192 versus 2469 for precipitated and crude methods, respectively. We found minimal differences in mass, cellular components, or hydrophobicity of proteins between methods. Intermethod variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming others. Age-related biological comparisons revealed upregulation of metabolic-related processes in younger donors and immune response and cell cycle-related processes in older donors. Variability between methods emphasizes the importance of cross-validation across multiple analytical approaches to ensure robust analysis. We recommend the in-gel crude method for its simplicity and efficiency, avoiding additional precipitation steps. Sample processing speed, cost, cleanliness, and reproducibility should be considered when a digestion method is selected for bile proteomics.

Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus

BackgroundDuring mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5—a critical MYC cofactor—regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein.ResultsUsing RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus.ConclusionsOverall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

Molecular Pathogenic Mechanisms of IgA Nephropathy Secondary to COVID-19 mRNA Vaccination

Introduction: Accumulating evidence has disclosed that IgA nephropathy (IgAN) could present shortly after the second dose of COVID-19 mRNA vaccine. However, the undying mechanism remains unclear and we aimed to investigate the potential molecular mechanisms. Methods: We downloaded gene expression datasets of COVID-19 mRNA vaccination (GSE201535) and IgAN (GSE104948). Weighted Gene Co-Expression Network Analysis (WGCNA) was performed to identify co-expression modules related to the second dose of COVID-19 mRNA vaccination and IgAN. Differentially expressed genes (DEGs) were screened, and a transcription factor (TF)-miRNA regulatory network and protein-drug interaction were constructed for the shared genes. Results: WGCNA identified one module associated with the second dose of COVID-19 mRNA vaccine and four modules associated with IgAN. Gene ontology (GO) analyses revealed enrichment of cell cycle-related processes for the COVID-19 mRNA vaccine hub genes and immune effector processes for the IgAN hub genes. We identified 74 DEGs for the second dose of COVID-19 mRNA vaccine and 574 DEGs for IgAN. Intersection analysis with COVID-19 vaccine-related genes led to the identification of two shared genes, TOP2A and CEP55. The TF-miRNA network analysis showed that hsa-miR-144 and ATF1 might regulate the shared hub genes. Conclusions: This study provides insights into the common pathogenesis of COVID-19 mRNA vaccination and IgAN. The identified pivotal genes may offer new directions for further mechanistic studies of IgAN secondary to COVID-19 mRNA vaccination.

Identification of potential key genes for colorectal cancer based on bioinformatics analysis.

This study aimed to explore key genes as potential biomarkers for colorectal cancer (CRC) diagnosis and prognosis in order to improve their clinical utility. To identify and screen candidate genes involved in CRC carcinogenesis and disease progression, we downloaded the microarray datasets GSE143939, GSE196006, and GSE200427 from the GEO database and applied the GEO2R tool to obtain differentially expressed genes (DEGs) between colorectal cancer tissue samples and normal tissue samples. Differentially expressed genes were analyzed using the DAVID online database for gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analyses. Protein-protein interaction network was constructed and related module analysis was performed using STRING and Cytoscape. In total, 241 DEGs were identified, including 127 downregulated and 114 upregulated genes. DEGs enriched functions and pathways included cellular response to chemical stimulus, extracellular region, carbonate dehydratase activity, cell division, spindle, and cell division. The abundant functions and pathways of DEGs included cellular response to chemical stimulus, extracellular region, carbonate dehydratase activity, cell division, spindle, cell adhesion molecule binding, Aldosterone-regulated sodium reabsorption, and Cell cycle-related processes. Fifteen key genes were identified, and bioprocess analyses showed that these genes were mainly enriched in cell cycle, cell division, mitotic spindle, and tubulin binding processes. It was found that CDK1, CEP55, MKI67, and TOP2A may be involved in CRC cancer invasion and recurrence. The pivotal genes identified in this study contribute to our understanding of the molecular and pathogenic mechanisms of CRC carcinogenesis and progression, and provide possible biomarkers for the diagnosis and treatment of CRC.

Mongolian HCC vs. Caucasian HCC: The Metabolic Reprogramming Process in Mongolian HCC is an Interesting Difference.

Racial/ethnic and region disparities in incidence and mortality are obviously in liver cancer. Mongolia has the highest reported incidence and mortality of hepatocellular carcinoma (HCC) in the world, while the incidence of HCC is relatively low in the United States, but differences in their molecular characteristics remain largely elusive. Here we report differentially expressed genes (DEGs) in Mongolian hepatocellular carcinoma and in Caucasian HCC and their intersection DEGs, as well as their corresponding signaling pathways in Mongolian and Caucasian hepatocellular carcinoma patients based on the transcriptome sequences from Gene Expression Omnibus (GEO) database. We got 908 up-regulated genes and 1946 down-regulated genes in Mongolian HCC, 1244 up-regulated genes and 1912 down-regulated genes in Caucasian HCC, 254 Co-upregulated genes and 1035 co-downregulated genes in Mongolian and Caucasian. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that most of the genes with altered expression levels in Mongolian HCC participate in biological processes that involve metabolic reprogramming of various substances, accounting for about one-third of all biological processes. In particular, multiple amino acid biosynthesis and metabolic processes appear to be specific in Mongolian HCC compared with Caucasian HCC. The biological processes they share include those in which most immune cells are involved and cell cycle-related biological processes. In addition, we also found the genes UPP2, PCK1, GLYAT, GNMT, ADH1B and HPD, encode for key metabolic enzymes, whose expression level up-regulated or down-regulated more than 5 times in Mongolian HCC and was dramatically correlated with survival in Mongolian HCC (p value < 0.01), More importantly, these molecules are potential targets for some metabolic antitumor drugs. This study not only makes up for the shortcomings of previous studies on liver cancer, which paid more attention to its commonality, but ignored the specificity of liver cancer in different races and regions. More importantly, the purpose of this study is to identify robust molecular subclasses and information with underlying unique tumor biology. And this study may have important implications for the study of the pathogenic factors and molecular mechanisms of hepatocellular carcinoma and the precise therapy of Mongolian HCC.

Hibernation-Induced microRNA Expression Promotes Signaling Pathways and Cell Cycle Dysregulation in Ictidomys tridecemlineatus Cardiac Tissue.

The thirteen-lined ground squirrel Ictidomys tridecemlineatus is a rodent that lives throughout the United States and Canada and uses metabolic rate depression to facilitate circannual hibernation which helps it survive the winter. Metabolic rate depression is the reorganization of cellular physiology and molecular biology to facilitate a global downregulation of nonessential genes and processes, which conserves endogenous fuel resources and prevents the buildup of waste byproducts. Facilitating metabolic rate depression requires a complex interplay of regulatory approaches, including post-transcriptional modes such as microRNA. MicroRNA are short, single-stranded RNA species that bind to mRNA transcripts and target them for degradation or translational suppression. Using next-generation sequencing, we analyzed euthermic vs. hibernating cardiac tissue in I. tridecemlineatus to predict seven miRNAs (let-7e-5p, miR-122-5p, miR-2355-3p, miR-6715b-3p, miR-378i, miR-9851-3p, and miR-454-3p) that may be differentially regulated during hibernation. Gene ontology and KEGG pathway analysis suggested that these miRNAs cause a strong activation of ErbB2 signaling which causes downstream effects, including the activation of MAPK and PI3K/Akt signaling and concurrent decreases in p53 signaling and cell cycle-related processes. Taken together, these results predict critical miRNAs that may change during hibernation in the hearts of I. tridecemlineatus and identify key signaling pathways that warrant further study in this species.

Identification of characteristic genes and construction of regulatory network in gallbladder carcinoma

BackgroundGallbladder carcinoma (GBC) is a highly malignant tumor with a poor overall prognosis. This study aimed to identify the characteristic microRNAs (miRNAs) of GBC and the competing endogenous RNA (ceRNA) regulatory mechanisms.MethodsThe microarray data of GBC tissue samples and normal gallbladder (NGB) tissue samples from the Gene Expression Omnibus (GEO) database was downloaded. GBC-related differentially expressed miRNAs (DE-miRNAs) were identified by inter-group differential expression analysis and weighted gene co-expression network analysis (WGCNA). Machine learning algorithms were used to screen the characteristic miRNA based on the intersect between least absolute shrinkage and selection operator (LASSO) and Support vector machine-recursive feature elimination (SVM-RFE). Based on the differential expression analysis of GEO database, the ceRNA network of characteristic miRNA was predicted and constructed. The biological functions of the ceRNA network were revealed by carrying out the gene enrichment analysis was implemented. We further screened the key genes of ceRNA network and constructed a protein-protein interaction (PPI) network, and predicted and generated the transcription factors (TFs) network of signature miRNAs. The expression of characteristic miRNA in clinical samples was verified by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsA total of 131 GBC-related DE-miRNAs were obtained. The hsa-miR-4770 was defined as characteristic miRNA for GBC. The ceRNA network containing 211 mRNAs, one miRNA, two lncRNAs, and 48 circRNAs was created. Gene enrichment analysis suggested that the downstream genes were mainly involved in actin filament organization, cell-substrate adhesion, cell-matrix adhesion, reactive oxygen species metabolic process, glutamine metabolic process and extracellular matrix (ECM)-receptor interaction pathway. 10 key genes in the network were found to be most correlated with disease, and involved in cell cycle-related processes, p53, and extrinsic apoptotic signaling pathways. The qRT-PCR result demonstrated that hsa-miR-4770 is down-regulated in GBC, and the expression trend is consistent with the public database.ConclusionsWe identified hsa-miR-4770 as the characteristic miRNA for GBC. The ceRNA network of hsa-miR-4770 may play key roles in GBC. This study provided some basis for potential pathogenesis of GBC.

Abstract A008: Cancer predisposition signaling pathways drive Beckwith-Wiedemann syndrome Wilms tumor oncogenesis

Abstract Wilms Tumor (WT), or nephroblastoma, is the most common pediatric renal cancer. The most prevalent epigenetic alterations in WT occur on chromosome 11p15, accounting for at least two-thirds of WT. These same changes on chromosome 11p15 also cause Beckwith-Wiedemann Syndrome (BWS). BWS is a pediatric overgrowth disorder affecting numerous tissues, including the kidney, and up to 28% of patients with BWS develop cancer. As patients with BWS have the same 11p15 epigenetic and structural changes that arise in some sporadic WT, BWS-WT provides a unique opportunity to investigate the specific molecular drivers by which 11p15 alterations induce oncogenesis. Using clinical data and patient samples from the International BWS Registry and Biorepository, we used whole exome sequencing, methylation array, and RNA-sequencing technologies to examine the genomic and epigenomic landscape of BWS-WT compared to control kidney samples. We found several large-scale recurrent copy number alterations (CNAs) in the BWS-WT cohort across cancer-associated chr1p36, WT-associated chr16q, and at a novel chr15q site. Two BWS-WT presented with an above average number of genomic alterations; one had mutations in telomere maintenance genes, and the other had mutations in mismatch repair genes. Recurrent mutations in genes associated with sporadic WT, such as TP53, CTNNB1, and WT1 were not present in BWS samples. We found other single nucleotide variants (SNVs) in BCORL1, ASXL1, ATM and AXL genes in the BWS cohort. We further compared the BWS data to publicly available non-syndromic/nonBWS data, stratified based on 11p15 status (normal or altered), and the molecular signaling differences were investigated. Differential methylation studies showed enrichment of biological processes related to the Wnt signaling, integrin signaling, insulin receptor signaling, and BMP signaling pathways in BWS-WT. Altered-11p15 nonBWS-WT showed enrichment of cell cycle and DNA damage checkpoint processes, as well as TORC1/TOR signaling and Wnt signaling pathways, as assayed by differential methylation. RNA sequencing data showed differential gene expression of Wnt signaling along with other distinct pathways including Notch, BMP, PPAR and, NIK/NF-kappaB signaling pathways in BWS-WT. Based on an interactome study, CTNNB1 exhibited the broadest range of interactions, and its overexpression was observed in BWS-WT, although not specifically mutated in the BWS samples. Weighted gene coexpression network analysis (WGCNA) showed that cell cycle-related processes and DNA repair mechanisms are shared features of WT with alterations in chromosome 11p15. While BWS-WT predisposition and 11p15 alterations in nonBWS-WT are well-established, this study is the first to focus on stratifying tumors by this characteristic. Data presented herein suggest a mechanism in which 11p15-altered genomes predispose cells to oncogenic transitions. Further investigation of our findings may serve to identify novel diagnostic or therapeutic targets in WT oncogenesis. Citation Format: Snehal Dinkar Nirgude, Natali Sobel Naveh, Sanam Kavai, Jennifer Kalish. Cancer predisposition signaling pathways drive Beckwith-Wiedemann syndrome Wilms tumor oncogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A008.

High throughput sequencing revealed enhanced cell cycle signaling in SLE patients

The multi-system involvement and high heterogeneity of systemic lupus erythematosus (SLE) pose great challenges to its diagnosis and treatment. The purpose of the current study is to identify genes and pathways involved in the pathogenesis of SLE. High throughput sequencing was performed on the PBMCs from SLE patients. We conducted differential gene analysis, gene ontology (GO) analysis, kyoto encyclopedia of genes and genomes (KEGG) analysis, and quantitative real-time PCR (qRT-PCR) verification. Protein–protein interaction (PPI) analysis, alternative splicing analysis, and disease correlation analysis were conducted on some key pathogenic genes as well. Furthermore, si-CDC6 was used for transfection and cell proliferation was monitored using a cell counting kit-8 (CCK-8) assay. We identified 2495 differential genes (1494 upregulated and 1001 downregulated) in SLE patients compared with healthy controls. The significantly upregulated genes were enriched in the biological process-related GO terms of the cell cycle, response to stress, and chromosome organization. KEGG enrichment analysis revealed 7 significantly upregulated pathways including SLE, alcoholism, viral carcinogenesis, cell cycle, proteasome, malaria, and transcriptional misregulation in cancer. We successfully verified some differential genes on the SLE pathway and the cell cycle pathway. CDC6, a key gene in the cell cycle pathway, had remarkably higher MXE alternative splicing events in SLE patients than that in controls, which may explain its significant upregulation in SLE patients. We found that CDC6 participates in the pathogenesis of many proliferation-related diseases and its levels are positively correlated with the severity of SLE. Knockdown of CDC6 suppressed the proliferation of Hela cells and PBMCs from SLE patients in vitro. We identified SLE-related genes and their alternative splicing events. The cell cycle pathway and the cell cycle-related biological processes are over-activated in SLE patients. We revealed a higher incidence of MXE events of CDC6, which may lead to its high expression in SLE patients. Upregulated cell cycle signaling and CDC6 may be related to the hyperproliferation and pathogenesis of SLE.

Hydroquinidine displays a significant anticarcinogenic activity in breast and ovarian cancer cells via inhibiting cell-cycle and stimulating apoptosis.

Breast and ovarian cancers are women's most commonly diagnosed cancers. Seeking an efficient anticarcinogenic compound is still a top priority regarding the aggressiveness of these cancers and the limited benefit of current therapies. Hydroquinidine (HQ) is a natural alkaloid used in arrhythmia and Brugada syndrome. As an ion channel blocker, HQ exhibits its activity by altering ion gradient and membrane potential. Considering the growing evidence of ion channel blockers' antineoplastic potential, we were prompted to test HQ's effect on breast and ovarian cancers. MCF-7 and SKOV-3 cell lines were used to inspect how HQ acts on survival, clonogenicity, migration, tumorigenicity, proliferation, and apoptosis. The molecular basis for the remarkable antiproliferative and proapoptotic effect of HQ in these cells was dissected by proteomics. CDK1, PSMB5, PSMC2, MCM2, MCM7, YWHAH, YWHAQ, and YWHAB proteins in HQ-treated MCF-7 cells, and RRM2, PSMD2, PSME2, COX2, COX4l1, and CDK6 proteins in HQ-treated SKOV-3 cells were found as low-abundant, which was noteworthy. Based on the in-depth analysis, upon HQ treatment, several cell cycle-related processes were found as suppressed, whereas apoptosis and ferroptosis pathways were found to be activated. The observed proteome alteration in cancer cells may provide mechanistic explanations for the growth-limiting effects of HQ at the cellular level.

Identification of pyroptosis-related gene signature for predicting prognosis of patients with pancreatic cancer using bioinformatics.

Pancreatic cancer, a common digestive system malignancy, is dubbed the “king of cancers”. The role of pyrophosis-related genes (PRGs) in pancreatic cancer prognosis is yet unknown. In pancreatic cancer and normal tissue, we discovered 9 PRGs that are expressed differently in pancreatic cancer and healthy tissue. Based on the differential expression of PRGs, 2 clusters of pancreatic cancer cases could be identified. The 2 groups had significant disparities in total survival time. The prognostic model of a 5-PRGs signature was created using least absolute shrinkage and selection operator (LASSO) method. The median risk score was used to split pancreatic cancer patients in The Cancer Genome Atlas (TCGA) cohort into 2 groups: low risk and high risk. Patients classified as low-risk had significantly higher survival rates than those classified as high-risk (P < .01). The same results were obtained by validating them against the Gene Expression Omnibus database (P = .030). Cox regression statistical analysis showed that risk score was an independent predictor of overall survival in pancreatic cancer patients. Functional enrichment analysis revealed that apoptosis, cell proliferation, and cell cycle-related biological processes and signaling pathways were enriched. Additionally, the immunological status of the high-risk group worsened. In conclusion, a novel pyroptosis-related gene signature can be used to predict pancreatic cancer patient prognosis.

Comparison of meiotic transcriptomes of three maize inbreds with different origins reveals differences in cell cycle and recombination

BackgroundCellular events during meiosis can differ between inbred lines in maize. Substantial differences in the average numbers of chiasmata and double-strand breaks (DSBs) per meiotic cell have been documented among diverse inbred lines of maize: CML228, a tropical maize inbred line, B73 and Mo17, temperate maize lines. To determine if gene expression might explain these observed differences, an RNA-Seq experiment was performed on CML228 male meiocytes which was compared to B73 and Mo17 male meiocytes, where plants were grown in the same controlled environment.ResultsWe found that a few DSB-repair/meiotic genes which promote class I crossovers (COs) and the Zyp1 gene which limits newly formed class I COs were up-regulated, whereas Mus81 homolog 2 which promotes class II COs was down-regulated in CML228. Although we did not find enriched gene ontology (GO) categories directly related to meiosis, we found that GO categories in membrane, localization, proteolysis, energy processes were up-regulated in CML228, while chromatin remodeling, epigenetic regulation, and cell cycle related processes including meiosis related cell cycle processes were down-regulated in CML228. The degree of similarity in expression patterns between the three maize lines reflect their genetic relatedness: B73 and Mo17 had similar meiotic expressions and CML228 had a more distinct expression profile.ConclusionsWe found that meiotic related genes were mostly conserved among the three maize inbreds except for a few DSB-repair/meiotic genes. The findings that the molecular players in limiting class I CO formation (once CO assurance is achieved) were up-regulated and those involved in promoting class II CO formation were down-regulated in CML228 agree with the lower chiasmata number observed in CML228 previously. In addition, epigenetics such as chromatin remodeling and histone modification might play a role. Transport and energy-related processes was up-regulated and Cyclin13 was down-regulated in CML228. The direction of gene expression of these processes agree with that previously found in meiotic tissues compared with vegetative tissues. In summary, we used different natural maize inbred lines from different climatic conditions and have shown their differences in expression landscape in male meiocytes.

COVID-19-specific transcriptomic signature detectable in blood across multiple cohorts.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading across the world despite vast global vaccination efforts. Consequently, many studies have looked for potential human host factors and immune mechanisms associated with the disease. However, most studies have focused on comparing COVID-19 patients to healthy controls, while fewer have elucidated the specific host factors distinguishing COVID-19 from other infections. To discover genes specifically related to COVID-19, we reanalyzed transcriptome data from nine independent cohort studies, covering multiple infections, including COVID-19, influenza, seasonal coronaviruses, and bacterial pneumonia. The identified COVID-19-specific signature consisted of 149 genes, involving many signals previously associated with the disease, such as induction of a strong immunoglobulin response and hemostasis, as well as dysregulation of cell cycle-related processes. Additionally, potential new gene candidates related to COVID-19 were discovered. To facilitate exploration of the signature with respect to disease severity, disease progression, and different cell types, we also offer an online tool for easy visualization of the selected genes across multiple datasets at both bulk and single-cell levels.

Healing Ability of Central Corneal Epithelium in Rabbit Ocular Surface Injury Models.

PurposeWound healing of the corneal epithelium mainly involves two types of cells: limbal stem/progenitor cells (LSCs) and differentiated central corneal epithelial cells (CECs). The healing ability of CECs is still debatable, and its correlated transcriptomic alterations during wound healing are yet to be elucidated. This study aimed to determine the healing ability and mechanisms underlying the actions of CECs using rabbit ocular surface injury models.MethodsA central corneal ring-like residual epithelium model was used to investigate the healing ability of CECs. Uninjured and injury-stimulated LSCs and CECs were collected for transcriptomic analysis. The analysis results were verified by quantitative reverse transcriptase polymerase chain reaction, immunofluorescence staining, and two types of rabbit corneal injury models.ResultsDuring wound healing, the upregulated genes in LSCs were mostly enriched in the mitotic cell cycle–related processes, but those in CECs were mostly enriched in cell adhesion and migration. CECs could repair the epithelial defects successfully at one-time injuries. However, after repetitive injuries, the CECs repaired notably slower and failed to completely heal the defect, but the LSCs repaired even faster than the one-time injury.ConclusionsOur results indicated rabbit CECs repair the epithelial defect mainly depending on migration and its proliferative ability is limited, and LSCs are the main source of regenerative epithelial cells.Translational RelevanceThis study provides information on gene expression in the corneal epithelium during wound healing, indicating that regulation of the cell cycle, cell adhesion, and migration may be the basis for future treatment strategies for corneal wound healing.

Identification and validation of HOXD3 and UNC5C as molecular signatures in keloid based on weighted gene co-expression network analysis

BackgroundKeloid is a benign proliferative disease characterized by excessive deposition of extracellular matrix collagen during skin wound healing. The mechanisms of keloid formation have not been fully elucidated, and the current treatment methods are not effective for all keloid patients. Therefore, there is an urgent need to find more effective therapies, and our research focused on identifying characteristic molecular signatures of keloid to explore potential therapeutic targets. MethodsGene expression profiles of keloid and control group samples were retrieved from the GEO database. Taking the GSE113619 dataset as the training set, the dataset collected skin tissues from non-lesion sites of healthy and keloid-prone individuals, denoted as Day0. The second sampling was performed 42 days later at the original sampling site of control and keloid groups, denoted as Day42.The 'limma' package and Venn diagram identified differentially expressed genes (DEGs) specific to keloid day42 versus day0 samples. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome pathway functional enrichment, and annotation of the characteristic genes were conducted on the Metascape website. Ingenuity canonical pathways, disease & function enrichment analysis and gene interaction network were performed and predicted in Ingenuity Pathway Analysis (IPA) software. Key module genes related to keloid were filtered out by Weighted Gene Co-expression Network Analysis (WGCNA). We utilized the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm to screen the characteristic genes in keloid by the ‘glmnet’ package. The area under the curve (AUC) of receiver operating characteristic (ROC) was utilized to determine the effectiveness of potential signatures in discriminating keloid samples from normal samples and performed by using the ‘pROC’ package. The enrich scores of 24 immune cells in each sample were calculated by the single-sample gene set enrichment analysis (ssGSEA) algorithm， and then the Gene Set Variation Analysis (GSVA) was performed. Finally, RNA from 4 normal and 6 keloid samples was extracted, and RT-qPCR and Western Blot validated the expression of characteristic genes. ResultsA total of 640 DEGs specific to keloid day42 versus day0 samples were detected. 69 key module genes were uncovered and implicated in ‘NCAM signaling for neurite out-growth’, ‘oncogenic MAPK signaling’, ‘transmission across chemical synapses’ pathways, and the mitotic cell cycle-related processes. Five characteristic genes (MTUS1, UNC5C, CEP57, NAA35, and HOXD3) of keloid were identified by LASSO, and among which UNC5C and HOXD3 were validated by ROC plot in external dataset, RT-qPCR and Western Blot in validation samples. The result of ssGSEA indicated that the infiltration of neutrophils showed a relatively higher abundance and natural killer cells with relatively low enrichment in the keloid group compared to the control group. UNC5C was correlated with more immune cells compared with other characteristic genes. ConclusionIn this study, characteristic genes associated with keloid were identified by bioinformatic approaches and verified in clinical validation samples, providing potential targets for the diagnosis and treatment of keloid.

Comparative transcriptional analysis uncovers molecular processes in early and mature somatic cyst cells of Drosophila testes

The tight interaction between somatic and germline cells is conserved in animal spermatogenesis. The testes of Drosophila melanogaster are the model of choice to identify processes responsible for mature gamete production. However, processes of differentiation and soma-germline interactions occurring in somatic cyst cells are currently understudied. Here we focused on the comparison of transcriptome expression patterns of early and mature somatic cyst cells to find out the developmental changes taking place in them. We employed a FACS-based approach for the isolation of early and mature somatic cyst cells from fly testes, subsequent preparation of RNA-Seq libraries, and analysis of gene differential expression in the sorted cells. We found increased expression of genes involved in cell cycle-related processes in early cyst cells, which is necessary for the proliferation and self-renewal of a crucial population of early cyst cells, cyst stem cells. Genes proposedly required for lamellipodium-like projection organization for proper cyst formation were also detected among the upregulated ones in early cyst cells. Gene Ontology and interactome analyses of upregulated genes in mature cyst cells revealed a striking over-representation of gene categories responsible for metabolic and catabolic cellular processes, as well as genes supporting the energetic state of the cells provided by oxidative phosphorylation that is carried out in mitochondria. Our comparative analyses of differentially expressed genes revealed major peculiarities in early and mature cyst cells and provide novel insight into their regulation, which is important for male fertility.

An analysis of lncRNA-miRNA-mRNA networks to investigate the effects of HDAC4 inhibition on skeletal muscle atrophy caused by peripheral nerve injury.

BackgroundMuscle atrophy caused by peripheral nerve injury is a common clinical disease, with no effective treatments currently available. Our previous studies have found that denervation-induced muscle atrophy can be alleviated by inhibiting histone deacetylase 4 (HDAC4). An increasing amount of evidence shows that microRNA (miRNA) and long noncoding RNA (lncRNA) are involved in the occurrence of muscle atrophy. This study aimed to find the mechanism by which HDAC4 regulates denervation-induced muscle atrophy based on lncRNA-associated competing endogenous RNA (ceRNA) networks.MethodsWe analyzed the influence of short hairpin RNA (shRNA) knockdown of HDAC4 on lncRNAs and miRNAs after denervated muscle atrophy using RNA sequencing. A Pearson’s correlation heat map and principal component analysis were employed to analyze differentially expressed miRNAs and lncRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of target genes were conducted. The ceRNA network of lncRNA-miRNA-mRNA was constructed, and the core regulatory molecules in the ceRNA network were analyzed.ResultsWe found 32 miRNAs and 111 lncRNAs related to denervated muscle atrophy regulated by HDAC4. Moreover, 15 downregulated lncRNAs, 14 upregulated miRNAs, and 61 downregulated mRNAs constituted a ceRNA regulatory network, participating in the biological processes including response to denervation involved in regulation of muscle adaptation, along with the signaling pathways including autophagy, FoxO signaling pathways, and Jak-STAT signaling pathways. Additionally, 6 upregulated lncRNAs, 8 downregulated miRNAs, and 66 upregulated mRNAs constituted another ceRNA regulatory network, which was mainly involved in cell cycle-related biological processes and pathways. Finally, 3 lncRNAs, 4 miRNAs, and 12 mRNAs constituted a ceRNA sub-network, and XR_377582.2 and ENSMUST00000143649 were considered to be the key lncRNAs.ConclusionsIn the ceRNA network, all nodes are directly or indirectly involved in the process by which HDAC4 regulates skeletal muscle atrophy caused by peripheral nerve injury. XR_377582.2 and ENSMUST00000143649 may be the key lncRNAs related to HDAC4 involved in the regulation of muscle atrophy.

Expression Profiles of HOXC6 Predict the Survival of Glioblastoma Patients and Correlate with Cell Cycle.

The goal of this study was to investigate the homeobox (HOX) gene expression status and its prognostic value in glioblastoma multiforme (GBM) and to uncover the biological processes related to its expression. The prognostic value of HOX genes in GBM was systematically investigated by a genome-wide analysis of HOX gene expression profiles in GBM patient samples in The Cancer Genome Atlas (TCGA) project (microarray dataset) and validation datasets. Using the differentially expressed gene (DEG) analysis and a Cox regression model, we discovered that the HOXC6 could stratify patients into significantly different survival (p = 0.0012, log-rank test) groups in the training cohort. TCGA RNA-seq and GSE16011 datasets were used for validation. Multivariate Cox and stratification analysis indicated that HOXC6 was an independent prognostic factor after adjusting for other clinical covariates. Bioinformatic analysis suggested that the HOXC6 might be involved in the cell cycle-related biological processes and pathways that are well established in the context of glioblastoma tumorigenesis. We further explored the bioinformatic implications by gene set enrichment analysis (GSEA). Tumor cell biology experiments verified the role of HOXC6 in proliferation and cell cycle progression. In conclusion, HOXC6 might be a candidate biomarker gene for individual treatment optimization of glioblastoma. HOXC6 expression has a significant prognostic value and is related to the cell cycle process in glioblastoma.

A Hypoxia-Related Signature for Predicting Prognosis, Cellular Processes, Immune Microenvironment and Targeted Compounds in Lung Squamous Cell Carcinoma.

BackgroundLung squamous cell carcinoma (LUSC) is a malignant tumour of the lung epithelium. A hypoxic environment can promote tumour cell proliferation and invasion. Therefore, this study aims to explore hypoxia-related genes and construct reliable models to predict the prognosis, cellular processes, immune microenvironment and target compounds of lung squamous carcinoma.MethodsThe transcriptome data and matched clinical information of LUSC were retrieved from The Cancer Genome Atlas (TCGA) database. The GSVA algorithm calculated each LUSC patient’s hypoxia score, and all LUSC patients were divided into the high hypoxia score group and low hypoxia score group. Weighted gene co-expression network analysis (WGCNA) and differential expression analysis were performed to screen out differentially expressed hypoxia-related genes (DE-HRGs) in LUSC microenvironment, and the underlying regulatory mechanism of DE-HRGs in LUSC was explored through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Hereafter, we established a prognosis-related genetic signature for DE-HRGs using univariate and multivariate Cox regression analyses. The relationship between gene signature and immune cells was further evaluated. Finally, the Comparative Toxicogenomics Database (CTD) was utilized to predict the targeted drugs for the prognostic genes.ResultsWe obtained 376 DE-HRGs. Functional enrichment analysis indicated that the DE-HRGs were involved in the cell cycle-related regulatory processes. Next, we developed and validated 3 HRGs-based prognostic signature for LUSC, including HELLS, GPRIN1, and FAM83A. Risk score is an independent prognostic factor for LUSC. Functional enrichment analysis and immune landscape analysis suggested that the risk scoring system might be involved in altering the immune microenvironment of LUSC patients to influence patient outcomes. Ultimately, a total of 92 potential compounds were predicted for the three prognostic genes.ConclusionIn summary, we developed and validated a hypoxia-related model for LUSC, reflecting the cellular processes and immune microenvironment characteristics and predicting the prognostic outcomes and targeted compounds.

Comprehensive Transcriptome Analysis of mRNA Expression Patterns Associated With Enhanced Biological Functions in Periodontal Ligament Stem Cells Subjected to Short-Term Hypoxia Pretreatment.

Short-term hypoxia pretreatment significantly enhances periodontal ligament stem cell (PDLSC)-based periodontal tissue regeneration by improving various cellular biological functions, but the underlying mechanisms remain unclear. In this study, based on RNA sequencing (RNA-seq), we comprehensively analyzed the possible regulatory mechanisms of the short-term hypoxic effects on the biological functions of healthy and inflammatory PDLSCs. A total of 134 and 164 differentially expressed genes (DEGs) were identified under healthy and inflammatory conditions, respectively. Functional enrichment analyses indicated that DEGs under both conditions share certain biological processes and pathways, including metabolic processes, developmental processes, reproductive processes, localization, immune system processes and the HIF-1 signaling pathway. The DEGs identified under inflammatory conditions were more significantly enriched in cell cycle-related processes and immune-related pathways, while DEGs identified under healthy condition were more significantly enriched in the TGF-β signaling pathway. A protein-protein interaction network analysis of the 59 DEGs in both conditions was performed, and 15 hub genes were identified. These hub genes were mainly involved in glycolysis, the cellular response to hypoxia, cell differentiation, and immune system processes. In addition, we found that hypoxia induced significant differential expression of genes associated with proliferation, differentiation, migration, apoptosis and immunoregulation under both healthy and inflammatory conditions. This study provides comprehensive insights into the effects of short-term hypoxia on the biological functions of PDLSCs and suggests a potentially feasible strategy for improving the clinical effectiveness of cell-based periodontal tissue engineering.