Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus and is a major cause of end-stage kidney disease. Cordyceps sinensis  is a widely applied ingredient for treating patients with DN in China, while the molecular mechanisms remain unclear. This study is aimed at revealing the therapeutic mechanisms of Cordyceps in DN by undertaking a network pharmacology analysis.  In this study, active ingredients and associated target proteins of Cordyceps sinensis were obtained via Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and Swiss Target Prediction platform, then reconfirmed by using PubChem databases. The collection of DN-related target genes was based on DisGeNET and GeneCards databases. A DN-Cordyceps common target interaction network was carried out via the STRING database, and the results were integrated and visualized by utilizing Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to determine the molecular mechanisms and therapeutic effects of Cordyceps on the treatment of DN.  Seven active ingredients were screened from Cordyceps, 293 putative target genes were identified, and 85 overlapping targets matched with DN were considered potential therapeutic targets, such as TNF, MAPK1, EGFR, ACE, and CASP3. The results of GO and KEGG analyses revealed that hub targets mainly participated in the AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, PI3K-Akt signaling pathway, and IL-17 signaling pathway. These targets were correlated with inflammatory response, apoptosis, oxidative stress, insulin resistance, and other biological processes.  Our study showed that Cordyceps is characterized as multicomponent, multitarget, and multichannel. Cordyceps may play a crucial role in the treatment of DN by targeting TNF, MAPK1, EGFR, ACE, and CASP3 signaling and involved in the inflammatory response, apoptosis, oxidative stress, and insulin resistance. Diabetic nephropathy (DN) is featured as hyperglycemia, hyperfiltration, proteinuria, and progressive renal function decline, which can cause end-stage kidney disease . It accoThis study is aimed at analyzing the pharmacological mechanisms of active ingredients of Cordyceps involved in the progression of DN via using the network pharmacology databases and biological analysis methods. It laid a stable foundation for further research on exploring pharmacological mechanisms of Cordyceps in treating DN. The study framework is showed in http://lsp.nwu.edu.cn/tcmsp.php), a systematic pharmacological platform that contains the relationships among herbal compounds, related targets, and diseases [https://www.ncbi.nlm.nih.gov/pccompound) to double-check the final compounds of Cordyceps [http://www.swisstargetprediction.ch.), a webserver to accurately identify the targets of active molecules [TCMSP , a human gene compendium with information about genomics, proteomics, and transcriptomics [https://www.disgenet.org/home/), a comprehensive platform including one of the largest publicly accessible collections of genes, to search for DN-associated targets [The keyword “diabetic nephropathy” was inputted in the GeneCards , which covers nearly all functional interactions among the expressed proteins [https://www.cytoscape.org/) software where the interaction information was integrated and analyzed.A protein-protein interaction (PPI) network was obtained based on the STRING platform (proteins . Target http://www.kegg.jp/) is an encyclopedia of genes and genomes; connecting genomic information to higher-order functional information to capture significantly enriched biological pathways is the major objective of the KEGG knowledgebase [GO is the most comprehensive and widely used knowledgebase for the classification of gene functions, including the biological process (BP), molecular function (MF), and cell component (CC) . KEGG  as the leading factors, chronic inflammation and infiltrated immune cells in renal tissue are considered to be the common pathological consequences , 21. Des pathway , 30. In  pathway . Besides pathway ; LXA4 cac effect , 32.We found that many targets can be regulated by multiple compounds from the compound-target network, such as CYP19A1, NOS2, NR1H3, SHBG, and PTGS2. When it comes to core targets, TNF (degree = 51), MAPK1 (degree = 51), EGFR (degree = 45), and CASP3 (degree = 45) played an important role in the process of Cordyceps in DN treatment. TNF and MAPK1 are correlated with inflammation response and deterioration of renal function , 34. CASβ1 mainly participate in the biological processes of inflammatory response, oxidative stress, and lipid metabolic process [We also selected 85 common targets between the components of Cordyceps and DN for performing GO enrichment, consisting of biological processes, molecular function, and cellular components, which is aimed at predicting the mechanism of Cordyceps in treating DN. We found that the candidate targets are involved in multiple biological processes, such as regulation of inflammatory response, response to lipopolysaccharide, response to nutrient levels, response to oxygen levels, cellular response to chemical stress, regulation of lipid metabolic process, and lipid localization. The active targets such as TNF, PPARG, MAPK1, EGFR, and TGF- process , 38, 39. process , 41. It κB, MAPK/ERK, and TGF-β/Smad [α-SMA, ending up with renal interstitial fibrosis [Using network pharmacological analysis and performing KEGG enrichment, we found that these Cordyceps components may relieve the symptoms of DN through the action of targets in various signaling pathways and multiple biological processes, including AGE-RAGE signaling pathway, TNF signaling pathway, apoptosis, IL-17 signaling pathway, PI3K-Akt signaling pathway, and insulin resistance. Activation of the receptor for AGEs or RAGE (receptor for advanced glycation end-products) is associated with the development of DN , which eF-β/Smad –45. Addifibrosis . Moreovefibrosis .α, NF-κB moves from the cytoplasm to the nucleus and activates the transcription of VCAM-1, ICAM-1, IL-6, and IL-8, which will result in endothelial inflammatory and DN pathological process acceleration [κB can be activated by several cytokines, which in turn induces the production of TNF-α, resulting in diabetic renal damage [TNF signaling is characterized as a well-known inflammatory cytokine associated with renal injury . Once stleration . Furtherl damage .β1, blood pressure, inflammation, TNF-α, IL-6, and oxidative stress [Insulin resistance is a critical process and one of the main symptoms in the initiation and progression of DN, which is closely related to microalbuminuria , 52. Hige stress –56.Therefore, these results of network pharmacological analyses not only verify that our screened targets are consistent with previous literature reports but also indicate that Cordyceps play a significant therapeutic role in DN by regulating several signaling pathways, including inflammatory response, insulin resistance, oxidative stress, apoptosis, and other pathways with unclear mechanisms. It will provide a novel methodology for further study of the therapeutic mechanism of Cordyceps in alleviating DN.In summary, our network pharmacological analyses have shown that Cordyceps play an indispensable supplementary role in the treatment of DN, which is consistent with previous studies. Moreover, the biological functions of active chemical molecules and their corresponding targets of Cordyceps analyzed by network pharmacological methods provide a unique and innovative path for the study of TCM and further reveal the molecular biological mechanism of Cordyceps in treating DN. However, in vivo and in vitro experiments should be undertaken to validate the relationship between key targets and pathways of Cordyceps for the treatment of DN. Despite the limitations of this study, the results of this study provide new evidence and theoretical basis which will be used in subsequent theoretical and clinical research studies of Cordyceps.


Bos taurus we created a gene identifier mapping database with bridgeDb and a pathway collection in WikiPathways. The regulation of marbling is possibly the result of the interplay between signaling pathways in muscle, fat, and intramuscular connective tissue. Pathway analysis revealed 17 pathways that were significantly different between well-marbled and lean-marbled beef. The MAPK signaling pathway was enriched, and the signaling pathways that play a role in tissue development were also affected. Interestingly, pathways related to immune response and insulin signaling were enriched.Red meat is an important dietary source that provides part of the nutritional requirements. Intramuscular fat, known as marbling, is located throughout skeletal muscle. Marbling is a trait of major economic relevance that positively influences sensory quality aspects. The aim of the present study was to identify and better understand biological pathways defining marbling in beef cattle. Pathway analysis was performed in PathVisio with publicly available transcriptomic data from semitendinosus muscle of well-marbled and lean-marbled beef. Moreover, for  Bos taurus cattle  can increase the tenderness, more than the Bos indicus cattle   . Althoug breeds) . Also, W breeds) . There i breeds) . Underst breeds) . Therefo breeds) . It has  breeds) . The pub breeds) .B. taurus at WikiPathways  4 x 44K Gene Expression Microarray oligonucleotide slides . Sadkowski and coworkers checked the quality of the data and performed LOWESS normalization. The normalized transcriptomic data compared well-marbled beef Holstein-Friesian (n = 4) or Hereford (n = 4) to lean-marbled beef (n = 4). The four log10 fold change (log10FC) values for each group comparison were averaged to obtain an estimate of the 10logFC between the entire groups. Furthermore, a one-sample t-test was performed on both sets of four values, comparing those to 0 . Bovine genes were considered to be significantly, differentially expressed with p ≤ 0.05 and an absolute FC ≥ 1.3 .B. taurus collection was missing. We updated and extended the pathway collection for B. taurus. We also created a B. taurus gene identifier (ID) mapping database based on mappings present in the Ensembl-based BridgeDb framework . Second, the WikiPathways homology based the homology mapper which is available at GitHub (https://github.com/PathVisio/homology.mapper) was updated to improve homology coverage for gene products that were annotated with different data sources. The pathways were converted from human pathways, with a required minimum successful conversion of at least 50% of the original human genes. Third, we manually curated all converted pathways to check whether the genes were correctly annotated and pathways are relevant in B. taurus. Finally, new pathways directly derived from cow breeding literature and not present in the WikiPathways collection were designed in PathVisio (v3.2.0) (https://www.pathvisio.org/plugin/wikipathways-plugin/) for PathVisio.The online biological pathway repository, WikiPathways , containramework . The new(v3.2.0) , the patB. taurus WikiPathways pathway collection, containing 286 pathways (6/30/2015), and the B. taurus ID mapping database, was used in the analysis. The pathways are ranked based on a standardized difference score (Z-score) based on the expected value and standard deviation of the number of significantly (p ≤ 0.05) and differentially (absolute FC ≥ 1.3) expressed genes in a pathway. Biological pathways significantly changed when (i) Z-score > 1.96, (ii) permuted p-value < 0.05 and (iii) minimum number of changed genes is 3. Additionally, alterations in gene expression  when comparing Hereford to Limousin were visualized on the B. taurus pathways with PathVisio.To analyze and visualize the molecular changes in marbling at biological process level a pathway-based over-representation analysis was performed in PathVisio (v3.2.0) . The B. B. taurus collection at WikiPathways we performed Gene Ontology (GO) analysis via the GO-Elite web-interface , was used . First, In the selected transcriptomic data set of beef marbling 42,990 microarray reporters were measured in both lean marbling beef (Limousin) and well marbling beef (Hereford and Holstein-Friesian) animals. Statistical analysis was performed on 29,677 reported genes that remained from the 42,990 reporters after quality control and annotation with Ensembl gene IDs. In the Hereford breed compared to the Limousin breed, 1,513 were higher expressed and 1,556 lower expressed . When comparing the Holstein-Friesian breed to the Limousin breed, 1,772 genes were higher expressed and 2,458 lower expressed in the Holstein Friesian breed. The genes that met these criteria were used for further analysis.https://www.pathvisio.org/downloads/download-pathways/). Moreover, 4 pathways were newly created based on the bovine breeding literature: Growth hormone signaling (WP2890) (Roudbari Z and Kutmon M: Growth Hormone (GH) Signaling (B. taurus); (https://www.wikipathways.org/instance/WP2890), Growth hormone receptor signaling (WP2891)  Signaling (B. taurus) (https://www.wikipathways.org/instance/WP2891), IGF1-signaling (WP2892)  (https://www.wikipathways.org/instance/WP2892), and Gonadotropin- releasing hormone signaling (WP2901)  signaling (B. taurus) (https://www.wikipathways.org/instance/WP2901). As an example, the newly designed GNRH signaling pathway is shown in In total, 282 human pathways were converted from human pathways to cattle pathways. All these pathways were manually checked and are available at  the overall gene expression was higher in skeletal muscle of Hereford and Holstein-Friesian compared to Limousin  signaling pathway has found to be responsible for transduction of extracellular signals to their intracellular targets in different types of cells, including skeletal muscle cells and which leads to several biological effects for example proliferation, differentiation, migration, growth, apoptosis, and more specifically to muscle cells, hypertrophy . The p38B. taurus IL-1 signaling pathway (http://www.wikipathways.org/instance/WP3271), such as IL-1α, IL-1β, MAP3K1, UBE2N, MAPK14, REL, ATF2, and JUN were significantly up-regulated in Hereford breed versus Limousin breed  showed significant expression differences. Fourteen genes were identified as up-regulated in Hereford breed , and twelve were down-regulated in Hereford breed  when compared to Limousin breed  under the accession number GSE46411 and in the wikipathways.org knowledge base under the accession numbers: WikiPathways: WP2890, WikiPathways: WP2891, WikiPathways: WP2891, and WikiPathways: WP2901.Ethical review and approval was not required for the animal study because this study is just analysis.ZR, SC, and CE conceptualized and designed the study. ZR, SC, MK, LE, and JM analyzed the data. ZR collected and assembled the data and wrote the manuscript and SC, CE, and TS contributed to reviewing and editing the manuscript and also read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilise its metabolism as a basis for bio-based production.The yeast K. marxianus. We use the phenylalanine biosynthetic pathway both as a prototype and because phenylalanine is a precursor for commercially valuable secondary metabolites. First, we modify and overexpress the pathway to be resistant to feedback inhibition so as to overproduce phenylalanine de novo from synthetic minimal medium. Second, we assess native and heterologous means to increase precursor supply to the biosynthetic pathway. Finally, we eliminate branch points and competing reactions in the pathway and rebalance precursors to redirect metabolic flux to a specific product, 2-phenylethanol (2-PE). As a result, we are able to construct robust strains capable of producing over 800 mg L−1 2-PE from minimal medium.To illustrate the synthetic biology strategies to be followed and showcase its potential, we describe a comprehensive approach to rationally engineer a metabolic pathway in The strains we constructed are a promising platform for the production of aromatic amino acid-based biochemicals, and our results illustrate challenges with attempting to combine individually beneficial modifications in an integrated platform. Saccharomyces cerevisiae has proved to be an excellent host for de novo synthesis of valuable secondary metabolites in the flavonoid, stilbenoid and alkaloid families . Figure S1. Strain construction flowchart. Engineering strategies and strains are coloured by the use of native or heterologous enzymes, and whether an improvement in 2-phenylethanol production was observed. Figure S2. Extracellular acetate production for strains using a KmASR.004 or b KmASR.046 as a base. Figure S3. Full expression of the non-oxidative pentose pathway allows for a faster production of 2-phenylethanol over short fermentation times. Figure S4. The effect of knocking down TYR1 expression and knocking out KmARO8 on KmASR.062 results in a smaller increase in 2-PE production than when the same modifications are made in KmASR.047. Figure S5. Overexpressing 2-PE producing genes form the Ehrlich pathway does not significantly improve 2-PE production in K. marxianus NBRC1777.Additional file 2. Supplementary data 1.Additional file 3. Supplementary data 2.


Yifei Sanjie Formula (YFSJF) is an effective formula on pulmonary fibrosis (PF), which has been used in clinic for more than 30 years. In order to investigate the molecular mechanism of YFSJF in treating PF, network pharmacology was used to predict the cooperative ingredients and associated pathways.  Firstly, we collected potential active ingredients of YFSJF by TCMSP databases. Secondly, we obtained PF-associated targets through OMIM and Genecards database. Finally, metascape was applied for the analysis of GO terms and KEGG pathways. P < 0.05), including 4632 biological processes, 444 cellular components, and 639 molecular functions. A total of 143 related KEGG pathways were enriched (P < 0.05), including IL-17 signaling pathway, T cell receptor signaling pathway, TNF signaling pathway, calcium signaling pathway, TH17 cell differentiation, HIF-1 signaling pathway, and PI3K-Akt signaling pathway.  We screened out 76 potential active ingredients and 98 associated proteins. A total of 5715 items were obtained by GO enrichment analysis ( YFSJF can interfere with immune and inflammatory response through multiple targets and pathways, which has a certain role in the treatment of PF. This study lays a foundation for future experimental research. Pulmonary fibrosis (PF) is a common and irreversible pathological outcome of many chronic lung diseases, which is characterized by progressive alveolar epithelial injury and abnormal repair. In recent years, the incidence rate and mortality rate of PF are increasing . It is rMori Cortex), Zhebeimu (Fritillariae Thunbrgii Bulbus), Jiezi (Sinapis Semen), E'zhu (Curcumae Rhizoma), and Sanqi (Panax Notoginseng (Burk.) F. H. Chen Ex C. Chow). In the whole formula, drugs are compatible with each other to achieve the effects of tonifying qi, eliminating phlegm and removing blood stasis. The preliminary research of the research group confirmed [Yifei Sanjie Formula (YFSJF) is an effective formula in treating PF, which is authorized by professor Qingshen Li, a famous physician of traditional Chinese medicine in Yunnan Province. The formula is based on Yupingfeng formula, which is composed of Sangbaipi  under the conditions of oral bioavailability (OB) ≥30% and drug-like (DL) ≥0.18. The targets data in the TCMSP database were collected from the DrugBank database [https://www.drugbank.ca/). The UniProt database [https://www.uniprot.org/) was utilized to convert the obtained target names of YFSJF to the gene names.The active ingredients and corresponding targets of YFSJF were collected from Traditional Chinese Medicine Systems Pharmacology database  (TCMSP ddatabase  (https:/database  (https:/http://www.biostars.org/p/260759) to construct the YFSJF-active ingredient-action target network and were analyzed with the tool Network Analyzer.The active ingredients in YFSJF and their corresponding gene names were import into Cytoscape software  (versionhttps://www.genecards.org/) and the Online Mendelian Inheritin man database [https://omim.org/) provide functional information for all known and putative human genes. Using the “PF” as the keyword, the species was designated as “Homo sapiens (Human)” to obtain the associated targets of the disease.The GeneCards database   to collect their corresponding diseases. Then, diseases were classified into groups according to the Medical Subject Headings Browser [https://www.ncbi.nlm.nih.gov/mesh/?term=). Finally, target-disease network was constructed based on potential targets and their corresponding diseases. The importance of nodes with “Degree” was constructed with Network Analyzer.In order to get a better understanding of diseases related to YFSJF, the potential targets were projected to the Therapeutic Target Database  , the protein species was set as “HOMA sapiens”, and the minimum interaction threshold was set as “medium confidence” (>0.4). Next, Network Analyzer was used to evaluate the importance of the nodes with “Degree”. The final network was constructed based on the nodes with positive degree, and then, the network was exported to an image.The obtained potential targets were imported to the STRING database  (https:/https://metascape.org/gp/index.html#/main/step1) for enrichment analysis. P < 0.05 was accepted to obtain main GO and KEGG pathways, and the first 20 pathways were screened out. Afterwards, Cytoscape software was used to analyze the enrichment of GO and KEGG pathways for potential targets, and the relationship between potential targets and biological functions or disease-related pathways was screened out and visualized.The potential targets of YFSJF on PF were introduced into the Metascape  (https:/Hedysarum Multijugum Maxim (HMM), 11 from Saposhnikoviae Radix (SR), 7 from Atractylodes Macrocephala Koidz (AMK), 7 from Fritillariae Thunbrgii Bulbus (FTB), 3 from Sinapis Semen (SS), 24 from Mori Cortex (MC), 8 from Panax Notoginseng (Burk.) F. H. Chen Ex C. Chow (PN), and 3 from Curcumae Rhizoma (CR)  network was constructed based on the potential targets and corresponding diseases. As shown in P < 0.05) was enriched with Metasape. Top 20 terms are shown in GO function of 98 action targets (P < 0.05), enrichment analysis yielded 144 pathways and visualization analysis of the top 20 signaling pathways , and voltage-gated potassium channel subfamily A member 3 and other ion channels [+ T cells, are raised in the airways and lungs of PF patients, upregulating tissue inflammation, and exacerbate alveolar destruction by producing IL-17, an important pathogenic factor. Th17 cells also exert direct influence on epithelial cells, smooth muscle cells, and airway fibroblasts to induce neutrophil chemokine secretion [β-D-glucopyranosyl-7-Oβ-D-gentiobioside (QGG) can significantly improve the survival conditions, growth ratio, and pulmonary functions of rats, which can reduce the ratio of CD4+ IL-17+/FOXP3+CD4+ in peripheral blood and reduce the apoptosis of lung tissues, repair damaged tissue, and maintain the integrity of organ [γ content [T cells are important immune cells, which mediate adaptive immune responses in humans and express calcium ions in T lymphocyte release activates Cachannels . The actchannels . Calciumecretion , 30. Queof organ . Th17 ceof organ . Baldeviof organ  have fouof organ . Anti-ILof organ . The stu content . TherefoPF is characterized by alveolar interstitial inflammatory cell infiltration, fibroblast proliferation, and alveolar interstitial fibrous connective tissue deposition. The aggregation of inflammatory cells and the release of cytokines will induce a large number of collagen fibers, resulting in the remodeling of lung tissue structure, reduction of alveolar number, deformation, atresia, and loss of lung function . Therefoα and IL-6 and decrease in the alveolar wall thickening, both in the inflammatory infiltrate and in the collagen fiber deposition [β-Sitosterol-D-glucoside can inhibit the growth and lung metastasis of animal tumors to some extent, upregulate miR-10a expression, inhibit PI3K-AKT signaling pathway, reverse the expression levels of EMT, and inhibit the expression of metastatic related cytokines, eventually inducing apoptosis and inhibit metastasis in tumor cells [Tumor necrosis factor (TNF) is a kind of cytokines, which can promote cell growth, differentiation, and apoptosis and induce inflammation by binding to specific receptors on the cell membrane . TNF-alpposition . Hypoxiaposition . Hae-Rimposition  have fouposition . PI3K caposition . Peiminiposition . β-Sitosor cells . Combineor cells , YFSJF wThis study shows that quercetin, kaempferol, beta-sitosterol, 7-o-methylosmucronulatol, and stigmasterol can regulate GSK3B, L2, IL6, MAPKs, NOS2, and VEGFA and participate in regulating PI3K-Akt signaling pathway, HIF-1 signaling pathway, and IL-17 signaling pathway. This may be the main material basis for YFSJF to alleviate inflammatory reaction, regulate immunity, inhibit fibroblast proliferation and promote apoptosis, reduce collagen deposition, and finally to alleviate PF. NOS2 and GSK3B are involved in multiple signal transduction pathways and have high degree of freedom in the network of component-target-pathway. Therefore, it can be speculated that YFSJF might regulate signal transduction through NOS2 and GSK3B, thus interfering with PF .YFSJF has a therapeutic effect on PF, and the antifibrosis mechanism mainly includes the effects on immunity, inflammatory factors and multiple signaling pathways. This study indicated that quercetin, kaempferol, and beta-sitosterol in the active ingredients of YFSJF could regulate PI3K/AKT signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, IL-17 signaling pathway, T cell receptor signaling pathway, calcium signaling pathway, and Th17 cell differentiation, then alleviate inflammatory response, enhance immunity, inhibit fibroblast proliferation and promote apoptosis, reduce collagen deposition, and eventually alleviate pulmonary fibrosis. The study further revealed the relevant mechanism of YFSJF in preventing and treating PF broadened the clinical application scope of YFSJF and also provided a new therapeutic idea for preventing and treating PF. Due to the limitations of various platform database data and corresponding analysis algorithms and software functions, the results may be biased. In the next step, we will carry out experimental verification based on the results of this study.


SACS KO SH-SY5Y neuroblastoma cell model. Single-stranded deoxyoligonucleotides, selected in vitro from large random libraries, bound and quantified molecular targets related to the neuroinflammation signaling pathway and to neuronal development. Changes in protein levels were further analyzed by bioinformatics and network approaches to identify biomarkers of ARSACS and functional pathways impaired in the disease. We identified novel significantly dysregulated biological processes related to neuroinflammation, synaptogenesis, and engulfment of cells in patients and in KO cells compared with controls. Among the differential expressed proteins found in this work, we identified several proteins encoded by genes already known to be mutated in other forms of neurodegeneration. This finding suggests that common dysfunctional networks could be therapeutic targets for future investigations.Although the genetic basis of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) has been uncovered, our poor understanding of disease mechanisms requires new light on functional pathways and modifying factors to improve early diagnostic strategies and offer alternative treatment options in a rare condition with no cure. Investigation of the pathologic state combining disease models and quantitative omic approach might improve biomarkers discovery with possible implications in patients' diagnoses. In this study, we analyzed proteomics data obtained using the SomaLogic technology, comparing cell lysates from ARSACS patients and from a  SACS, the gene encoding sacsin  is a rare disease caused by mutations in g sacsin . ARSACS g sacsin –4. The dg sacsin .The availability of disease biomarkers in peripheral tissues, assessable using simple, non-invasive techniques, would help to shed light on the impaired biological pathways underlying the disease and could be important for early diagnosis.Proteomics, the massive analysis of proteins, is now feasible using various technologies, such as mass spectrometry, gel-based techniques, antibody-based arrays, and recently developed aptamer-based technologies –8. DespiIn this study, capitalizing on our early work investigating the mRNA signature in neuronal models of ARSACS , we usedSacs−/− mouse models , 2 mM l-glutamine, and 1% penicillin/streptomycin. Cells were treated with 20 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) for 2 h to induce mitochondrial damage and fragmentation. Human fibroblasts were collected from diagnostic punch-skin biopsies using standard procedures and with signed informed consent. Primary fibroblast cell lines from three ARSACS patients presenting severe disease status and carrying different mutations in SACS . All samples (fibroblasts and cell models) were washed twice with PBS 1X and then homogenized in M-PER™ Mammalian Protein Extraction Reagent , Italy) containing inhibitors of proteases , Italy), following the manufacturer's standard protocol. About 200 μg/mL of cell proteins, measured by BCA assay  were placed in a final volume of 75 μL of PBS, ready for SOMAscan assay.In a previous work, we generated and characterized the SH-SY5Y neuroblastoma cell line and derived KO and WT clones . In brie in SACS  and fromhttps://somalogic.com/technology/publications). A SOMAmer-protein binding step was followed by partitioning and wash steps that convert relative protein concentrations into measurable nucleic acid signals; these signals were quantified using hybridization to custom DNA microarrays, as already reported   were normalized separately and processed at the Genomic Laboratory, Fondazione Edo ed Elvo Tempia, Biella, Italy.siggenes R package was used to identify lists of differentially expressed proteins in cultured skin fibroblasts from ARSACS patients vs. healthy controls and KO vs. WT neuronal-like cell lines, treated or untreated. Lists of statistically significant positive (upregulated) and negative (downregulated) differentially expressed proteins (DEPs) were retrieved by setting the fold-change (FC) threshold higher than 1.25 and the p-value threshold lower than 0.01.The significant analysis of microarray method was used based on a set of sample permutations and performed as described . The sighttps://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) to interpret datasets in the context of biological processes, pathways, and molecular networks. Expression analysis was based on protein FC, to calculate directionality (z-score) in the analysis, whereas the Ingenuity Knowledge Base reference set was used for p-value calculation.Sets of DEPs in each sample were categorized using the Ingenuity Pathway Analysis (IPA) molecular suite  and queried the GENESIS 2.0 platform (https://www.tgp-foundation.org/genesis-log-in) (https://cadd.gs.washington.edu/) >25 for missense mutations (https://gnomad.broadinstitute.org/); and (5) homozygous count <2 in either gnomAD or GENESIS 2.0.To link DEPs belonging to the different ARSACS experimental models, we used the IPA rer tool , setting-log-in) , an affo-log-in) , we perfutations ; (3) adautations ; (4) minWe ran three technical replicates per sample on the multiplexed proteomic platform using the SOMAScan Assay technology to investigate a targeted set of 1,300 proteins and compare their expression in skin fibroblasts from ARSACS patients and healthy subjects and in neuroblastoma WT and KO cell lines, either untreated or treated with FCCP, the uncoupler of mitochondrial oxidation and phosphorylation leading to impaired mitochondrial fragmentation .p-value < 0.01) for all comparisons. By means of this strategy, we quantified differentially expressed proteins filtered by a FC threshold (>25% both in upregulation and downregulation) and a strict statistical significance threshold  functional networks in the Disease & Function category, together with impairment of two Canonical Pathways—Neuroinflammation Signaling Pathway  and Synaptogenesis Signaling Pathway .Following further scrutiny of the main functional annotations in KO cells treated with the uncoupling FCCP, we also noticed an overall reduction of the dysregulation level of proteins involved in Synaptogenesis Signaling Pathway DEPs were found to be distributed along the presynaptic  as well as the post-synaptic terminal  and the synaptic cleft . We also identified four proteins  with a neuronal role in the Engulfment of Cells functional network, and two cytokines (IL-6 and CXCL12) involved in neuromodulation , was found to be downregulated both in cell models and in skin cells from patients. NAGK is implicated in sugar metabolism and was recently found to be highly expressed in neuronal dendrites; it appears important in axonal growth of developing neurons . HoweverEngulfment of Cells pathway  occurred in patients with an already genetically characterized form of spastic ataxia (SPAX) , giving EPHA3/EPHB2, EPHB2/SRC, EPHA3/MPO, APOE/PIK3CA), encoding proteins that are mostly part of the Synaptogenesis Signaling Pathway.It is worth noting that 36 individuals with undiagnosed ataxia harbored at least two putative pathogenetic mutations in DEP-encoding genes and that nine of these share the same combination of multiple mutated genes , pointing to pathophysiological features shared with other neurodegenerative conditions –33. To mAlthough only a single DEP was found to be common to the two cell models and its significance in ARSACS remain unclear, we showed an overlap of more than 50% of the DEPs and significant agreement with data generated in an earlier quantitative transcriptome analysis , furtherin vivo (KO mice) to substantiate the specific biological function/pathway impairment. Affected pathways and related functions of Synaptogenesis and Neuronal Cell Death related to sacsin loss-of-function are known in mice, and they indicate impairment of Purkinje cells and axonal development  or elopment , 14. It elopment , and SNCelopment –37, suggEngulfment of Cells was also shown to be impaired in our models. Based on recent data showing that engulfment of dendrites by microglia precedes Purkinje cell degeneration in a mouse model of Niemann Pick Type-C disease  under accession number GSE162132'.The studies involving human participants were reviewed and approved by Tuscany Pediatric Ethic Committee. The patients/participants provided their written informed consent to participate in this study.FM, SD, GC, DG, and ES contributed to technical data. FM, SD, and FS set experiments and interpretation of results. FF, ES, EB, and SZ provided intellectual contribution. FM, FS, and FG supervised the study. FM and FS provided funds. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tripterygium wilfordii Hook (TWH) in the treatment of Crohn’s disease (CD) by network pharmacology.To explore the mechanism of action of Traditional Chinese Medicine Systems Pharmacology database and analysis platform (TCMSP) was used to obtain the active constituents and targets of TWH. “Crohn’s disease” was used as a search term to search for related targets of CD from GeneCards database and OMIM database, thereby obtaining the targets of TWH against CD. The Cytoscape 3.7.1 software was used to construct a Chinese medicine compound-target network and STRING database to construct a protein-protein interaction network (PPI). The DAVID 6.8 online tool was used to perform gene ontology (GO) and kyoto encyclopedia of genes and genome (KEGG) pathway enrichment analysis of overlapping targets.The database results showed that there were 30 active ingredients (14 key active ingredients) in TWH and 36 targets were screened out for CD treatment. Network analysis indicated that main targets of main active components of TWH were target genes such as VEGFA, MAPK8 and CASP3, which are involved in the regulation of cancer pathway, TNF signal pathway, hepatitis B pathway, apoptosis pathway, NF-kappa B signal pathway and so forth.TWH can play a multi-target and multi-channel synergistic treatment of CD by anti-angiogenesis, anti-apoptosis, anti-inflammation and immune regulation. Crohn’s disease (CD) is a chronic inflammatory bowel disease with multi-factors that genetics and environment interact to manifest the disease . It is tTripterygium wilfordii Hook (TWH) is a woody vine of the genus Tripterygium. It contains a variety of chemical components such as alkaloids, diterpenoids, triterpenoids and sesquiterpenes. As early as in Li Shizhen’s book “Compendium of Materia Medical” of the sixteenth century, there are records that TWH can anti-rheumatism, promote blood circulation, reduce swelling, and relieve pain. In recent years, clinical studies have found that TWH can effectively induce CD remission and maintain remission [emission , 5, howeemission . CompareThis study aimed to screen the bioactive components of TWH and elucidate the targets that contribute to its therapeutic effect, thereby further explaining its mechanism of action in CD treatment by network pharmacology. The study design and workflow are presented in Fig. http://lsp.nwu.edu.cn/tcmsp.php), the bioactive ingredients and targets of TWH were obtained. Bioactive ingredients (OB) refers to the rate and extent that a drug is absorbed into the body’s circulation. Drug-like properties (DL) reflects the nature of a drug which has a specific functional group or contains the same or similar physical characteristics. The human intestinal cell line Caco-2 is a useful tool for studying the absorption and transport of drugs in intestinal epithelial cells. The drug half-life reflects the concentration of the drug in the blood or body and is an important parameter for calculating the dosing interval, the dose administered, and the drug accumulation. Bioactive ingredients were collected based on the condition that OB > 30%, DL > 0.18, Caco-2 permeability > − 0.4, and half-life > 3 h as described previously [The Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) is a platform for integrating pharmacokinetics, medicinal chemistry, and drug-target-disease networks . Accordihttps://www.genecards.org/) and the Online Mendelian Inheritance in Man (OMIM) database (http://omim.org/). The overlapping targets from CD treatment and from bioactive ingredients of TWH then allowed identification of targets of TWH in the treatment of CD.Search for CD related targets with “Crohn’s disease” as a search term using GeneCards database (https://string-db.org/) can be used to analyze the interaction between proteins and proteins [Homo sapiens”, the lowest interaction score was set to medium confidence (0.400), then discrete targets were hidden and the remaining parameters remained the default settings. Subsequently, the PPI network obtained in the STRING database was visualized and further analyzed using Cytoscape software.The STRING database  provides systematic, comprehensive biological annotation information for large-scale genes or proteins, and provides the most significantly enriched biological annotations [The Database for Annotation, Visualization and Integrated Discovery  is a member of a large family of proteins and receptors involved in immune regulation which interacts with two different receptors, and then initiates downstream signaling pathways to exert pro-inflammatory biological effects. In the inflammatory process of CD, TNF-α is an early pro-inflammatory cytokine and has been observed to be involved in the pathological processes in CD  . Serum THowever, due to the reported risk of toxic action on such organs as the liver, kidney, spleen, gastrointestinal tract or heart , applicaIn this paper, network pharmacology was used to explore the active components, potential targets and mechanisms of TWH in treating CD. The results showed that 14 ingredients of TWH may play important roles in biological processes of anti-angiogenesis, anti-apoptosis, anti-inflammatory and immune regulation by acting on VEGF, MAPK8, and CASP3 through TNF-α and NF-κB signaling pathways. More importantly, the potential targets and biological processes mentioned above may provide valuable information for further investigation into the mechanism of TWH for treating CD.


Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6.The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate and pyridoxine-5′-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria • Known vitamin B6 routes have been genetically engineered.• Underground metabolism facilitates the emergence of novel vitamin B6 biosynthetic pathways.• These pathways may be suitable to engineer bacteria overproducing vitamin B6. Bacillus subtilis is likely to have 65 PLP-dependent proteins , pyridoxamine (PM), pyridoxine (PN) and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate (PMP) and pyridoxine-5′-phosphate (PNP) . Previous studies showed that the inactivation of the yggS gene in E. coli and Salmonella enterica causes pleiotropic phenotypes, amongst them the accumulation of PNP  to PN  relives the PLP auxotrophy of a pdxB mutant strain lacking the 4PE dehydrogenase PdxB E. coli . However, independent lineages of the B. subtilis suppressor mutants were subjected to an adaptive laboratory evolution experiment to isolate variants with enhanced growth rates (Rosenberg et al. ytoQ gene of unknown function is required to promote rapid growth of the B. subtilis ΔpdxST pdxJH mutants lacking the bacillithiol biosynthetic genes (Rosenberg et al. B. subtilis facilitates the emergence of a pathway for synthesis of the essential cofactor PLP using parts of a non-native metabolic pathway (Rosenberg et al. As described above, Belitsky . RecentlE. coli, the novel vitamin B6 pathway in the evolved B. subtilis ΔpdxST pdxJH suppressor mutants could also contain enzymes of the serine metabolic pathway (Fig. B. subtilis (Sachla and Helmann E. coli (Fig. B. subtilis produces an intermediate of the DXP-dependent vitamin B6 pathway that could serve as a precursor for the PLP synthesis of the truncated pathway that was implemented in the ΔpdxST mutant. Similar to E. coli, the generated erythronate could be converted via 3,4-dihydroxy-2-oxobutyrate and 4HT to 4HTP by the promiscuous activities of the 3PG dehydrogenase SerA, the serine aminotransferase SerC and the homoserine kinase ThrB (Fig. B. subtilis. However, several native enzymes are present in B. subtilis that could be part of the novel serendipitous DXP-dependent vitamin B6 pathway (Fig. B. subtilis to employ the PdxH and PdxJ enzyme to produce PLP, the lack of bacillithiol could be beneficial because the thiol was shown to inactivate the 3PG dehydrogenase SerA by binding to the cysteine residue 410 in the active site (Chi et al. How could the new vitamin B6 pathway be structured? Certainly, there must be intermediates either generated by non-enzymatic catalysis of by promiscuous enzymes that feed into the truncated non-native pathway to allow synthesis of PLP. Similar to the serendipitous pathway, which connects serine biosynthesis with PLP synthesis in way Fig. . Recentloli Fig. . Thus, αhrB Fig. , 2015. TE. coli and B. subtilis, for overproducing the B6 vitamers PN and PL were unsuccessful. In fact, major genetic engineering efforts seem to be required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Moreover, the suppressor analyses using E. coli and B. subtilis mutants carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis consisting of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. These serendipitous pathways could serve as a promising starting point for engineering the bacteria for overproducing vitamin B6 at commercially attractive levels.Previous attempts to engineer bacteria, including the Gram-negative and Gram-positive model bacteria


Chordoma is a rare bone cancer originating from embryologic notochordal remnants. Clival chordomas show different dural penetration ability, with serious dural penetration exhibiting poorer prognosis. The molecular mechanism of dural penetration is not clear. We analyzed lncRNA and mRNA profiles in 12 chordoma patients with different degrees of dural penetration using expression microarrays. The differentially expressed lncRNAs and mRNAs were used to construct a lncRNA-mRNA co-expression network. LncRNAs were classified into lincRNA, enhancer-like lncRNA, or antisense lncRNA. Biological functions for lncRNAs were predicted according to the lncRNA-mRNA network and adjacent coding genes by pathway analysis. The 2760 lncRNAs and 3988 mRNAs were differentially expressed in chordomas between two groups of patients with and without dural penetration. Possible pathway involvement of the significance among the 55 lncRNAs located in the lncRNA-mRNA network, 24 lincRNAs, 7 enhancer-like lncRNAs, and 14 antisense lncRNAs include cell adhesion, metastasis, invasion, proliferation, and apoptosis. Expression of 10 lncRNAs and mRNAs, and epidermal growth factor mRNA with two identified lncRNAs were subsequently verified by qRT-PCR in chordoma tissues. Our report predicts the biological functions of many lncRNAs which may be used as diagnostic and prognostic biomarkers as well as therapeutic targets during the process of dural penetration in chordoma. Chordomas are rare, malignant, primary bone tumors, with an incidence of approximately 0.1/100,000/year . The staChordomas are characterized as being aggressive and locally invasive, and clivus chordomas have different degrees of dural penetration. Some tumors severely penetrate the dura mater, with little bone invasion, while others show little or no dura mater penetration with extensive skull base bone invasion. Our previous study showed chordoma patients with serious dural penetration have poorer prognosis and patients without dural penetration have longer overall survival and progressive-free survival .Long non-coding RNAs (lncRNAs) are greater than 200 nucleotides in length and regulate gene expression at transcriptional and post-transcriptional levels , 12. TraLncRNAs participate in various aspects of cell biology, including cell proliferation, apoptosis, differentiation, invasion and metastasis, and are commonly dysregulated in multiple types of cancers . Recent In the present study, we identified differentially expressed lncRNAs and gene expression profiles of chordomas with varying degrees of dural penetration and constructed lncRNA-gene co-expression networks to reveal the functional role of lncRNAs in regulating dural penetration of clivus chordoma. Among the top differentially expressed lncRNAs, we identified lincRNA, antisense lncRNA, and enhancer lncRNA which may contribute to the dural penetration biological processes in chordoma.Twenty patients were enrolled in the study and were equally classified into no dural penetration group and serious dural penetration group. The tumor with no dural penetration demonstrated expansive growth, like a “bubble” in magnetic resonance imaging (MRI) and dural perforation was not found in surgical procedure , 1B. In P < 0.05, FDR < 0.05; We compared the lncRNA and mRNA expression patterns in the tissue samples between clival chordoma patients with no dural penetration (n = 6) and with serious dural penetration (n = 6). The 2760 lncRNAs and 3988 mRNAs were identified that had significantly differential expression in no dural penetration samples compared to serious dural penetration samples , overlapping exons or intron in sense or antisense orientation, and bidirectional . Our resThe lncRNA-mRNA co-expression network was constructed following the workflow shown in P < 0.05, The identification of target genes is important for exploring the molecular mechanisms underlying lncRNAs function. The KEGG pathway analysis was applied to analyze the significant pathway of the altered 3988 mRNAs. The pathway analysis revealed that there are 62 and 38 significant pathways corresponding to the up and down-regulated mRNAs respectively P < 0.0, 5. ThesAccording to the lncRNA-mRNA co-expression network and pathway analysis five down-regulated pathways were identified, and they contributed to various cellular processes, including cell adhesion, migration, invasion, apoptosis and proliferation, and affected the dural penetration ability of chordoma cells . The 27 To further explore the possible roles of differentially expressed lncRNAs during the process of dural penetration in chordoma patients, we analyzed their genomic locations and classified them into lincRNA, enhancer-like lncRNA or antisense lncRNA . Among tLincRNAs are large intergenic non-coding RNA . The funWe then predicted the roles of enhancer-like lncRNAs  in the dLastly, we analyzed 143 antisense lncRNAs, 63 of which are intronic antisense and 80 of which were natural antisense. Based on KEGG analysis, the biological roles of 15 antisense lncRNAs  were predicted, and the results indicated that 14 coding-genes participated in nine down-regulated pathways and two up-regulated pathway , 8B. TheTo validate the microarray analysis results, we randomly selected 10 lncRNAs and mRNAs with larger fold changes including up-regulated and down-regulated expression from the microarray results and analyzed their expression levels by qRT-PCR in all chordoma tissues, 10 samples with serious dural penetration, and 10 samples without dural penetration. The results showed that the changes were reasonably consistent between microarray and qPCR data, and further confirmed the findings of the lncRNA and mRNA microarray data , 9B.Next, we analyzed the expression of EGF mRNA and the lncRNAs ABCC6P1 and AL078621.4 located in the lncRNA-mRNA co-expression network to explore whether EGF was regulated by these differentially expressed lncRNAs in dural penetration chordoma. EGF plays an important role in chordoma. As shown in Our previous study showed that clival chordoma patients with dural penetration prone to have worse outcomes . The aggWe identified a set of 2760 lncRNAs and 3988 mRNAs with differential expression between no dural penetration and serious dural penetration samples. This indicates that RNA homeostasis might be essential for the invasion of chordoma, and their disruption might lead to dural penetration. We found that 1773 out of the 2760 (64.2%) lncRNAs were down regulated, suggesting that decreased lncRNAs play a more important role than increased lncRNAs in dural penetration.LncRNA-mRNA expression combined with KEGG pathway analysis showed that 55 altered lncRNAs related to migration and invasion, cell apoptosis and proliferation and participated in 5 down-regulated pathways. The pathways proteoglycans in cancer, focal adhesion, and ECM-receptor interaction are major migratory and invasive pathways. The pathway in cancer modulates important biological processes, including evading apoptosis and proliferation, and the PI3K-AKT signaling plays a crucial role in the cell cycle regulation. All of these pathways showed decreased expression in chordoma without dural penetration. The result illustrated that chordoma with serious dural penetration has strong invasion, proliferation, and decreased apoptosis ability.We also observed differential expression of 24 lincRNAs, 7 enhancer-like lncRNAs, and 14 antisense RNAs modulating dural penetration in chordomas by altering various pathological processes, including cell adhesion, metastasis, invasion, and apoptosis. In addition to the above 5 signaling pathways, 45 of these lncRNAs also participate in the regulation of 4 other down-regulated signaling pathways, including Wnt signaling, Hedgehog signaling, hippo signaling, P53 signaling, and 3 up-regulated pathways, including cell adhesion molecules (CAMs), apoptosis, and MAPK signaling.Previous reports of receptor tyrosine kinase (RTK) expression in chordomas suggest that these tumors may respond to kinase inhibitor therapy . ChordomAdditionally, mRNA for EGF and its regulatory lncRNAs levels of ABCC6P1 and AL078621.4 in chordoma tissues with serious dural penetration or without dural penetration were detected by RT-qPCR. Therefore, together with the data presented herein, these findings strongly suggest that EGF was activated during the process of dural penetration in chordoma. Further studies are warranted to determine the role of EGF and these lncRNA in dural penetration of chordoma.The main limitation of our present study is the small sample size due to the extreme low incidence of clival chordomas. Although we used rigorous inclusion criteria in this exploratory study, further studies with more chordoma samples are still needed to validate this work.In conclusion, our study found a multitude of differential expression of lncRNAs and mRNAs between chordomas with serious dural penetration and those with no dural penetration, and explored the potential roles of these lncRNAs associated with dural penetration of chordoma. Our data suggested that these dysregulated lncRNAs could serve as useful therapeutic targets and prognostic biomarkers of chordoma with dural penetration.The chordoma samples included in this study were collected from 20 patients with clival chordomas who underwent endoscopic endonasal transsphenoidal surgery at Beijing Tiantan Hospital, among which 10 cases had serious dural penetration and 10 cases did not have dural penetration. Six samples with serious dural penetration and 6 samples with no dural penetration were used in microarray detection. All of the samples were included in the qRT-PCR validation. No patients received chemotherapy or radiotherapy prior to the surgery. Tumor tissues were collected immediately after resection, snap frozen in liquid nitrogen and stored at -80°C. Histological diagnoses were made on formalin-fixed, paraffin-embedded hematoxylin and eosin sections by reviewers blinded to the original diagnosis. The use of the patient samples was approved by the Ethics Committee of Beijing Tiantan Hospital. Written informed consent for the study was obtained from all subjects.Total RNA was isolated with Trizol reagents  and RNeasy Mini Kit . The purity and quantity of RNA were measured using Nanodrop 2000c . Each sample was amplified and transcribed into fluorescent cRNA (complementary RNA) . The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). One μg of labeled cRNAs were fragmented and hybridized with the slide of Human LncRNA V3.0 Array (Arraystar). The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized array was washed, fixed and scanned using the Agilent DNA Microarray Scanner.Based on the NetAffx annotation of the probe sets, the Refseq_NR, Ensemble, Gencode, RNAdb, UCSC known gene and lincRNA Cabili of lncRNAs and the RefSeq, Ensemble, and GenBank annotation of mRNAs, a total of 58,944 distinct probes  were detected by the Arraystar Human LncRNA Microarray V3.0.P < 0.05 were considered the criteria for significantly differential expression.The acquired array images were analyzed by Agilent Feature Extraction software . Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package (Agilent Technologies). After normalization of the data, lncRNAs and mRNAs that were flagged as Present or Marginal  in at least six out of 12 samples were chosen for further data analysis. Differentially expressed lncRNAs and mRNAs between the two groups were identified through fold-change filtering and two-tailed Student’s t-test. Multiple testing correction was performed by calculating the Benjamini-Hochberg false discovery rate (FDR). Fold-change (FC) ≥ 2 and t-tests were used for age comparisons and follow-up time comparisons between the two groups. Overall survival and progression-free survival were estimated using Kaplan-Meier survival analysis and a 2-sided log-rank test was used to compare between groups.Clinical information was analyzed using SPSS . The Pearson Chi-Square test was used for sex comparisons, and two-sided Student’s http://www.genome.jp/kegg/). Differentially expressed genes were mapped to defined KEGG pathways. Enrichment scoring, which is equal to -log10 , was used to measure pathway significance and specificity. Specifically, a two-sided Fisher’s exact test was used to classify the pathway and we computed P values for the pathway of each of the differential genes, P < 0.05 was considered statistically significant.Pathway analysis was used to identify the significant pathways of differentially expressed genes according to Kyoto Encyclopedia of Genes and Genomes (KEGG)  were selected to construct the network.−ΔΔCT method.We used qPCR to confirm the expression of lncRNAs and mRNAs in chordoma tissues. First-strand cDNA was synthesized from total RNA (1μg) using the SuperScript III First-Strand Synthesis System . qRT-PCR was performed on an ABI 7500 Fast system  using the Power SYBR Green PCR Master Mix (Applied Biosystems). The amplification conditions were 95°C for 10 minutes, then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. All of the primers used are listed in Supplementary Figure 1Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Tables 9, 10


Carcinogenesis is linked with massive changes in regulation of gene networks. We used high throughput mutation and gene expression data to interrogate involvement of 278 signaling, 72 metabolic, 48 DNA repair and 47 cytoskeleton molecular pathways in cancer. Totally, we analyzed 4910 primary tumor samples with individual cancer RNA sequencing and whole exome sequencing profiles including ~1.3 million DNA mutations and representing thirteen cancer types. Gene expression in cancers was compared with the corresponding 655 normal tissue profiles. For the first time, we calculated mutation enrichment values and activation levels for these pathways. We found that pathway activation profiles were largely congruent among the different cancer types. However, we observed no correlation between mutation enrichment and expression changes both at the gene and at the pathway levels. Overall, positive median cancer-specific activation levels were seen in the DNA repair, versus similar slightly negative values in the other types of pathways. The DNA repair pathways also demonstrated the highest values of mutation enrichment. However, the signaling and cytoskeleton pathways had the biggest proportions of representatives among the outstandingly frequently mutated genes thus suggesting their initiator roles in carcinogenesis and the auxiliary/supporting roles for the other groups of molecular pathways. Cancer has complex pathogenesis  and moleper se is not enough for understanding mechanisms of carcinogenesis because each gene product only serves as a component of complex biological processes determining cell fate. One of the key systemic genetic approaches in cancer is data analysis at the level of molecular pathways [However, the analysis of individual genes pathways ,9,10,11.pathways ,13,14.signaling, metabolic, cytoskeleton and DNA repair processes .Several databases of molecular pathways were published, frequently specifically devoted to the pathways with specific functions, such as MetaCyc and SynSysNet ,16. On tsignaling pathways transmit different types of signals into the cell, e.g., signals from external cell surface receptors. They control all major biological processes in the cell such as proliferation, cell growth, migration, differentiation and death. Impaired molecular signaling can lead to acquisition of cancer phenotype by the cell and further disease progression [The gression ,26,27,28Metabolic pathways are responsible for the whole repertoire of biochemical reactions in organism. In the living cell they are dynamically controlled by the external signals, concentrations of biomolecules and differentiation programs [programs . In cancprograms ,31. For programs . On the programs .cytoskeleton and DNA repair pathways may be considered as the specific sort of signaling pathways because many of them if not all are directly controlled by external or internal stimuli [cytoskeleton pathways can lead to defects in mitosis and cellular morphogenesis, altered intercellular contacts and cell motility. This can promote cancer progression, invasion and metastasis [In turn, the  stimuli ,35. Imbatastasis . DNA repair pathways are most likely the key mechanisms in the emergence and development of malignant tumors. Mutations in genes responsible for genome integrity frequently point on individuals with a predisposition to cancer, but also may serve as biomarkers of response on specific anticancer therapies [Finally, mutations in herapies . These mherapies ,39. Moreherapies ,41.In this study we for the first time scrutinized mutation frequencies and expression profiles of genes included in 419 molecular pathways belonging to the above four groups with different biological functions. Totally, we analyzed 4910 matched individual cancer gene expression and whole exome sequencing profiles collectively covering 1,252,669 mutations from the TCGA project database  in thirtFor all the biosamples, we present a database of mutation and expression data at the individual gene and at the molecular pathway levels. At the gene level, normalized mutation rate and case-to-normal ratio data are given. Conversely, at the pathway level we present algorithmically calculated pathway mutation instability rates  and pathWe extracted molecular pathway gene contents and architecture from the publicly available databases ,45,46,47We then measured mutation rates of genes belonging to these functional groups of pathways . To thisThe tumor samples analyzed represented thirteen tumor types and were originally profiled within the The Cancer Genome Atlas (TCGA) project  while thsignaling and cytoskeleton pathways had highest representations in the first decile which was decreasing further to the minimal values in the last decile, thus evidencing high proportion of especially frequently mutated genes in these groups , each representing 10% of genes. In each decile, we measured proportions of genes belonging to the four functional groups of pathways under investigation . We obsee groups . In cont deciles .We then re-analyzed these representations and confirmed the above two trends separately for the thirteen cancer types . These tTo characterize in brief gene compositions of the molecular pathways, we intersected them for the different groups of pathways . JaccardSignaling pathways included 2295 genes, of them 1513 were specific for this group and the rest were the interface genes. Metabolic pathways had totally 635 genes and 574 specific genes; cytoskeleton pathways—824 and 307 genes, respectively; DNA repair pathways—385 and 98 genes, respectively. The highest intersection level was seen for the signaling and cytoskeleton pathway genes  and for the signaling and DNA repair genes .In total, all the 419 pathways contained 3281 unique genes, of them 809 were the interface genes, i.e., simultaneously members of two or more groups of pathways. signaling/cytoskeleton vs metabolic/DNA repair groups of pathways were also clearly detectable for the pathway type-specific genes  values were calculated to characterize molecular pathway activation using differential gene expression data [PAL scores can take positive and negative values and are congruent with the expected differential activation of a pathway [We then used tumor and normal RNA sequencing profiles to analyze differential gene expression and to assess activation levels of molecular pathways. For every comparison, the tumor profiles were normalized on the corresponding normal tissues, e.g., breast cancer profiles were normalized on the normal breast samples, etc. ion data ,50,51, S pathway .Pathway instability (PI) metric that characterizes average mutation load per gene in a pathway [PI score can take only positive values and is congruent with the pathway mutation burden.The mutation enrichment of the same molecular pathways in 4910 tumors was assessed using  pathway , Supplemsignaling pathways, 30.4% of cytoskeleton pathways, 29.2% of DNA repair pathways, and 9.6% of metabolic pathways had mutations in their genes and, therefore, had PI values greater than zero. Interestingly, the distribution of PI scores demonstrated that overall the DNA repair pathways had the highest relative mutation rates per gene, the signaling pathways had intermediate scores and the metabolic and cytoskeleton pathways had the lowest relative mutation levels , most of the pathway types were mostly downregulated in cancer, except for the egulated B. The gepathways B. OveralWe then investigated in more detail up- and downregulated pathways in each group. Because the same pathway can be differently regulated in different samples, we tried to focus on cancer type-specific changes. To this end, for every pathway in every cancer type, we calculated percentage of samples in which the pathway was upregulated, as reflected by PAL > 0 . Most ofcytoskeleton, metabolic, signaling, DNA repair pathways.We then performed Gene Ontology analyses of up/downregulated pathways. To this end, we took fractions of top pathways with PAL > 0 in more than 80% of cancer samples under investigation, and of bottom pathways with PAL < 0 in more than 80% of the samples. In this way we selected totally 8 up/14 downregulated signaling pathways, 4/2 metabolic pathways, 15/0 DNA repair pathways, and 0/2 cytoskeleton pathways, accordingly. As before, the genes from these pathways were subjected for gene ontology analysis using GOrilla software  the resucytoskeleton pathways, whereas the others dealt with  and the respective activation of the specific molecular processes (PAL scores). For example, the processes of transcription by RNA polymerase II, chromosomal organization, processing and presentation of antigens via MHC class II and activation of immune cells were featured for the genes from For all types of the pathways, we found no correlation between mutation burden and expression changes at the level of individual genes, i.e., case-to-normal ratio (CNR) and normalized mutation rate (nMR) values . We alsoWe then assessed correlations between all tumor samples for mutation burden at gene (nMR) and pathway (PI) levels, and also for expression changes at gene (CNR) and pathway (PAL) levels . To thisTaken together, these data suggest that for both mutation and gene expression data, the intertumoral similarities are much higher at the pathway level . On the Juxtaposition of clinical and molecular tumor phenotypes provides a basis for further improvement of cancer treatments. High-throughput cancer gene expression and mutation data can help identifying new oncogenes and driver mutations . In turnNowadays, many molecular pathways were reported and collected in specific databases. Despite the existence of universal methods to pathway annotation like PathwayCommons, these databases were mostly generated separately and utilize different approaches to pathways nomenclature, and the same or very similar pathways can be included in different databases under different names . FunctioHowever, most studies of cancer biology focus on only single pathways or small groups of pathways ,62. For signaling, cytoskeleton, metabolic and DNA repair pathways. In this study, we performed more thorough analysis in terms of number of molecular pathways investigated. We explored 419 molecular pathways using new instruments to algorithmically assess pathway activation levels  and pathWe then compared our results with the previous study “Oncogenic Signaling Pathways in The Cancer Genome Atlas” on genetic alterations scoring in ten signaling pathways using TCGA data . The patWe also compared our results with the trends previously revealed for BAS in different tumor subtypes. To this end, we calculated on the pathway level average BAS, PAL and PI scores per tumor subtype . At the signaling/cytoskeleton and metabolism/DNA repair groups of pathways at the level of mutation data. Most importantly, the signaling/cytoskeleton group members were outstandingly enriched by the most highly mutated genes and deficient by the genes with the low mutation levels. This suggests their initiator roles in carcinogenesis and the auxiliary/supporting roles for the other groups of molecular pathways. Totally, in our study we analyzed 4910 individual cancer gene expression and matching whole exome sequencing profiles collectively covering ~1.3 million mutations in thirteen cancer types. We found that for both mutation and gene expression data, the intertumoral similarities were much higher at the pathway level than at the level of individual genes. We identified common trends for the representatives of DNA repair group showed markedly upregulated activation levels in cancers, whereas for the other groups of pathways, the downregulated members prevailed. On the other hand, DNA repair pathways also demonstrated the most congruent activation patterns among all the tumor samples.However, at the level of gene expression, the metabolic molecular pathways in cancer. In many reports, a focus was made on the metabolic alterations in tumors, so that cancer is even called a metabolic disorder [Our results also confirmed largely downregulated activities of the disorder ,66,67,68disorder ,66,67,68cytoskeleton) with membrane organization, receptor-mediated endocytosis; (DNA repair) with regulation of DNA quality and associated DNA repair, and with rearrangement of immunoglobulin loci; (metabolic) with biosynthesis of nucleotides and DNA replication;  with negative regulation of glucocorticoid receptor pathway, regulation of hormone levels, circadian rhythms, regulation of H3 histone deacetylation and protein catabolism. More specifically, the least mutated pathways dealt (cytoskeleton) exocytosis, cell cycle progression, processing and presentation of antigens via MHC class II; (DNA repair) cellular response to stress, chromosome organization, hydrolysis of phosphodiester bonds; (metabolic) drug response and catabolism, arachidonic acid secretion, fatty acid derivative pathways, glycosylation, sulfur and benzene-containing compound pathways, production of mitochondrial RNA, and tRNA aminoacetylation for protein translation;  regulation through phosphatidyl inositol, cell surface receptor pathways, growth and proliferation, transport of calcium, transcription by RNA polymerase II, and regulation of immune cell activation. On the other hand, the most strongly cancer- downregulated pathways related to (cytoskeleton) movement of cell or subcellular components and locomotion, regulation of development and cell proliferation; (metabolic) cellular ketone body metabolism, neurotransmitter catabolic processes, sulfur pathways, drug response pathways;  transcription by RNA polymerase II, interleukin-7-mediated pathway.In turn, the most strongly mutated pathways were responsible for  rearrangement of immune receptor loci, protein localization on chromosomes and chromosomal organization; (metabolic) oligosaccharide biosynthesis, nucleotide biosynthesis and DNA replication;  cell migration and adhesion, regulation of cell death, calcium ion transmembrane transport, extracellular matrix organization, regulation of mRNA stability, processing and presentation of exogenous peptide antigen via MHC class I, and activation of immune cells.Among the most strongly cancer-upregulated pathways, there were no Finally, our study poses a challenge of analyzing the whole cancer interactome model where separate pathways would be connected into a single graph with known connectivity and functional relationships between its nodes. In the future, this type of analysis could be beneficial for personalized finding of causative cancer mutations and clinically actionable therapeutic molecular targets.DNA mutation data were extracted from the Catalogue Of Somatic Mutations In Cancer (COSMIC) project database . We usedGene expression profiles were obtained from GDC data portal . We downThe gene structures and molecular architectures of 419 intracellular pathways were extracted from the public databases Reactome , NCI PatTo increase statistical accuracy, we considered only 419 molecular pathways including ten or more genes. These pathways were classified into four functional groups by expert supervision: 278 signaling, 72 metabolic, 48 DNA repair and 47 cytoskeleton pathways . 26 pathPathway instability (PI) reflects mutation enrichment of a molecular pathway [PI calculation utilizes mutation frequencies of genes forming a pathway. To assess mutation frequencies of individual genes, we introduced normalized mutation rate (nMR) value expressed by the formula:nnMR is normalized mutation rate of gene n; N mut is total number of mutations for gene n in group of samples g; N samples (g) is number of samples in group g; Length CDS (n) is length of coding DNA sequence (CDS) of gene n in nucleotides. PI levels were calculated as follows:pPI is PI for pathway p; nnMR is as described above; n belongs to pathway p, or equals to zero if not; p. The calculations were made using Amazon and Microsoft Azure cloud services. pathway . PI calcPathway activation level (PAL) characterizes cumulative changes of expression levels of genes belonging to a certain molecular pathway [pPAL is PAL for pathway p, nCNR is case-to-normal ratio, the ratio of gene n expression level in a tumor sample under study to an average level for the control group; ARR (activator/repressor role) is Boolean flag that depends on function of gene n product in pathway p. ARR is −1 if gene product n inhibits pathway p; 1 if n activates apthway; 0 if n has ambiguous or unclear role in a pathway; 0.5 or −0.5, if n is rather activator of a pathway or its inhibitor, respectively. The calculations were made using Amazon and Microsoft Azure cloud services. pathway ,50,72,73 pathway :(3)PALp=target dataset was the gene set of interest and background was list of protein coding human genes from Human Genome Organisation (HUGO) Gene Nomenclature Committee (HGNC) database, version 20170717 [p-value threshold was set as 10−3. We performed analysis of biological processes involving gene sets under investigation using GOrilla tool for Gene Ontology terms analysis . We used20170717 , p-valueallowed similarity = 0.7, semantic similarity measure was SimRel), except for dispensability cut-off level (0.25). We excluded from visualization the non-informative general terms like “biological processes” and irrelevant terms like “kidney morphogenesis” by expert curation. Gorilla outputs were visualized using REVIGO viewer with the default settings (We used Genomic Alteration matrixes of gene and pathway levels from the study “Oncogenic Signaling Pathways in The Cancer Genome Atlas”  to compaThe study  analyzedUsing high throughput mutation and gene expression data, we established pathway activation and mutation enrichment metrics of 278 signaling, 72 metabolic, 48 DNA repair and 47 cytoskeleton molecular pathways in 4910 individual cancer samples RNA sequencing and whole exome sequencing profiles representing thirteen cancer types. We found no correlation between mutation enrichment and expression changes both at the individual gene and molecular pathway levels. We observed generally congruent pathway activation profiles for different cancer types. The highest cancer-specific activation levels were seen in the DNA repair pathways, which had also the highest mutation enrichment levels. Moreover, the DNA repair pathways also demonstrated the most congruent activation patterns among all the tumor samples. However, the signaling and cytoskeleton pathways had the highest proportions of outstandingly frequently mutated genes thus suggesting their initiator roles in carcinogenesis and the supporting roles for the other groups of molecular pathways.


Zingiber officinale), as a commonly used herbal medicine, has been suggested as a potential agent for colon cancer. This study was aimed to identify the bioactive components and potential mechanisms of ginger for colon cancer prevention by an integrated network pharmacology approach.Colon cancer is occurring at an increasing rate and ginger (The putative ingredients of ginger and its related targets were discerned from the TCMSP  and Swiss target prediction database. After that, the targets interacting with colon cancer were collected using Genecards, OMIM, and Drugbank databases. KEGG pathway and GO enrichment analysis were performed to explore the signaling pathways related to ginger for colon cancer treatments. The PPI and compound-target-disease networks were constructed using Cytoscape 3.8.1. Finally, Discovery studio software was employed to confirm the key genes and active components from ginger.Six potential active compounds, 285 interacting targets in addition to 1356 disease-related targets were collected, of which 118 intersection targets were obtained. A total of 34 key targets including PIK3CA, SRC, and TP53 were identified through PPI network analysis. These targets were mainly focused on the biological processes of phosphatidylinositol 3-kinase signaling, cellular response to oxidative stress, and cellular response to peptide hormone stimulus. The KEGG enrichment manifested that three signaling pathways were closely related to colon cancer prevention of ginger, cancer, endocrine resistance, and hepatitis B pathways. TP53, HSP90AA1, and JAK2 were viewed as the most important genes, which were validated by molecular docking simulation.This study demonstrated that ginger produced preventive effects against colon cancer by regulating multi-targets and multi-pathways with multi-components. And, the combined data provide novel insight for ginger compounds developed as new drug for anti-colon cancer. Currently, increasing therapeutic approaches for colon cancer treatment are available, not only the conventional treatments of surgical resection and radiotherapy but also new interventional therapies like drug therapy. However, high costs of molecular targeted therapy and chemotherapy-induced adverse effects such as nausea, vomiting, and digestive tract irritation reaction always leads to treatment drop out.With the rapid development of human society and the improvement of life standard, cancer as a growing threat, is the second leading noncommunicable disease of death globally next only to cardiovascular disease . ColorecZingiber officinale), belong to Zingiberaceae family, has long been used as a traditional Chinese medicine (TCM), which mainly distributed in India, China, and Nigeria [Historically medicinal botanicals, as one part of complementary medicine in the USA, have provided an important resource for discovering anticancer drug agents, and more than half of currently available drugs are related to them . Ginger  Nigeria . Ginger  Nigeria , 6. Furt Nigeria , 8. HoweWhile moving ahead with computer technology, network pharmacology incorporating a series of disciplines and techniques has emerged. Network pharmacology has been successfully introduced to reveal the therapeutic mechanisms of TCM, as it in accordance with the connotation of holistic strategy of multi-components, multi-targets and multi-pathways –11.In this study, network pharmacology was conducted to establish the components-targets-pathways-disease network to investigate the potential mechanisms of ginger in colon cancer prevention. The detailed flowchart of the study design was shown in Fig. http://tcmspw.com/tcmsp.php) database is a platform containing many compounds from herbal medicine, related protein targets as well as its pharmacokinetic properties [http://www.swisstargetprediction.ch/) databases [Traditional Chinese medicine systems pharmacology  [https://omim.org/) [https://www.drugbank.ca/). To reduce the false positives, only the curated targets that directly associated with the disease were included, and the repetitive targets were removed. Then, the protein targets related to disease and ginger compounds were both imported into Uniprot database (https://www.uniprot.org/) to acquire its related gene name, gene ID and functions, respectively. The obtained genes were both inputted to draw Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/), and the intersection genes were collected as candidate genes.Keywords such as “colon cancer” or “colon tumor” were used to search the known targets related to the pathogenesis of colon cancer from three major databases, GeneCards (rds.org) , OMIM (him.org/) , and Druhttps://string-db.org/) database, with a highest confidence of 0.9 [Based on the candidate genes, a PPI network was constructed by importing the candidate genes to the Search Tool for the Retrieval of Interacting Genes . GO functionally annotates key genes into three main terms, including cellular components (CCs), molecular functions (MFs), and biological processes (BPs). Besides, Cytoscape ClueGO plugin was employed to further analyze BPs enrichment. KEGG enrichment analysis unveils the possible biological process with key genes. In addition, the bubble chart of GO and KEGG enrichment analysis were performed on the bioinformatics platform (http://www.bioinformatics.com.cn/).The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed by the Database for Metascape  database. In brief, Discovery studio software  was employed, and the screened active components were prepared using “Prepare Ligands” module to obtain an effective three-dismensional conformation. After removing crystallographic water molecules, the “Prepare Protein” module was used to remove the polyconformation of target protein and to supplement the incomplete amino acid residues. Subsequently, molecular docking was performed in “LibDock” module, and LibDockScore was required to evaluate affinity of the target proteins and active components. The LibDockScore of the target protein and its corresponding prototype ligand was viewed as the threshold, and the components with higher scores were regarded as the active ones for interacting with this protein.After searching, filtering, and removal of the duplicates, 6 putative components  with OB ≥ 30% and DL ≥ 0.18 were selected from TCMSP database , with transmembrane receptor protein tyrosine kinase GO: 0007169, cellular response to nitrogen compound GO: 1901699, and peptidyl-serine phosphorylation GO: 0018105 as the top ones. The size of the dot in bubble chart indicates the number of target genes in the corresponding function pathway, and the enrichment expresses the ratio of the number of target genes belonging to all the annotated genes located in the pathway. Besides, further analysis of the network was performed using Cytoscape plugin ClueGO, and we found that these BPs terms were mainly associated with the phosphatidylinositol 3-kinase signaling, cellular response to oxidative stress, cellular response to peptide hormone stimulus, and peptidyl-serine phosphorylation , endocrine resistance (hsa01522), EGFR tyrosine kinase inhibitor resistance (hsa01521), and PI3K-Akt signaling pathway (hsa04151) as the top ones. Among these pathways, pathways in cancer was identified as a set of important key pathway with the most target enrichment and lowest p value.The KEGG enrichment showed how ginger acts on the pathway, thereby playing a therapeutic role in colon cancer. Here, based on the 34 core target genes, 20 significant signaling pathways  hydrolase expression and induction of G2/M arrest –21. It iBy target fishing, a total of 34 key genes were screened as the most important ones that contribute to the colon cancer prevention of ginger. These potential target genes were placed in the Metascape for KEGG pathway analysis, and 20 significant pathways that may be regulated by ginger in the treatment of colon cancer were identified, including pathways in cancer, endocrine resistance, hepatitis B, PI3K-Akt signaling pathway, and EGFR tyrosine kinase inhibitor resistance. Importantly, PI3K-Akt pathway is a critical signal cascade focusing on serine/threonine kinase Akt, and AKT1, PIK3CA, MAPK3, TP53 genes were involved in this pathway. It perhaps the most commonly activated signaling pathway in human cancer and misregulation of the PI3K-Akt pathway has been revealed to be closely associated with pathogenetic process of colon cancer , 24. By According to the compound-target-pathway-disease interaction network, each active component acts on at least one target genes and most of the target acts on at least one pathway. For example, TP53 as a tumor suppressor gene was found to be involved in 11 pathways, such as EGFR tyrosine kinase inhibitor resistance and PI3K-Akt signaling pathway. Another important gene of PIK3R1 was associated with all the significant pathways. Furthermore, the core targets TP53, HSP90AA1, and JAK2 were verified to be the potential targets of ginger for treating colon cancer. These findings revealed that the anti-cancer effect of ginger was played through regulating multi-targets by multi-components. However, the illustration in the molecular levels of ginger prevented colon cancer need to be provided in the future. Meanwhile, another limitation of this investigation is that it was focused on lipid substances of ginger, and large molecular compounds such as polysaccharides and proteins were not included. Further research is required to supplement this data.Collectively, 6 active components were identified from ginger and 285 targets in addition to 1356 disease-related targets in the treatment of colon cancer were collected. In the String analysis, a total of 34 candidate targets were yielded, and 10 signaling pathways including pathways in cancer, PI3K-Akt signaling pathway, and EGFR tyrosine kinase inhibitor resistance were observed to play an important role in the mechanism of ginger for colon cancer prevention. In addition, TP53, HSP90AA1, and JAK2 were speculated to be the most important target proteins. These results might be promising to searching for leading compounds and the developments of new drug for colon cancer, however, continued experiments are demanded to available the findings.