Expression analysis of transcription factors in sugarcane during cold stress / Análise de expressão de fatores de transcrição em cana-de-açúcar sob estresse por frio

Abstract Transcription factors (TF) are a wide class of genes in plants, and these can regulate the expression of other genes in response to various environmental stresses (biotic and abiotic). In the current study, transcription factor activity in sugarcane was examined during cold stress. Initially, RNA transcript reads of two sugarcane cultivars (ROC22 and GT08-1108) under cold stress were downloaded from SRA NCBI database. The reads were aligned into a reference genome and the differential expression analyses were performed with the R/Bioconductor edgeR package. Based on our analyses in the ROC22 cultivar, 963 TF genes were significantly upregulated under cold stress among a total of 5649 upregulated genes, while 293 TF genes were downregulated among a total of 3,289 downregulated genes. In the GT08-1108 cultivar, 974 TF genes were identified among 5,649 upregulated genes and 283 TF genes were found among 3,289 downregulated genes. Most transcription factors were annotated with GO categories related to protein binding, transcription factor binding, DNA-sequence-specific binding, transcription factor complex, transcription factor activity in RNA polymerase II, the activity of nucleic acid binding transcription factor, transcription corepressor activity, sequence-specific regulatory region, the activity of transcription factor of RNA polymerase II, transcription factor cofactor activity, transcription factor activity from plastid promoter, transcription factor activity from RNA polymerase I promoter, polymerase II and RNA polymerase III. The findings of above results will help to identify differentially expressed transcription factors during cold stress. It also provides a comprehensive analysis of the regulation of the transcription activity of many genes. Therefore, this study provides the molecular basis for improving cold tolerance in sugarcane and other economically important grasses.

Resumo Fatores de transcrição (FT) são uma ampla classe de genes em plantas e podem regular a expressão de outros genes em resposta a vários estresses ambientais (estresses bióticos e abióticos). No presente estudo, a atividade do fator de transcrição na cana-de-açúcar foi examinada durante o estresse pelo frio. Inicialmente, as leituras de transcrição de RNA de duas cultivares de cana-de-açúcar (ROC22 e GT08-1108) sob estresse frio foram baixadas do banco de dados SRA NCBI. As leituras foram alinhadas em um genoma de referência e as análises de expressão diferencial foram realizadas com o pacote R / Bioconductor edgeR. Com base em nossas análises no cultivar ROC22, 963 genes TF foram significativamente regulados positivamente sob estresse pelo frio entre um total de 5.649 genes regulados positivamente, enquanto 293 genes TF foram regulados negativamente entre um total de 3.289 genes regulados negativamente. No cultivar GT08-1108, 974 genes TF foram identificados entre 5.649 genes regulados positivamente e 283 genes TF foram encontrados entre 3.289 genes regulados negativamente. Os fatores de transcrição, em sua maioria, foram anotados com categorias GO relacionadas à ligação de proteína, ligação de fator de transcrição, ligação específica de sequência de DNA, complexo de fator de transcrição, atividade de fator de transcrição em RNA polimerase II, atividade de fator de transcrição de ligação de ácido nucleico, atividade de corepressor de transcrição, sequência específica da região reguladora, atividade do fator de transcrição da RNA polimerase II, atividade do cofator do fator de transcrição, atividade do fator de transcrição do promotor do plastídio, atividade do fator de transcrição do promotor da RNA polimerase I, polimerase II e RNA polimerase III. As descobertas dos resultados acima ajudarão a identificar fatores de transcrição expressos diferencialmente durante o estresse pelo frio. Ele também fornece uma análise abrangente da regulação da atividade de transcrição de muitos genes. Portanto, este estudo fornece base molecular para melhorar a tolerância ao frio em cana-de-açúcar e outras gramíneas economicamente importantes.

Análise do perfil de mRNAs diferencialmente traduzidos no carcinoma renal de células claras / Analysis of differentially translated mRNA profile in clear cell renal cell carcinoma

O carcinoma renal de células claras (CRCC) é o tipo de neoplasia renal com maior incidência, cerca de 80%. A maioria dos casos são curados após cirurgia, porém, cerca de um terço dos pacientes apresentam recidiva da doença com metástase à distância. O tratamento para este tumor evoluiu muito nas últimas duas décadas, entretanto, pacientes metastáticos ainda apresentam baixas taxas de resposta aos tratamentos devido a resistência adquirida pelo tumor para escapar da terapia alvo. Identificar os mecanismos moleculares associados à carcinogênese do CRCC é essencial para entender as características tumorais que estão associadas a progressão da doença e resistência aos tratamentos. Entre as alterações mais frequentes no CRCC está a perda do gene VHL, um supressor tumoral e principal regulador da resposta à hipóxia. VHL tem dois principais alvos, o fator induzido por hipóxia 1α (HIF-1α) e o fator induzido por hipóxia α (HIF-2α). Em normóxia, VHL é responsável pela degradação das subunidades de HIF. Em hipóxia, VHL deixa de reconhecer e marcar HIF-1α e HIF-2α para degradação e, uma vez estabilizadas, ativam vias de sinalização associadas a sobrevivência celular. As informações sobre alterações encontradas em tumores normalmente são estudadas a partir do sequenciamento da população total de mRNAs, oferecendo uma visão do transcriptoma. Nossa abordagem metodológica coleta e analisa apenas a população de mRNAs ativamente traduzidos, oferecendo uma visão mais próxima da expressão proteica final. A via de mTOR regula o início da tradução de mRNAs e está frequentemente mutada em CRCC. A hipóxia afeta a expressão de genes tanto via transcrição quanto via tradução. Alterações no controle traducional em CRCC afetam a expressão gênica contribuindo para a formação do tumor e progressão da doença. Assim, nosso objetivo principal foi identificar o perfil de genes diferencialmente traduzidos dependendo do status de VHL e da via de mTOR. Para isso utilizamos um modelo celular de CRCC deficiente em VHL e sua contraparte onde VHL foi restituído. Realizamos o perfil polissomal em modelos celulares de CRCC para separar e coletar a população de mRNAs ativamente traduzidos que foram posteriormente sequenciados. Nossos dados mostraram perfis distintos de tradução entre as células VHL- deficientes e VHL-proficientes. Além disso, após a inibição de mTOR, ambas as células também apresentaram respostas diferentes ao tratamento. Além disso, observamos alterações na resposta imune e aumento do ciclo celular na ausência de VHL, que podem contribuir para a progressão tumoral. Em modelo com tecido tumoral congelado, nossos resultados parciais indicam que alterações na tradução global podem interferir principalmente no estadiamento clínico de pacientes com CRCC. Por fim, também analisamos a expressão de HIF-2α, um dos alvos de VHL, em tecidos de pacientes com CRCC. Nossos resultados mostram que HIF-2α pode ser utilizado na estratificação de pacientes com maior risco de recidiva, dependendo do estadiamento clínico.

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal neoplasia with 80% of incidence. Most cases are cured after surgery, however, one third of all patients will have disease recurrence with distant metastasis. ccRCC treatment had evolved in the past two decades, however, metastatic patients still have low response rates due to tumor resistance. The identification of molecular mechanisms associated with ccRCC carcinogenesis is essential to understand the characteristics associated with disease progression and treatment resistance. The most frequent alteration in ccRCC is the loss of VHL gene, a tumor suppressor and the main regulator in response to hypoxia. VHL has two main target, hypoxia-induced factor 1 α (HIF-1 α) and hypoxia-induced factor α (HIF-2 α). In normoxic conditions, VHL can lead HIF subunits to degradation. In hypoxia, HIF-1α and HIF-2α stabilize and activate cell survival associated signaling pathways. Studies about tumor alterations usually provides a view of the transcriptome. Our approach is based on the actively translated mRNAs collection and analysis, which provides a closer view from protein expression. mTOR pathway regulates translation initiation and is frequently mutated in ccRCC. Hypoxia affects gene expression in both transcriptional and translational regulation. Alteration in translational control in ccRCC affect gene expression which contributes to tumor progression. Our main objective was to identify the differentially translated gene profile depending on VHL status and mTOR pathway activation. To assess this, we used a VHL-deficient and a VHL-proficient ccRCC cell line. We used the polysome profiling technique to separate and collect the population of mRNAs actively translated that were subsequently sequenced. Our data showed distinct translation profiles between VHL-deficient and VHL-proficient cells. In addition, after mTOR inhibition, both cells showed different responses to treatment. We observed changes in immune response and increased cell cycle pathways in VHL deficient cells, which may contribute to tumor progression. In tumor tissue, our polysome profiling analysis indicate that changes in global translation may interfere in clinical staging of ccRCC patients. Finally, we analyzed the expression of HIF-2α, a VHL target, in ccRCC patient's tissues. Our results showed that HIF-2α can distinct patients at higher recurrence risk depending on clinical staging.

Whole-transcriptome sequencing analysis of placental differential miRNA expression profile in Down syndrome / 南方医科大学学报

OBJECTIVE@#To identify new biomarkers and molecular pathogenesis of Down syndrome (DS) by analyzing differentially expressed miRNAs in the placentas and their biological pathways.@*METHODS@#Whole transcriptome sequencing was used to identify the differentially expressed miRNAs in DS (n=3) and normal placental samples (n=3) diagnosed by prenatal diagnosis. The target genes were predicted using miRWalk, Targetscan and miRDB, and GO and KEGG pathway analyses were performed for gene enrichment studies.@*RESULTS@#We identified a total of 82 differentially expressed miRNAs in the placental tissues of DS, including 29 up-regulated miRNAs (fold change ≥2, P < 0.05) and 15 down-regulated miRNAs (fold change ≥2, P < 0.05), among which 10 miRNAs with relatively high expression abundance were selected for further analysis, including 4 up-regulated and 6 down-regulated miRNAs. These selected miRNAs shared the common target genes BTBD3 and AUTS2, both of which were associated with neurodevelopment. GO analysis showed that the target genes of the selected miRNAs were mainly enriched in protein binding, hydrolytic enzymes, metal ion binding protein combining, transferase activity, nucleotide, cytoplasmic constituents, nucleus composition, transcriptional regulation, RNA metabolism regulation, DNA-dependent RNA polymerase Ⅱ promoter transcriptional regulation, eye development, and sensory organ development. KEGG enrichment analysis showed that the target genes of these differentially expressed miRNAs were involved in the signaling pathways including tumor-related signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, Rap1 signaling pathway, cytoskeletal regulatory signaling pathway, purine metabolization-related signaling pathway and P53 signaling pathway.@*CONCLUSION@#The differentially expressed miRNAs may play important roles in placental damage and pregnancy pathology in DS and provide clues for the prevention and treatment of mental retardation-related diseases.

Bioinformatics screening and analysis of key differentially expressed genes characteristics in nonalcoholic fatty liver disease / 中华肝脏病杂志

Objective: To screen and analyze the key differentially expressed genes characteristics in nonalcoholic fatty liver disease (NAFLD) with bioinformatics method. Methods: NAFLD-related expression matrix GSE89632 was downloaded from the GEO database. Limma package was used to screen differentially expressed genes (DEGs) in healthy, steatosis (SS), and nonalcoholic steatohepatitis (NASH) samples. WGCNA was used to analyze the output gene module. The intersection of module genes and differential genes was used to determine the differential genes characteristic, and then GO function and KEGG signaling pathway enrichment analysis were performed. The protein-protein interaction network (PPI) was constructed using the online website STRING and Cytoscape software, and the key (Hub) genes were screened. Finally, R software was used to analyze the receiver operating characteristic curve (ROC) of the Hub gene. Results: 92 differentially expressed genes characteristic were obtained through screening, which were mainly enriched in inflammatory response-related functions of "lipopolysaccharide response and molecular response of bacterial origin", as well as cancer signaling pathways of "proteoglycan in cancer" and "T-cell leukemia virus infection-related". 10 hub genes (FOS, CXCL8, SERPINE1, CYR61, THBS1, FOSL1, CCL2, MYC, SOCS3 and ATF3) had good diagnostic value. Conclusion: The differentially expressed hub genes among the 10 NAFLD disease-related characteristics obtained with bioinformatics analysis may become a diagnostic and prognostic marker and potential therapeutic target for NAFLD. However, further basic and clinical studies are needed to validate.

Application of single-cell transcriptome sequencing in mechanistic toxicology / 中华预防医学杂志

Traditional bulk RNA sequencing assesses the average expression level of genes in tissues rather than the differences in cellular responses. Accordingly, it is hard to differentiate sensitive responding cells, leading to inaccurate identification of toxicity pathways. Single-cell RNA sequencing (scRNA-seq) isolated single cells from tissue and subjected them to cell subtypes-specific transcriptome analysis. This technique in toxicological studies realizes the heterogeneous cellular responses in the tissue microenvironment upon chemical exposure. Thus it helps to identify sensitive responding cells and key molecular events, providing a powerful tool and a new perspective for exploring the mechanisms of toxicity and the modes of action. This review summarizes the development, principle, method, application and limitations of scRNA-seq in mechanistic toxicological researches, and discusses the prospect of multi-directional applications.

Bioinformatics analysis of differently expressed genes in osteoblastic sarcoma and screening of key genes / 中华肿瘤杂志

Objective: To screen the different expressed genes between osteosarcoma and normal osteoblasts, and find the key genes for the occurrence and development of osteosarcoma. Methods: The gene expression dataset GSE33382 of normal osteoblasts and osteosarcoma was obtained from Gene Expression Omnibus (GEO) database. The different expressed genes between normal osteoblasts and osteosarcoma were screened by limma package of R language, and the different expressed genes were analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The protein interaction network was constructed by the String database, and the network modules in the interaction network were screened by the molecular complex detection (MCODE) plug-in of Cytoscape software. The different expressed genes contained in the first three main modules screened by MCODE were analyzed by gene ontology (GO) using the BiNGO module of Cytoscape software. The MCC algorithm was used to screen the top 10 key genes in the protein interaction network. The gene expression and survival dataset GSE39055 of osteosarcoma was obtained from GEO database, and the survival analysis was performed by Kaplan-Meier method. The data of 48 patients with osteosarcoma treated in the First Affiliated Hospital of Fujian Medical University from January 2005 to December 2015 were selected for verification. The expression of STC2 protein in osteosarcoma was detected by immunohistochemical method, and the survival analysis was carried out combined with the clinical data of the patients. Results: A total of 874 different expressed genes were identified from GSE33382 dataset, including 402 down-regulated genes and 472 up-regulated genes. KEGG enrichment analysis showed that different expressed genes were mainly related to p53 signal pathway, glutathione metabolism, extracellular matrix receptor interaction, cell adhesion molecules, folate tolerance, and cell senescence. The top 10 key genes in the interaction network were GAS6, IL6, RCN1, MXRA8, STC2, EVA1A, PNPLA2, CYR61, SPARCL1 and FSTL3. STC2 was related to the survival rate of patients with osteosarcoma (P<0.05). The results showed that the expression of STC2 protein was related to tumor size and Enneking stage in 48 cases of osteosarcoma. The median survival time of 25 cases with STC2 high expression was 21.4 months, and that of 23 cases with STC2 low expression was 65.4 months. The survival rate of patients with high expression of STC2 was lower than that of patients with low expression of STC2 (P<0.05). Conclusions: Bioinformatics analysis can effectively screen the different expressed genes between osteosarcoma and normal osteoblasts. STC2 is one of the important predictors for the prognosis of osteosarcoma.

Investigation on the differentially expressed circular RNAs in myocardium of mice with diabetic cardiomyopathy / 中华心血管病杂志

Objective: To identify the differentially expressed circular RNA (circRNA) in the myocardium of diabetic cardiomyopathy (DCM) mice, and analyze their possible biological functions and related regulatory network. Methods: C57BL/6 mice, aged 8 weeks, and weighing were 21-27 g. Eight mice were selected as the control group and 15 mice were selected as the experimental group. The diabetic mice model was established by intraperitoneal injection of streptozotocin in the experimental group. One week after injection, the fasting blood glucose level of mice was measured, and 12 diabetic mice were included in the final experimental group. All mice were fed for 12 weeks under the same laboratory conditions. The cardiac structure and function were detected by echocardiography. Diabetic mice with the left ventricular ejection fraction less than 60% and the E/A less than 1.6 were selected as DCM group (n=3). Mice in DCM group and control group were then sacrificed under deep anesthesia. RNA was extracted from myocardial tissue. High-throughput RNA sequencing technology was used to sequence and identify the RNA in the myocardial tissue of DCM group and normal control group, and the difference was analyzed by DeSeq2. The analysis results were verified at the tissue level by RT-qPCR, and the differential circRNA were analyzed by GO and KEGG pathway analysis. The differentially expressed circRNA-microRNA(miRNA) interaction was predicted by the miRNA target gene prediction software. Results: A total of 63 differentially expressed circRNAs were found in the myocardium of DCM mice. The results of RT-qPCR showed that the tissue level expression of 8 differentially expressed circRNAs was consistent with the sequencing results, of which 7 were up-regulated and 1 was down-regulated. KEGG pathway analysis showed that the up-regulated circRNAs was mainly related to AMPK signal pathway and intercellular adhesion junction pathway, and the down-regulated circRNA was mainly related to cardiomyopathy. Go analysis showed that the up-regulated circRNA was mainly related to the binding process of ions, proteins, kinases and other factors in terms of molecular function, and was involved in regulating the intracellular structure, especially the composition of organelles in terms of cell components. The functional analysis of molecular function and cell components showed that the up-regulated circRNA were related to the cell component origin, recruitment and tissue, and thus participated in the regulation of cell biological process. The down regulated circRNA was related to catalytic activity in terms of molecular function, protein kinase binding process, transferase and calmodulin activity, and was closely related to the components of contractile fibers and the composition of myofibrils. These differentially expressed circRNAs were also related to biological processes such as lysine peptide modification, sarcomere composition, myofibril assembly, morphological development of myocardial tissue, myocardial hypertrophy and so on. Conclusions: In this study, we detected the novel differentially expressed circRNAs in the myocardium of DCM mice, and bioinformatics analysis confirmed that these circRNAs are related to oxidative stress, fibrosis and death of cardiomyocytes, and finally participate in the pathophysiological process of DCM.

Screening and identification of key genes ATP1B3 and ENAH in the progression of hepatocellular carcinoma: based on data mining and clinical validation / 南方医科大学学报

OBJECTIVE@#To explore the marker genes correlated with the prognosis, progression and clinical diagnosis of hepatocellular carcinoma (HCC) based on bioinformatics methods.@*METHODS@#The TCGA-LIHC, GSE84432, GSE143233 and GSE63898 datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were analyzed. The differentially expressed genes (DEGs) shared by different disease types were obtained using GEO2R and edge R packages, and Gene Ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) enrichment analyses of the DEGs were performed. The expression levels of these DEGs in normal and cancerous tissues were verified in TCGA-LIHC to identify the upregulated genes in HCC. Survival analysis, receiver-operating characteristic (ROC) curve analysis, and correlation analysis between the key genes and the clinical features of the patients were carried out using the R language. The differential expressions of 15 key genes were verified in clinical samples of HCC and adjacent tissues using RT-qPCR.@*RESULTS@#A total of 118 common DEGs were obtained in the database, and among them two genes, namely ATPase Na +/K + transport subunit beta 3 (ATP1B3) and actin regulator (ENAH), showed increased expressions with disease progression. Survival analysis combined with the TCGA-LIHC dataset suggested that high expressions of ATP1B3 and ENAH were both significantly correlated with a poor prognosis of HCC patients (P < 0.05), and their AUC values were 0.821 and 0.933, respectively. A high expression of ATP1B3 was correlated with T stage, pathological stage and pathological grade of the tumors (P < 0.05), while that of ENAH was associated only with an advanced tumor grade (P < 0.05). The results of RT-qPCR showed that ATP1B3 and ENAH were both significantly upregulated in clinical HCC tissues (P < 0.05).@*CONCLUSION@#ATPIB3 and ENAH are both upregulated in HCC, and their high expressions may serve as biomarkers of progression of liver diseases and a poor prognosis of HCC.

Study on mechanisms of Th17/Treg imbalance in patients with cystic echinococcosis based on miRNA expression profiles / 中国血吸虫病防治杂志

OBJECTIVE@#To investigate the serum microRNA (miRNA) expression and examine the impact of miRNA expression profiles on T helper type 17 (Th17)/regulatory T cells (Treg) imbalance among patients with cystic echinococcosis, so as to provide insights into the illustration of the mechanisms underlying chronic Echinococcus granulosus infections, and long-term pathogenesis.@*METHODS@#Total RNA was extracted from the sera of cystic echinococcosis patients and healthy controls, and subjected to high-throughput sequencing with the Illumina sequencing platform. Known miRNAs were annotated and new miRNAs were predicted using the miRBase database and the miRDeep2 tool, and differentially expressed miRNAs were identified. The target genes of differentially expressed miRNAs were predicted using the software miRanda and TargetScan, and the intersection was selected for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Among the differentially expressed miRNAs with the 20 highest fold changes, miRNAs that targeted genes relating to key transcription factors RORC and FOXP3 that determine the production of Th17 and Treg cells or their important regulatory pathways (PI3K-Akt and mTOR pathways) were matched.@*RESULTS@#A total of 53 differentially expressed miRNAs were screened in sera of cystic echinococcosis patients and healthy controls, including 47 up-regulated miRNAs and 6 down-regulated miRNAs. GO enrichment analysis showed that these differentially expressed miRNA were involved DNA transcription and translation, cell components, cell morphology, neurodevelopment and metabolic decomposition, and KEGG pathway analysis showed that the differentially expressed miRNA were mainly involved in MAPK, PI3K-Akt and mTOR signaling pathways. Among the differentially expressed miRNAs with the 20 highest fold changes, there were 3 miRNAs that had a potential for target regulation of RORC, and 15 miRNAs that had a potential to target the PI3K-Akt and mTOR signaling pathways.@*CONCLUSIONS@#Significant changes are found in serum miRNA expression profiles among patients with E. granulosus infections, and differentially expressed miRNAs may lead to Th17/Treg imbalance through targeting the key transcription factors of Th17/Treg or PI3K-Akt and mTOR pathways, which facilitates the long-term parasitism of E. granulosus in hosts and causes a chronic disease.

Transcriptome Analysis of Schwann Cells at Various Stages of Myelination Implicates Chromatin Regulator Sin3A in Control of Myelination Identity / 神经科学通报·英文版

Enhancing remyelination after injury is of utmost importance for optimizing the recovery of nerve function. While the formation of myelin by Schwann cells (SCs) is critical for the function of the peripheral nervous system, the temporal dynamics and regulatory mechanisms that control the progress of the SC lineage through myelination require further elucidation. Here, using in vitro co-culture models, gene expression profiling of laser capture-microdissected SCs at various stages of myelination, and multilevel bioinformatic analysis, we demonstrated that SCs exhibit three distinct transcriptional characteristics during myelination: the immature, promyelinating, and myelinating states. We showed that suppressor interacting 3a (Sin3A) and 16 other transcription factors and chromatin regulators play important roles in the progress of myelination. Sin3A knockdown in the sciatic nerve or specifically in SCs reduced or delayed the myelination of regenerating axons in a rat crushed sciatic nerve model, while overexpression of Sin3A greatly promoted the remyelination of axons. Further, in vitro experiments revealed that Sin3A silencing inhibited SC migration and differentiation at the promyelination stage and promoted SC proliferation at the immature stage. In addition, SC differentiation and maturation may be regulated by the Sin3A/histone deacetylase2 (HDAC2) complex functionally cooperating with Sox10, as demonstrated by rescue assays. Together, these results complement the recent genome and proteome analyses of SCs during peripheral nerve myelin formation. The results also reveal a key role of Sin3A-dependent chromatin organization in promoting myelinogenic programs and SC differentiation to control peripheral myelination and repair. These findings may inform new treatments for enhancing remyelination and nerve regeneration.

The Expression of RTN1 in Lung Adenocarcinoma and Its Effect on Immune Microenvironment / 中国肺癌杂志

BACKGROUND@#Reticulosome family gene 1 (RTN1) is a reticulosome-encoding gene associated with the endoplasmic reticulum. RTN1 plays a key role in membrane trafficking or neuroendocrine secretion of neuroendocrine cells, while RTN1 serves as a potential diagnostic/therapeutic marker for neurological diseases and cancer. However, the expression of RTN1 and its effect on the immune microenvironment in patients with lung adenocarcinoma have not been reported. In this study, we aimed to investigate the expression of RTN1 in lung adenocarcinoma and its correlation with immune infiltration and survival in lung adenocarcinoma using public databases and bioinformatics network tools.@*METHODS@#Expression levels of RTN1 mRNA in tumor and normal tissues were analyzed using Tumor Immune Estimation Resource 2.0 (TIMER 2.0) and Gene Expression Profiling Interactive Analysis 2 (GEPIA 2). RTN1 protein expression was examined using the Human Protein Atlas. The clinical prognostic significance of RTN1 was analyzed using the GEPIA2 plotter database. To further confirm the potential function of RTN1, the data were analyzed using gene set enrichment analysis. In addition, We performed dimensionality-reduced clustering analysis at the single-cell sequencing level on two datasets from the Tumor Immune Single-cell Hub (TISCH) database to observe the cellular clustering of RTN1 in different types of immune cells. Using the TIMER online tool to analyze and predict the infiltration abundance of different types of immune cells in the immune microenvironment of lung adenocarcinoma patients in the TCGA cohort; TIMER and CIBERSORT were used to study the relationship between genes co-expressed with RTN1 and its associated tumor-infiltrating immune cells; finally, TIMER was used to analyze the relationship between RTN1 and immune correlations between immune checkpoints.@*RESULTS@#We found that RTN1 expression was decreased in patients with lung adenocarcinoma and was closely related to patient prognosis. RTN1 is involved in the process of phagosome formation, hematopoietic cell formation and cell adhesion, and plays an important role in T cell activation. Using cBioPortal and TCGA data to analyze, it is found that RTN1 is significantly associated with BTK, CD4, ECSF1R, MNDA, NCKAP1L and SNX20. High expression of the above genes may cause significant upregulation of CD4+ T cells, mast cells, monocytes, myeloid dendritic cells and M1 macrophages. The expression of RTN1 is closely related to the common immune checkpoints CD274, CTLA4, HAVCR2, LAG3, PDCD1, PDCD1LG2, TIGIT and SIGLEC15 immune checkpoints.@*CONCLUSIONS@#RTN1 may act as a tumor suppressor gene and indicate better prognosis. Furthermore, RTN1 is associated with immune infiltration that may be involved in the immunotherapy response in LUAD. However, the related mechanism needs further research.

Bioinformatics Analysis of Core Genes and Key Pathways in Myelodysplastic Syndrome / 中国实验血液学杂志

OBJECTIVE@#To screen differentially expressed gene (DEG) related to myelodysplastic syndrome (MDS) based on Gene Expression Omnibus (GEO) database, and explore the core genes and pathogenesis of MDS by analyzing the biological functions and related signaling pathways of DEG.@*METHODS@#The expression profiles of GSE4619, GSE19429, GSE58831 including MDS patients and normal controls were downloaded from GEO database. The gene expression analysis tool (GEO2R) of GEO database was used to screen DEG according to | log FC (fold change) |≥1 and P<0.01. David online database was used to annotate gene ontology function (GO). Metascape online database was used to enrich and analyze differential genes in Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein-protein interaction network (PPI) was constructed by using STRING database. CytoHubba and Mcode plug-ins of Cytoscape were used to analyze the key gene clusters and hub genes. R language was used to diagnose hub genes and draw the ROC curve. GSEA enrichment analysis was performed on GSE19429 according to the expression of LEF1.@*RESULTS@#A total of 74 co-DEG were identified, including 14 up-regulated genes and 60 down regulated genes. GO enrichment analysis indicated that BP of down regulated genes was mainly enriched in the transcription and regulation of RNA polymerase II promoter, negative regulation of cell proliferation, and immune response. CC of down regulated genes was mainly enriched in the nucleus, transcription factor complexes, and adhesion spots. MF was mainly enriched in protein binding, DNA binding, and β-catenin binding. KEGG pathway was enriched in primary immunodeficiency, Hippo signaling pathway, cAMP signaling pathway, transcriptional mis-regulation in cancer and hematopoietic cell lineage. BP of up-regulated genes was mainly enriched in type I interferon signaling pathway and viral response. CC was mainly enriched in cytoplasm. MF was mainly enriched in RNA binding. Ten hub genes and three important gene clusters were screened by STRING database and Cytoscape software. The functions of the three key gene clusters were closely related to immune regulation. ROC analysis showed that the hub genes had a good diagnostic significance for MDS. GSEA analysis indicated that LEF1 may affect the normal function of hematopoietic stem cells by regulating inflammatory reaction, which further revealed the pathogenesis of MDS.@*CONCLUSION@#Bioinformatics can effectively screen the core genes and key signaling pathways of MDS, which provides a new strategy for the diagnosis and treatment of MDS.

Cancer biology deciphered by single-cell transcriptomic sequencing

Tumors are complex ecosystems in which heterogeneous cancer cells interact with their microenvironment composed of diverse immune, endothelial, and stromal cells. Cancer biology had been studied using bulk genomic and gene expression profiling, which however mask the cellular diversity and average the variability among individual molecular programs. Recent advances in single-cell transcriptomic sequencing have enabled a detailed dissection of tumor ecosystems and promoted our understanding of tumorigenesis at single-cell resolution. In the present review, we discuss the main topics of recent cancer studies that have implemented single-cell RNA sequencing (scRNA-seq). To study cancer cells, scRNA-seq has provided novel insights into the cancer stem-cell model, treatment resistance, and cancer metastasis. To study the tumor microenvironment, scRNA-seq has portrayed the diverse cell types and complex cellular states of both immune and non-immune cells interacting with cancer cells, with the promise to discover novel targets for future immunotherapy.

Comparative transcriptome analysis of candidate genes involved in chlorogenic acid biosynthesis during fruit development in three pear varieties of Xinjiang Uygur Autonomous Region / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Pear is one of the main fruits with thousands of years of cultivation history in China. There are more than 2000 varieties of pear cultivars around the world, including more than 1200 varieties or cultivars in China (Legrand et al., 2016). Xinjiang Uygur Autonomous Region is an important pear production region in China with 30 of varieties or cultivars. Pyrus sinkiangensis is the most popular variety, which is mainly distributed in Xinjiang (Zhou et al., 2018). Chlorogenic acid (CGA), p-coumaric acid, and arbutin are the main polyphenols in pear fruit, and their levels show great differences among different varieties (Li et al., 2014). CGA is a potential chemo-preventive agent, which possesses many important bioactivities including antioxidant, diabetes attenuating, and anti-obesity (Wang et al., 2021). Therefore, the specific CGA content of a variety is considered the embodiment of the functional nutritional value of pears.

Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)‍-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.

Bioinformatics Analysis of Microarray Data in Myelodysplastic Syndrome Based on Gene Expression Omnibus Database / 中国实验血液学杂志

OBJECTIVE@#To identify the key genes and explore mechanisms in the development of myelodysplastic syndrome (MDS) by bioinformatics analysis.@*METHODS@#Two cohorts profile datasets of MDS were downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed gene (DEG) was screened by GEO2R, functional annotation of DEG was gained from GO database, gene ontology (GO) enrichment analysis was performed via Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and key genes were screened by Matthews correlation coefficient (MCC) based on STRING database.@*RESULTS@#There were 112 DEGs identified, including 85 up-regulated genes and 27 down-regulated genes. GO enrichment analysis showed that biological processes were mainly enriched in immune response, etc, cellular component in cell membrane, etc, and molecular function in protein binding, etc. KEGG signaling pathway analysis showed that main gene enrichment pathways were primary immunodeficiency, hematopoietic cell lineage, B cell receptor signaling pathway, Hippo signaling pathway, and asthma. Three significant modules were screened by Cytoscape software MCODE plug-in, while 10 key node genes (CD19, CD79A, CD79B, EBF1, VPREB1, IRF4, BLNK, RAG1, POU2AF1, IRF8) in protein-protein interaction (PPI) network were screened based on STRING database.@*CONCLUSION@#These screened key genes and signaling pathways are helpful to better understand molecular mechanism of MDS, and provide theoretical basis for clinical targeted therapy.

Expression Analysis of miRNA Profiles in Apheresis Platelets during Storage / 中国实验血液学杂志

OBJECTIVE@#To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage.@*METHODS@#The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value < 0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR).@*RESULTS@#By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P>0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing.@*CONCLUSION@#The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.

Bioinformatics-based identification of key genes CDC5L and related pathways in osteosarcoma and Ewing's sarcoma / 中国骨伤

OBJECTIVE@#Osteosarcoma(OS) and Ewing's sarcoma (EWS) are the two most common primary malignant bone tumors in children. The aim of the study was to identify key genes in OS and EWS and investigate their potential pathways.@*METHODS@#Expression profiling (GSE16088 and GSE45544) were obtained from GEO DataSets. Differentially expressed genes were identified using GEO2R and key genes involved in the occurrence of both OS and EWS were selected using venn diagram. Gene ontology and pathway enrichment analyses were performed for the ensembl. Protein-protein interaction (PPI) networks were established by STRING. Further, UCSC was used to predict the transcription factors of the cell division cycke 5-like(CDC5L) gene, and GEPIA was used to analyze the correlation between the transcription factors and the CDC5L gene.@*RESULTS@#The results showed that CDC5L gene was the key gene involved in the pathogenesis of OS and EWS. The gene is mainly involved in mitosis, and is related to RNA metabolism, processing of capped intron-containing pre-mRNA, mRNA and pre-mRNA splicing.@*CONCLUSION@#CDC5L, as a key gene, plays a role in development of OS and EWS, which may be reliable targets for diagnosis and treatment of these primary malignant tumors.

Resistance of different ecotypes of Gastrodia elata to tuber rot / 中国中药杂志

Tuber rot has become a serious problem in the large-scale cultivation of Gastrodia elata. In this study, we compared the resistance of different ecotypes of G. elata to tuber rot by field experiments on the basis of the investigation of G. elata diseases. The histological observation and transcriptome analysis were conducted to reveal the resistance differences and the underlying mechanisms among different ecotypes. In the field, G. elata f. glauca had the highest incidence of tuber rot, followed by G. elata f. viridis, and G. elata f. elata and G. elata f. glauca×G. elata f. elata showed the lowest incidence. Tuber rot showcased obvious plant source specificity and mainly occurred in the buds and bottom of G. elata plants. After infection, the pathogen spread hyphae in host cortex cells, which can change the endophytic fungal community structure in the cortex and parenchyma of G. elata. G. elata f. glauca had thinner lytic layer and more sugar lumps in the parenchyma than G. elata f. elata. The transcription of genes involved in immune defense, enzyme synthesis, polysaccharide synthesis, carbohydrate transport and metabolism, hydroxylase activity, and aromatic compound synthesis had significant differences between G. elata f. glauca and G. elata f. elata. These findings suggested that the differences in resis-tance to tuber rot among different ecotypes of G. elata may be related to the varied gene expression patterns and secondary metabolites. This study provides basic data for the prevention and control of tuber rot and the improvement of planting technology for G. elata.

Screening of differentially expressed genes for colorectal cancer and prediction of potential traditional Chinese medicine: based on bioinformatics / 中国中药杂志

This study screened and analyzed the differentially expressed genes(DEGs) between colorectal cancer(CRC) tissues and normal tissues with bioinformatics techniques to predict biomarkers and Chinese medicinals for the diagnosis and treatment of CRC. The microarray data sets GSE21815, GSE106582, and GSE41657 were downloaded from the Gene Expression Omnibus(GEO), and the DEGs were screened by GEO2 R, followed by the Gene Ontology(GO) tern enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis of the DEGs based on DAVID. The protein-protein interaction network was constructed by STRING, and MCODE and Cytohubba plug-ins were used to screen the significant modules and hub genes in the network. UCSC, cBioPortal, and Oncomine were employed for hierarchical clustering, survival analysis, Oncomine analysis, and correlation analysis of clinical data. Coremine Medical was applied to predict the Chinese medicinals acting on hub genes. A total of 284 DEGs were screened out, with 146 up-regulated and 138 down-regulated. The up-regulated genes were mainly involved in cell cycle, NLRs pathway, and TNF signaling pathway, and the down-regulated genes were related to mineral absorption, nitrogen metabolism, and bicarbonate reabsorption in proximal tubules. The 15 hub genes were CDK1, CDC20, AURKA, MELK, TOP2 A, PTTG1, BUB1, CDCA5, CDC45, TPX2, NEK2, CEP55, CENPN, TRIP13, and GINS2, among which CDK1 and CDC20 were regarded as core genes. The high expression of CDK1 and CDC20 suggested poor prognosis, and they significantly expressed in many cancers, especially breast cancer, lung cancer, and CRC. The expression of CDK1 and CDC20 was correlated with gender, tumor type, TNM stage, and KRAS gene mutation. The potential effective medicinals against CRC were Scutellariae Radix, Scutellariae Barbatae Herba, Arnebiae Radix, etc. The significant expression of CDK1 and CDC20 can help distinguish tumor tissues from normal tissues, and is related to survival prognosis. Thus, the two can be used as biomarkers for the diagnosis and treatment of CRC. This study provides a reference for related drug development.