Identification of core pathogenic genes and pathways in elderly osteoporosis based on bioinformatics analysis / 中华预防医学杂志

Objective: Using bioinformatics methods to analyze the core pathogenic genes and related pathways in elderly osteoporosis. Methods: From November 2020 and August 2021, eight elderly osteoporosis patients who received treatment and five healthy participants who underwent physical examinations in Beijing Jishuitan Hospital were selected as subjects. The expression level of RNA in the peripheral blood of eight elderly osteoporosis patients and five healthy participants was collected for high-throughput transcriptome sequencing and analysis. The gene ontology (GO) analysis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed for the differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was constructed using the STRING website and Cytoscape software, and the most significant modules and hub genes were screened out. Results: Among the eight elderly osteoporosis patients, there were seven females and one male, with an average age of 72.4 years (SD=4.2). Among the five healthy participants, there were four females and one male, with an average age of 68.2 years (SD=5.7). A total of 1 635 DEGs (847 up-regulated and 788 down-regulated) were identified. GO analysis revealed that the molecular functions of DEGs were mainly enriched in structural constituents of the ribosome, protein dimerization activity, and cellular components were mainly enriched in the nucleosome, DNA packaging complex, cytosolic part, protein-DNA complex and the cytosolic ribosome. KEGG pathway analysis showed that DEGs were mainly enriched in systemic lupus erythematosus and ribosome. Gene UBA52, UBB, RPS27A, RPS15, RPS12, RPL13A, RPL23A, RPL10A, RPS25 and RPS6 were selected and seven of them could encode ribosome proteins. Conclusion: The pathogenesis of elderly osteoporosis may be associated with ribosome-related genes and pathways.

New strategies for the treatment of carcinoma of unknown primary / 中华肿瘤杂志

Carcinoma of unknown primary (CUP) is a kind of metastatic tumor whose primary origin cannot be identified after adequate examination and evaluation. The main treatment modality of CUP is empiric chemotherapy, and the median overall survival time is less than 1 year. Compared with immunohistochemistry, novel method based on gene expression profiling have improved the sensitivity and specificity of CUP detection, but its guiding value for treatment is still controversial. The approval of immune checkpoint inhibitors and pan-cancer antitumor agents has improved the prognosis of patients with CUP, and targeted therapy and immunotherapy based on specific molecular characteristics are the main directions of future research. Given the high heterogeneity and unique clinicopathological characteristics of CUP, "basket trial" is more suitable for clinical trial design in CUP.

Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis / 生物工程学报

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

Comparative analysis of differentially expressed genes for biosynthesis of active ingredients in fruits of different cultivars of Lycium barbarum L. based on transcriptome sequencing / 生物工程学报

To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of 'Ningqi No.1' and 'Ningqi No.7' during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of 'Ningqi No. 1' and 'Ningqi No. 7', respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.

Analysis of Significant Genes and Pathways in Esophageal Cancer Based on Gene Expression Omnibus Database / 中国医学科学杂志(英文版)

Objective To screen antigen targets for immunotherapy by analyzing over-expressed genes, and to identify significant pathways and molecular mechanisms in esophageal cancer by using bioinformatic methods such as enrichment analysis, protein-protein interaction (PPI) network, and survival analysis based on the Gene Expression Omnibus (GEO) database.Methods By screening with highly expressed genes, we mainly analyzed proteins MUC13 and EPCAM with transmembrane domain and antigen epitope from TMHMM and IEDB websites. Significant genes and pathways associated with the pathogenesis of esophageal cancer were identified using enrichment analysis, PPI network, and survival analysis. Several software and platforms including Prism 8, R language, Cytoscape, DAVID, STRING, and GEPIA platform were used in the search and/or figure creation.Results Genes MUC13 and EPCAM were over-expressed with several antigen epitopes in esophageal squamous cell carcinoma (ESCC) tissue. Enrichment analysis revealed that the process of keratinization was focused and a series of genes were related with the development of esophageal cancer. Four genes including ALDH3A1, C2, SLC6A1,and ZBTB7C were screened with significant P value of survival curve.Conclusions Genes MUC13 and EPCAM may be promising antigen targets or biomarkers for esophageal cancer. Keratinization may greatly impact the pathogenesis of esophageal cancer. Genes ALDH3A1, C2, SLC6A1,and ZBTB7C may play important roles in the development of esophageal cancer.

Bioinformatics analysis and identification to immune-related markers of osteoporosis / 细胞与分子免疫学杂志

Objective To identify immune-related dysregulation mechanisms and potential diagnostic predictive biomarkers in osteoporosis. Methods Gene expression data for both osteoporosis and control populations were retrieved from the GSE35958 and GSE56815 datasets. Immune-related differentially expressed genes (DEGs) were obtained by screening DEGs and were compared with the immunology database and analysis portal (ImmPort) database. Enrichment analysis of these immune-related DEGs was conducted using the Clusterprofiler software package. A protein-protein interaction network was built with the STRING database, which is a search tool for finding interacting genes/proteins, and the top 10 genes with the highest network connectivity were identified as candidate genes. Subsequently, the diagnostic predictive effect of candidate genes was evaluated using receiver operating characteristic (ROC) curves, logistic regression, and column plots. Finally, PCR and Western blot analysis were applied to detect the differential expression of these genes in bone marrow tissue of patients with osteoporosis. Results A total of 138 immune-related DEGs were obtained through intersection analysis. The results of the enrichment analysis indicated that these genes were involved in biological functions such as immune inflammation and signaling pathways including T cell receptors, mitogen activated protein kinase (MAPK), rat sarcoma virus oncogene homologs (Ras), osteoclast differentiation, and B cell receptors. In addition, among the candidate genes, upregulated vascular endothelial growth factor A (VEGFA) and epidermal growth factor receptor (EGFR) and downregulated AKT1, SRC, and JUN in osteoporosis showed the highest connectivity. Among them, VEGFA, EGFR, JUN, and AKT1 demonstrated the best diagnostic predictive value. Conclusion The screening of immune-related DEGs will enhance the understanding of osteoporosis and facilitate the development of immunotherapy targets.

Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii / 中国中药杂志

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Identification and validation of novel biomarkers for cold-dampness syndrome of rheumatoid arthritis based on integration of multiple bioinformatics methods / 中国中药杂志

This study aims to identify the novel biomarkers of cold-dampness syndrome(RA-Cold) of rheumatoid arthritis(RA) by gene set enrichment analysis(GSEA), weighted gene correlation network analysis(WGCNA), and clinical validation. Firstly, transcriptome sequencing was carried out for the whole blood samples from RA-Cold patients, RA patients with other traditional Chinese medicine(TCM) syndromes, and healthy volunteers. The differentially expressed gene(DEG) sets of RA-Cold were screened by comparison with the RA patients with other TCM syndromes and healthy volunteers. Then, GSEA and WGCNA were carried out to screen the key DEGs as candidate biomarkers for RA-Cold. Experimentally, the expression levels of the candidate biomarkers were determined by RT-qPCR for an independent clinical cohort(not less than 10 cases/group), and the clinical efficacy of the candidates was assessed using the receiver operating characteristic(ROC) curve. The results showed that 3 601 DEGs associated with RA-Cold were obtained, including 106 up-regulated genes and 3 495 down-regulated genes. The DEGs of RA-Cold were mainly enriched in the pathways associated with inflammation-immunity regulation, hormone regulation, substance and energy metabolism, cell function regulation, and synovial pannus formation. GSEA and WGCNA showed that recombinant proteasome 26S subunit, ATPase 2(PSMC2), which ranked in the top 50% in terms of coefficient of variation, representativeness of pathway, and biological modules, was a candidate biomarker of RA-Cold. Furthermore, the validation results based on the clinical independent sample set showed that the F1 value, specificity, accuracy, and precision of PSMC2 for RA-Cold were 70.3%, 61.9%, 64.5%, and 81.3%, respectively, and the area under the curve(AUC) value was 0.96. In summary, this study employed the "GSEA-WGCNA-validation" integrated strategy to identify novel biomarkers of RA-Cold, which helped to improve the TCM clinical diagnosis and treatment of core syndromes in RA and provided an experimental basis for TCM syndrome differentiation.

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.

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.

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.

Bioinformatics Analysis on Key Genes and Immune Infiltration of Osteosarcoma / 中国医学科学院学报

Objective To screen the potential key genes of osteosarcoma by bioinformatics methods and analyze their immune infiltration patterns. Methods The gene expression profiles GSE16088 and GSE12865 associated with osteosarcoma were obtained from the Gene Expression Omnibus(GEO),and the differentially expressed genes(DEGs)related to osteosarcoma were screened by bioinformatics tools.Gene Ontology(GO)annotation,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,and analysis of immune cell infiltration were then carried out for the DEGs.The potential Hub genes of osteosarcoma were identified by protein-protein interaction network,and the expression of Hub genes in osteosarcoma and normal tissue samples was verified via the Cancer Genome Atlas(TCGA). Results A total of 108 DEGs were screened out.GO annotation and KEGG pathway enrichment revealed that the DEGs were mainly involved in integrin binding,extracellular matrix (ECM) structural components,ECM receptor interactions,and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway.Macrophages were the predominant infiltrating immune cells in osteosarcoma.Secreted phosphoprotein 1(SPP1),matrix metallopeptidase 2(MMP2),lysyl oxidase(LOX),collagen type V alpha(II)chain(COL5A2),and melanoma cell adhesion molecule(MCAM)presented differential expression between osteosarcoma and normal tissue samples(all P<0.05). Conclusions SPP1,MMP2,LOX,COL5A2,and MCAM are all up-regulated in osteosarcoma,which may serve as potential biomarkers of osteosarcoma.Macrophages are the key infiltrating immune cells in osteosarcoma,which may provide new perspectives for the treatment of osteosarcoma.

Transcriptome-wide identification and expression pattern analysis for Dof gene family in Panax ginseng from Jilin / 中国中药杂志

Dof(DNA binding with one finger), a unique class of transcription factors in plants, play an important role in seed development, tissue differentiation, and metabolic regulation. To identify the number and function of Dof gene family members in Panax ginseng, this study identified the members of Dof gene family in P. ginseng and systematically analyzed their structures, evolution, functional differentiation, expression patterns, and interactions using bioinformatics methods at the transcriptome level. At the same time, the association analysis of Dof genes from P. ginseng with key enzyme genes for ginsenoside synthesis was carried out to screen the candidate PgDof genes involved in the regulation of ginsenoside biosynthesis. The results showed that there were 54 genes belonging to the Dof gene family in P. ginseng from Jilin. All PgDof genes had Zf-Dof conserved motifs, implying that they were evolutionarily conserved and could be divided into five groups. Expression pattern analysis confirmed that the expression of PgDof gene family members in different tissues, different year-old P. ginseng, and different farm varieties varied significantly. Simultaneously, as revealed by "gene-saponin content" and "gene-gene" linkage analysis, an important candidate PgDof14-1 gene involved in the regulation of ginsenoside biosynthesis was obtained. From the established genetic transformation system of this gene in the hairy roots of P. ginseng, a positive hairy root clone was determined. This study has laid a theoretical foundation for the study of Dof gene family in P. ginseng.

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.

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.

Gene Expression Profiles at Different Time Points after Acute Myocardial Infarction in Mice / 法医学杂志

OBJECTIVES@#To explore the mRNA differential expressions and the sequential change pattern in acute myocardial infarction (AMI) mice.@*METHODS@#The AMI mice relevant dataset GSE4648 was downloaded from Gene Expression Omnibus (GEO). In the dataset, 6 left ventricular myocardial tissue samples were selected at 0.25, 1, 4, 12, 24 and 48 h after operation in AMI group and sham control group, and 6 left ventricular myocardial tissue samples were selected in blank control group, a total of 78 samples were analyzed. Differentially expressed genes (DEGs) were analyzed by R/Bioconductor package limma, functional pathway enrichment analysis was performed by clusterProfiler, protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape software, the key genes were identified by Degree topological algorithm, cluster sequential changes on DEGs were analyzed by Mfuzz.@*RESULTS@#A total of 1 320 DEGs were associated with the development of AMI. Functional enrichment results included cellular catabolic process, regulation of inflammatory response, development of muscle system and vasculature system, cell adhesion and signaling pathways mainly enriched in mitogen-activated protein kinase (MAPK) signaling pathway. The key genes of AMI included MYL7, TSC22D2, HSPA1A, BTG2, NR4A1, RYR2 were up-regulated or down-regulated at 0.25-48 h after the occurrence of AMI.@*CONCLUSIONS@#The functional signaling pathway of DEGs and the sequential expression of key genes in AMI may provide a reference for the forensic identification of AMI.

Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion In Vitro / 中国医学科学杂志(英文版)

Objective To discover critical genes contributing to the stemness and maintenance of spermatogonial stem cells (SSCs) and provide new insights into the function of the leucine-rich repeat (LRR) family member Lrrc34 (leucine-rich repeat-containing 34) in SSCs from mice. Methods Bioinformatic methods, including differentially expressed gene (DEG), gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were used to uncover latent pluripotency-related genes. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence analyses were utilized to verify the mRNA and protein expression levels, respectively. RNA interference of Lrrc34 using siRNA was performed to detect its transient impact on SSCs. Results Eight DEGs between ID4-EGFP+ (G) and ID4-EGFP+/TSPAN8High (TH), eight DEGs between G and ID4-EGFP+/TSPAN8Low (TL) and eleven DEGs between TH and TL were discovered, and eleven protein-protein interaction (PPI) modules were found to be significant in the PPI network of DEGs. One of the DEGs, Lrrc34, was selected as a potential pluripotency-related gene due to its differential expression among ID4-EGFP+ spermatogonia subsets and its interaction with fibroblast growth factor 2 in the fifth module. Immunofluorescence experiments exhibited specific expression of Lrrc34 in a subpopulation of undifferentiated spermatogonia marked by LIN28A, and RT-PCR experiments confirmed the high expression of Lrrc34 in SSCs from P7 and adult mice. The transient knockdown of Lrrc34 in SSCs resulted in reduced colony sizes and significant changes in the transcriptome and apoptotic pathways. Conclusion Lrrc34 is highly expressed in mouse SSCs and is required for SSC proliferation in vitro through effects on transcriptome and signaling transduction pathways.

Vascular Transcriptome Profiling Reveals Aging-Related Genes in Angiotensin Ⅱ-Induced Hypertensive Mouse Aortas / 中国医学科学杂志(英文版)

Objective Angiotensin Ⅱ (Ang Ⅱ)-induced vascular damage is a major risk of hypertension. However, the underlying molecular mechanism of AngⅡ-induced vascular damage is still unclear. In this study, we explored the novel mechanism associated with Ang II-induced hypertension. Methods We treated 8- to 12-week-old C57BL/6J male mice with saline and Ang Ⅱ(0.72 mg/kg·d) for 28 days, respectively. Then the RNA of the media from the collected mice aortas was extracted for transcriptome sequencing. Principal component analysis was applied to show a clear separation of different samples and the distribution of differentially expressed genes was manifested by Volcano plot. Functional annotations including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed to reveal the molecular mechanism of Ang Ⅱ-induced hypertension. Finally, the differentially expressed genes were validated by using quantitative real-time PCR. Results The result revealed that a total of 773 genes, including 599 up-regulated genes and 174 down-regulated genes, were differentially expressed in the aorta of Ang Ⅱ-induced hypertension mice model. Functional analysis of differentially expressed genes manifested that various cellular processes may be involved in the Ang Ⅱ-induced hypertension, including some pathways associated with hypertension such as extracellular matrix, inflammation and immune response. Interestingly, we also found that the differentially expressed genes were enriched in vascular aging pathway, and further validated that the expression levels of insulin-like growth factor 1 and adiponectin were significantly increased (P<0.05). Conclusion We identify that vascular aging is involved in Ang Ⅱ-induced hypertension, and insulin-like growth factor 1 and adiponectin may be important candidate genes leading to vascular aging.

Transcriptional Profile Alteration of Peripheral Blood in Chronic Hypoxia / 中国医学科学杂志(英文版)

Objective Many physiological and pathological conditions, including cyanotic congenital heart diseases (CCHD), are accompanied by chronic hypoxia, which might interfere with the transcription process. However, the transcriptome profile in peripheral blood under hypoxia is still unidentified. The present work aimed to explore the transcriptional profile alteration of peripheral blood in chronic hypoxia. Methods The present study used a chronic hypoxia rat model to simulate the hypoxic state of CCHD patients. Two groups of Sprague-Dawley rats (n=6 per group) were either exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks. Body weight was measured weekly. Peripheral blood was collected and total RNA was extracted for RNA-Seq at the end of the hypoxia treatment. After quality assessment, the library was sequenced by the Illumina Hiseq platform. The differentially expressed genes were screened (false discovery rate<0.05 and fold change>2). The functional annotation analysis and cluster analysis of differentially expressed genes were performed based on the adjusted P-value (padj<0.05). Results Compared with the control group, the body weight of the rats in the hypoxia group was significantly lowered (P<0.01). RNA-Seq results showed that the transcriptome patterns of the two groups had significant differences. In total, 872 genes were identified as differentially expressed. Among all, 803 genes were down-regulated, while only 69 genes were up-regulated in the hypoxia group. The functional enrichment analysis of the 872 genes showed that multiple biological processes involved, such as porphyrin-containing compound metabolic process, hemoglobin complex and oxygen transporter activity. Conclusions Our study demonstrated the transcriptional profile alteration in peripheral blood of chronic hypoxia rat model. This study provided basic data and directions to further understand the physiological and pathological changes in patients with CCHD.

Transcriptome profiling and digital gene expression analysis of genes associated with salinity resistance in peanut

Background: Soil salinity can significantly reduce crop production, but the molecular mechanism of salinity tolerance in peanut is poorly understood. A mutant (S1) with higher salinity resistance than its mutagenic parent HY22 (S3) was obtained. Transcriptome sequencing and digital gene expression (DGE) analysis were performed with leaves of S1 and S3 before and after plants were irrigated with 250 mM NaCl. Results: A total of 107,725 comprehensive transcripts were assembled into 67,738 unigenes using TIGR Gene Indices clustering tools (TGICL). All unigenes were searched against the euKaryotic Ortholog Groups (KOG), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and these unigenes were assigned to 26 functional KOG categories, 56 GO terms, 32 KEGG groups, respectively. In total 112 differentially expressed genes (DEGs) between S1 and S3 after salinity stress were screened, among them, 86 were responsive to salinity stress in S1 and/or S3. These 86 DEGs included genes that encoded the following kinds of proteins that are known to be involved in resistance to salinity stress: late embryogenesis abundant proteins (LEAs), major intrinsic proteins (MIPs) or aquaporins, metallothioneins (MTs), lipid transfer protein (LTP), calcineurin B-like protein-interacting protein kinases (CIPKs), 9-cis-epoxycarotenoid dioxygenase (NCED) and oleosins, etc. Of these 86 DEGs, 18 could not be matched with known proteins. Conclusion: The results from this study will be useful for further research on the mechanism of salinity resistance and will provide a useful gene resource for the variety breeding of salinity resistance in peanut.