High-throughput screening of differential expression of exosomal miRNAs in DeBakey typeⅠacute aortic dissection patients / 中国胸心血管外科临床杂志

@#Objective    To evaluate the changes in the expression and significance of serum exosomal miRNAs in patients with DeBakey typeⅠacute aortic dissection (AAD). Methods    Twelve male patients with AAD and six healthy male medical examiners from our hospital were retrospectively included in this study. According to the time of chest pain, the AAD patients were divided into an AAD group within 24 h of chest pain onset, aged 47.00±8.79 years and an AAD group within 48 h of chest pain onset, aged 50.17±9.99 years. The healthy males were allocated to a control group, aged 49.17±4.26 years. Serum exosomal miRNAs were isolated, identified and quantified, and then differentially expressed exosomal miRNAs were screened. The bioinformatic analyses such as GO and KEGG were performed on the differentially expressed exosomal miRNAs. Results    High-throughput screening results revealed differential expression of AAD serum exosomal miRNAs. The upregulated miRNAs of AAD groups was hsa-miR-574-5p (P<0.05), and downregulated miRNAs were hsa-miR-223-3p, hsa-miR-146b-5p, hsa-miR-15b-5p, and hsa-miR-155-5p (P<0.05). Further bioinformatic analysis of the above miRNAs revealed that they were mainly enriched in signaling pathways such as transforming growth factor-β, cell cycle and endoplasmic reticulum protein synthesis. Conclusion    Differential expressions of serum exosomal miRNAs in AAD patients may be related to the pathogenesis of AAD, providing new ideas and clues for further exploration of AAD diagnostic markers and pathogenesis.

Gene cloning and enzymatic activity analysis of phenylalanine ammonia-lyase from Sinopodophyllum hexandrum (Royle) Ying / 生物工程学报

Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.

Bioinformatic analysis identifies potential key genes in the pathogenesis of age-related macular degeneration

Purpose: Age?related macular degeneration (AMD) is the leading cause of irreversible blindness in older individuals. More studies focused on screening the genes, which may be correlated with the development of AMD. With advances in various technologies like multiple microarray datasets, researchers could identify differentially expressed genes (DEGs) more accurately. Exploring abnormal gene expression in disease status can help to understand pathophysiological changes in complex diseases. This study aims to identify the key genes and upstream regulators in AMD and reveal factors, especially genetic association, and the prognosis of the development of this disease. Methods: Data from expression profile GSE125564 and profile GSE29801 were obtained from the Gene Expression Omnibus (GEO) database. We analyzed DEGs using R software (version 3.6.3). Functional enrichment and PPI network analysis were performed using the R package and online database STRING (version 11.0). Results: We compared AMD with normal and found 68 up?regulated genes (URGs) and 25 down?regulated genes (DRGs). We also compared wet AMD with dry AMD and found 41 DRGs in dry AMD. Further work including PPI network analysis, GO classification, and KEGG analysis was done to find connections with AMD. The URGs were mainly enriched in the biological process such as DNA replication, nucleoplasm, extracellular exosome, and cadherin binding. Besides, DRGs were mainly enriched in these functions such as an integral component of membrane and formation of the blood?aqueous barrier (BAB). Conclusion: This study implied that core genes might involve in the process of AMD. Our findings may contribute to revealing the pathogenesis, developing new biomarkers, and raising strategies of treatment for AMD

Identification of onco-miRNAs in hepatocellular carcinoma and analysis of their regulatory network / 南方医科大学学报

OBJECTIVE@#To construct the regulatory network of survival-related onco-miRNAs and their target genes in hepatocellular carcinoma (HCC) and verify the interactions between the key miRNAs and their targets.@*METHODS@#We screened survival-related miRNAs in HCC in OncomiR and Oncolnc databases, predicted their target genes using miRNet, and conducted survival and expression analysis using GEPIA2 and Ualcan, respectively. The miRNA-target gene co-expression analysis was performed and the miRNA-target network was constructed. Enrichment analysis was performed in Enrichr and protein-protein interaction analysis in STRING database. We tested the effects of transfection with the mimic or inhibitor of hsa-miR-1226-3p or hsa-miR-221-5p on proliferation of HepG2 cells using CCK8 assay and examined the changes in the expressions of the target genes using RT-qPCR. The effect of transfection with hsa-miR-221-5p mimic or inhibitor on protein expressions of the target genes was examined using Western blotting in. A dual luciferase reporter assay was used to test the interaction between hsa-miR-221-5p and its potential target gene GCDH. We further examined the effect of transfection with hsa-miR-221-5p mimic and pEGFP N1-GCDH, alone or in combination, on proliferation, migration and invasion of HepG2 cells.@*RESULTS@#We identified 223 survival-related miRNAs in HCC from OncomiR and 146 miRNAs from Oncolnc with an intersection of 131 miRNAs, and 48 miRNAs were identified as onco-miRNAs in HCC after survival and expression analysis. Twenty-seven eligible target genes were identified after miRNA-mRNA co-expression analysis. The constructed miRNA-target gene network consisted of 25 miRNAs and 27 target genes. The most enriched term was fatty acid metabolism for the target genes. In HepG2 cells, transfection with the mimic or inhibitor of hsa-miR-1226-3p or hsa-miR-221-5p caused significant changes of the mRNA and protein levels of their respective target genes (P < 0.05). The results of dual luciferase reporter assay confirmed the targeting relationship between hsa-miR-221-5p and GCDH gene (P < 0.05). Transfection with hsa-miR-221-5p mimic significantly suppressed the proliferation, migration and invasion of HepG2 cells, but this effect was obviously relieved by co-transformation with pEGFP N1-GCDH (P < 0.05).@*CONCLUSION@#Fatty acid metabolism might be one of the most crucial pathways that mediate the effect of the oncomiRNAs in HCC, and the hsa-miR-221-5p/GCDH axis is an important molecular mechanism for HCC progression.

Identification of laccase gene family members in peach and its relationship with chilling induced browning / 生物工程学报

The laccase (PpLAC) gene family members in peach fruit were identified and the relationship between their expression pattern and chilling induced browning were investigated. The study was performed using two varieties of peaches with different chilling tolerance, treated with or without exogenous γ-aminobutyric acid (GABA) during cold storage. Twenty-six genes were screened from the peach fruit genome. These genes were distributed on 6 chromosomes and each contained 5-7 exons. The PpLAC gene family members shared relatively similar gene structure and conserved motifs, and they were classified into 7 subgroups based on the cluster analysis. Transcriptome sequencing revealed that the expression levels of PpLAC7 and PpLAC9 exhibited an increasing pattern under low temperature storage, and displayed a similar trend with the browning index of peach fruit. Notably, GABA treatment reduced the degree of browning and inhibited the expression of PpLAC7 and PpLAC9. These results suggested that PpLAC7 and PpLAC9 might be involved in the browning of peach fruit during cold storage.

Identifying and Validating a Novel miRNA-mRNA Regulatory Network Associated with Prognosis in Lung Adenocarcinoma / 中国医学科学杂志(英文版)

Objective Many studies have revealed the crucial roles of miRNA in multiple human cancers, including lung adenocarcinoma (LUAD). In this study, we sought to explore new miRNA-mRNA pairs that are associated with LUAD prognosis. Methods A novel miRNA-mRNA regulatory network associated with prognosis in LUAD was identified and validated using the bioinformatic tools including OncomiR database, StarBase, miRnet, GEPIA2, UALCAN. Results Twenty key miRNAs were compiled after the analysis of the expression and prognostic value in OncomiR and StarBase. Targeted mRNAs of these key miRNAs were predicted in miRnet, and the resulting mRNAs were also analyzed for their prognostic values and expression patterns in GEPIA2 and UALCAN, respectively. Further expression correlation analysis was performed in StarBase. Subsequently, a new miRNA-mRNA network was built, of which each RNA pair showed negative expression correlation, opposite expression pattern, and prognostic value. Protein-protein interaction network was under construction for the mRNAs, and 19 hub genes were determined. Enrichment analysis showed that "Cell Cycle, Mitotic" was the most significantly enriched term. Then, a miRNA-hub gene sub-network was built. We selected and validated the regulatory relationship of some miRNA-hub pairs, including hsa-miR-1976/RFC2, hsa-let-7c-5p/RFC2, hsa-let-7c-5p/ESPL1, hsa-let-7c-5p/CDC25A, and hsa-miR-101-3p/KIF2C. Moreover, over-expression of hsa-miR-1976 and hsa-let-7c-5p resulted in significant cell cycle arrest. Conclusions Our results determined new prognosis-associated miRNA-mRNA pairs and might shed further light on the mechanism via which miRNA-mRNA network influences prognosis in LUAD.

Expression and significance of microRNA-129-1 in colon cancer based on database mining / 中华消化杂志

Objective:To investigate the expression, regulation, potential mechanism and clinical significance of microRNA(miRNA)-129-1 in colon cancer.Methods:The changes of expression and methylation of miRNA-129-1 were analyzed from the methylation, mRNA expression and miRNA expression data of colon cancer in the cancer genome atlas (TCGA) database. The target genes of miRNA-129-1 were predicted from miRwalk 2.0 and TargetScan database. DAVID 6.7 online software was used for gene oncology and Kyoto encyclopedia of genes and genomes enrichment analysis. STRING database was used for protein-protein interaction analysis. TCGA data were applied again to analyze the differential expression and prognosis of key target genes of miRNA-129-1. Paired t test and independent sample t test were used for statistical analysis. The receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of miRNA-129-1 gene methylation in colon cancer. Kaplan-Meier method and log-rank test were used to analyze the effects of miRNA-129-1 expression on survival. Results:The sequence of miRNA-129-1 among different species was conserved. After all colon cancer samples, and control samples of TCGA database were analyzed, the results showed that compared with those of control samples, the expression of miRNA-129-1 decreased in cancer samples (0.98±0.81 vs. 5.74±0.59), and the methylation levels of cg04524088, cg04840800, cg11364290, cg20734982 and cg24044186 locus of miRNA-129-1 significantly decreased (0.321±0.130 vs. 0.563±0.051, 0.432±0.123 vs. 0.624±0.064, 0.475±0.153 vs. 0.768±0.033, 0.659±0.180 vs. 0.816±0.037 and 0.862±0.096 vs. 0.916±0.019, respectively) in colon cancer tissues, and the differences were all statistically significant ( t=14.95, 11.36, 9.39, 11.74, 5.32 and 3.47, all P<0.01). The results of ROC analysis showed that the methylation levels of the above five locus of miRNA-129-1 gene had high diagnostic efficiency in colon cancer (area under curve=0.946, 0.915, 0.950, 0.758 and 0.667, all P<0.01). The results of survival analysis indicated that low expression of miRNA-129-1 was associated with poor prognosis (hazard ratio ( HR)=0.55, P=0.018). The results of bioinformatics analysis demonstrated that the target genes of miRNA-129-1 were enriched in serine / threonine kinase receptor, mitogen-activated protein kinase and other functional gene clusters closely related to tumor, and there was a complex interaction network among the target genes proteins. The high expression of ephrin type-B receptor2 ( EPHB2) gene, a potential key target gene of miRNA-129-1, was associated with the short overall survival and disease-free survival time ( HR=1.9 and 1.6, both P<0.01). Conclusions:The expression and methylation of miRNA-129-1 play an important regulatory role in the development and development of colon cancer. The methylation of miRNA-129-1 has potential value in the diagnosis of colon cancer, and miRNA-129-1 is an influencing factor for the prognosis of patients with colon cancer. EPHB2 may be a potential key target gene of miRNA-129-1.

Cloning and functional characterization of isopentenyl diphosphate isomerase genes from Panax vietnamensis var. fuscidiscus / 药学学报

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the regulation of triterpenes biosynthesis and plays an important role in ginsenoside biosynthesis. In this study, two IDI genes, PvfIDI1 (GenBank No. MZ736417) and PvfIDI2 (GenBank No. MZ736418) were cloned from Panax vietnamensis var. fuscidiscus. The open reading frame of both PvfIDI1 and PvfIDI2 was 924 bp encoding 307 amino acids. The molecular weights of PvfIDI1 and PvfIDI2 were 34.84 kDa and 34.66 kDa, respectively, with theoretical pIs of 6.01 and 5.66. Bioinformatic analysis indicated that PvfIDI1 and PvfIDI2 contained two conserved sequences: TNTCCSHPL and WGEHELDY. Phylogenetic analysis showed that PvfIDI1 and PvfIDI2 were closely related to Panax notoginseng IDI. Expression analysis showed that both PvfIDI1 and PvfIDI2 genes are expressed in root, rhizome, stem and leaf of P. vietnamensis var. fuscidiscus. However, PvfIDI1 is highly expressed in the rhizome and PvfIDI2 is highly expressed in the stem. PvfIDI1 and PvfIDI2 recombinant proteins were expressed in E. coli; a functional coloration experiment showed that PvfIDI1 and PvfIDI2 could promote the accumulation of lycopene, indicating that both PvfIDI1 and PvfIDI2 encode functional IDI enzymes. The cloning and functional studies on PvfIDI1 and PvfIDI2 provide a foundation for the further study of IDI and the regulation of ginsenoside biosynthesis in P. vietnamensis var. fuscidiscus.

Cloning and expression analysis of delta-12 fatty acid desaturase gene in Coix lacryma-jobi L / 药学学报

In this study, the fatty acid desaturase gene FAD2 was cloned from Coix lacryma-jobi L. and its molecular structure and function were studied. The results showed that the full-length cDNA sequence of FAD2 gene was 936 bp encoding 311 amino acid residues. Bioinformatics prediction results showed that the protein encoded by the FAD2 gene was an alkaline hydrophilic unstable protein with a molecular weight of 34.87 kDa. It contained three transmembrane helix domain, and did not contain the signal peptide splicing site, and was most likely to be located in plasmid membrane. Compared with other similar genes in plants, it has only a histidine conserved site, His Box Ⅲ histidine site (HXXHH), suggesting its activity may be reduced. Phylogenetic tree analysis showed that FAD2 was closely related to monocotyledonous plants, especially Maize and Oryza sativa japonica Group, but farther from dicotyledonous plants. Therefore, it was inferred that FAD2 might have similar functions with similar genes in Maize and Oryza sativa japonica Group. In addition, the expression of FAD2 gene could be detected in Coix lacryma-jobi L. with high oil content, but not in low oil content of Coix lacryma-jobi L. In order to clarify the function of FAD2, the gene was heterologously expressed in sporomyces cerevisiae. The results showed that the protein encoded by FAD2 gene did not catalyze the formation of C18∶1 unsaturated fatty acid into C18∶2 unsaturated fatty acid. Therefore, it was speculated that the deletion of histidinine conserved site of FAD2 gene might lead to the decrease of protein activity or even inactivation. This study provides reference value for further understanding the molecular structure characteristics of fatty acid desaturase. At the same time, it laid a foundation for elucidating the biosynthetic pathway of Coix lacryma-jobi L.

Screening and bioinformatic analysis of trehalase in Thelazia callipaeda / 中国血吸虫病防治杂志

Objective To characterize the trehalase gene in Thelazia callipaeda through screening the annotated data of the T. callipaeda genome, and to investigate the biological characteristics of the trehalase gene-coding protein. Methods The trehalase gene was screened from the T. callipaeda genome and subjected to validation by using a PCR assay. The structural features of the coding protein were analyzed with bioinformatics tools, including hydrophobicity, transmembrane region, signal peptides, conserved domains, as well as the secondary and tertiary structures and the antigen epitope. Homology analysis of the amino acid sequences was performed, and the phylogenetic tree was built by the MEGA X software. In addition, the protein-protein interaction network was deduced from the STRING database. Results The sequence of the trehalase gene with the complete CDS region was obtained from T. callipaeda genome, which had a length of 1 638 bp and encoded 545 amino acids. The encoded protein was predicted to have a molecular weight of 63 478.48 ku and be a secretory protein. The 5′ domain of the encoded protein contained a signal peptide without transmembrane regions, and was predicted to contain 7 antigen epitopes. Based on the protein-protein interaction network of nematodes in the STRING database, the protein-protein interaction network of the trehalase gene of T. callipaeda was deduced, and 27 interactions covering 10 genes were identified. Conclusions A trehalase gene is successfully identified in T. callipaeda genome and its coding protein receives a bioinformatics analysis, which provides insights into the research on the biological functions of the protein and the screening of vaccine candidates for thelaziasis callipaeda.

Virome: the next hotspot in microbiome research / 生物工程学报

Virome is the collective term for the viral collection or viral metagenomes that are distributed in various environments. Viruses can be found in bodies of water, glaciers, plants, animals, and even some viruses, which are classified as eukaryotes, prokaryotes and subviruses. Viruses play very important role in maintaining environmental homeostasis and ecosystem balance, and are especially closely related to human health. In recent years, with the advancement of sequencing technology and data analysis, we are able to gain more insights into the virome and explore its potential role in the ecological niche by metagenomic sequencing. A large amount of viral data have been obtained from glaciers, oceans, and various plants and animals, and numerous unknown viruses have been discovered. Virome has been studied mainly through metagenomic data mining, as well as virus-like particles separation and enrichment. To date, several different methods for viral isolation and enrichment exist, and numerous bioinformatic analyses of the virome have been performed. However, there is a lack of specific and complete reviews on the enrichment and data analysis methods for the virome. Thus, our review will summarize viral isolation and enrichment methods and data analysis, and present some of the landmark research conducted by the enrichment method, to provide a reference for researchers of interest and further advance the field of virome research.

Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

BACKGROUND: Kidney cancer is one of the most common cancers in the world. It is necessary to clarify its underlying mechanism and find its prognostic biomarkers. Current studies showed that SHMT2 may be participated in several kinds of cancer. METHODS: Our studies investigated the expression of SHMT2 in kidney cancer by Oncomine, Human Protein Atlas database and ULCAN database. Meanwhile, we found its co-expression gene by cBioPortal online tool and validated their relationship in A498 and ACHN cells by cell transfection, western blot and qRT-PCR. Besides these, we also explored their prognostic values via the Kaplan-Meier plotter database in different types of kidney cancer patients. RESULTS: SHMT2 was found to be increased in 7 kidney cancer datasets, compared to normal renal tissues. For the cancer stages, ages and races, there existed significant difference in the expression of SHMT2 among different groups by mining of the UALCAN database. High SHMT2 expression is associated with poor overall survival in patients with kidney cancer. Among all co-expressed genes, NDUFA4L2 and SHMT2 had a high co-expression efficient. SHMT2 overexpression led to the increased expression of NDUFA4L2 at both mRNA and protein levels. Like SHMT2, overexpressed NDUFA4L2 also was associated with worse overall survival in patients with kidney cancer. CONCLUSION: Based on above results, overexpressed SHMT2 and its co-expressed gene NDUFA4L2 were all correlated with the prognosis in kidney cancer. The present study might be benefit for better understanding the clinical significance of SHMT2 and provided a potential therapeutic target for kidney cancer in future.

Molecular Cloning and Sequence Analysis of GgUGE Gene in Glycyrrhiza glabra / 中国实验方剂学杂志

Objective::To clone the cDNA sequence of UDP-glucose 4-epimerase (UGE) in Glycyrrhiza glabra and analyze its sequence, so as to explore the potential relationship between the UGE gene and the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis. Method::The cDNA sequence of UGE was cloned from the root of G. glabra by reverse transcription polymerase chain reaction (RT-PCR), then sequenced and analyzed by bioinformatics software. Results::A GgUGE cDNA sequence with the full length of 1 121 bp was obtained. The open reading fame (ORF) of GgUGE was 1 053 bp, encoding 350 amino acid residues. The GgUGE cDNA sequence was submitted to GenBank, and the accession No. was MK638908. Sequence analysis showed that GgUGE was an unstable hydrophilic protein, its average relative molecular weight was 39.02 kDa, and isoelectric point was 6.13. It contained no signal peptides or transmembrane domains. Its secondary structure mainly constituted of α-helix and had a conversed domain of UDP-glucose 4-epimerase superfamily. The homologoue analysis showed that the cDNA and amino acid sequences of GgUGE had the closest evolutionary relationship to Leguminosae and relatively distant evolutionary relationship to Salicaceae. Conclusion::In this study, GgUGE cDNA sequence is successfully cloned from G. glabra for the very first time, which will provide reference for studying the function of GgUGE and explaining the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis in G. glabra.

Expression of superoxide dismutase 2 in breast cancer and its clinical significance / 南方医科大学学报

OBJECTIVE@#To evaluate the expression and prognostic value of superoxide dismutase 2 (SOD2) in breast cancer and explore its possible role in the occurrence and progression of breast cancer.@*METHODS@#We performed bioinformatics analysis of the TCGA data for the expression and clinical relevance of SOD2 in patients with breast cancer. Gene enrichment analysis (GSEA) was performed using the KEGG gene set, the protein interaction network was constructed using the STRING database, and the key genes were screened using Cytoscape software. We also collected 60 pairs of primary breast cancer tissue samples and adjacent samples for detecting SOD2 expressions using immunohistochemistry and RT-qPCR and analyzed the correlation of SOD2 expression with the clinicopathological parameters of the patients.@*RESULTS@#The expression of SOD2 was significantly lower in breast cancer tissue than in adjacent tissues with significant correlation with TNM stage and axillary lymph node metastasis ( < 0.05). Kaplan-Meier survival analysis showed that the recurrence-free survival, distant metastasis-free survival (RFS) and post-progressive survival were significantly shorted in patients with high SOD2 expression than in those with low SOD2 expression ( < 0.05). GSEA enrichment analysis indicated that SOD2 played an important role in the JAK-STAT signaling pathway. IL10 and STAT4 were identified as the key genes in the PPI network, and they were both positively correlated with SOD2. In the 60 pairs of clinical samples, SOD2 was highly expressed in breast cancer tissues with close correlation with axillary lymph node metastasis and the expressions of estrogen receptor and androgen receptor ( < 0.05).@*CONCLUSIONS@#The expression of SOD2 in breast cancer is significantly correlated with TNM stage and axillary lymph node metastasis. SOD2 may affect the proliferation, invasion and metastasis of breast cancer cells possibly by regulating IL10 and/or STAT4 to affect the JAK/STAT signaling pathway.

Cloning and Expression Analysis of PaAACT Gene from Phytolacca americana / 中国实验方剂学杂志

Objective: To clone the key enzyme gene involved in the biosynthesis of esculentoside A(EsA),acetoacetyl-CoA transferase(AACT) gene was cloned from Phytolacca americana for bioinformatics analysis and prokaryotic expression. Method: Total RNA was extracted from the root of P. americana, and then cDNA was synthesized through the reverse transcription. Based on analysis of the transcriptome data of P. americana, the specific primers of PaAACT gene were designed,and the cDNA sequence of PaAACT gene was amplified by polymerase chain reaction(PCR) method. Prokaryotic induction,expression and purification of the target protein were induced through the construction of the prokaryotic expression vector pET-32a-PaAACT. Result: The open reading frame (ORF) of PaAACT gene was 1 254 bp,and encoded 417 amino acid residues. Bioinformatic analysis showed that the molecular formula of PaAACT protein was C1 914H3 120N538O576S17,inferring that its molecular weight was 43.43 kDa,the theoretical isoelectric point was 8.90,and the instability index of PaAACT protein was 32.27,which was a stable protein. According to bioinformatics analysis,PaAACT protein was a member of the thiolase family and contained one conserved site and one active site of the thiolase family at the C-terminal. PaAACT protein may be located in the cytoplasm,without a signal peptide or transmembrane domain. The phylogenetic analysis indicated that PaAACT protein showed the highest homology with AACT protein from polygonaceae plants (such as Beta vulgaris). The recombinant PaAACT protein was successfully expressed in Escherichia coli BL21(DE3) strain through IPTG induction, and the purified target protein was obtained by Ni2+ affinity chromatography. Conclusion: In this study,the PaAACT gene was cloned from P. americana,which lays a foundation for further determination of enzyme activity assay of PaAACT and preparation of antibody,and provides the theoretical basis for studying its role in the biosynthesis pathway of EsA.

Identification and bioinformatic analysis of target genes of lncRNA LOC102606465 induced by ionizing radiation / 中华放射医学与防护杂志

Objective@#To screen the target genes of long non-coding RNA LOC102606465, which was previously identified to be induced by ionizing radiation, in order to examine its potential biological role.@*Methods@#The downstream differentially expressed genes (DEGs) of LOC102606465 were detected by microarray and partially verified by qRT-PCR. GO and KEGG enrichment analysis was performed, and PPI protein interaction network was constructed to screen significant modules and hub genes.@*Results@#The expression of LOC102606465 targeted by siRNA-447 and siRNA-541 was significantly lower than that of siRNA-NC (t=29.095, 13.751, P<0.01). A total of 374 common DEGs were identified(112 up-regulated/262 down-regulated) in both siRNA-447 and siRNA-541. The qRT-PCR was used to validate the expression of DEGs, which was consistent with the microarray result. In GO enrichment analysis, down-regulated DEGs were significantly enriched in " oxidoreductase activity, acting on the CH-CH group of donors, NAD or NADP as acceptor" (molecular function), " basal lamina" (cellular component), " ammonium ion metabolic process" (biological process). Up-regulated DEGs were mainly enriched in " protein phosphatase inhibitor activity" (molecular function), " SNARE complex" (cellular component), " negative regulation of fibrinolysis" (biological process). In addition, the KEGG enrichment analysis revealed that DEGs were significantly enriched in " metabolism of xenobiotics by cytochrome P450" , " dorso-ventral axis formation" , " lysosome glycerophospholipid metabolism" and " p53 signaling pathway" . Based on the STRING database, the PPI network was constructed (including 194 nodes and 268 edges), and one significant module and five key hub genes ACTRT3, CDKN1A, DPYD, TMP4, and PRKACB were identified.@*Conclusions@#LOC102606465 could be a potential biomarker for the regulation of ionizing radiation sensitivity, and the down-regulation of LOC102606465 plays an important role in the response to radiation, which would be an important target for regulating radiation sensitivity.

Bioinformatics analysis of COL1A1 regulated by miR-129-5p as a potential therapeutic target for gastric cancer / 南方医科大学学报

OBJECTIVE@#To explore the pathogenesis of gastric cancer through a bioinformatic approach to provide evidence for the prevention and treatment of gastric cancer.@*METHODS@#The differentially expressed genes (DEGs) in gastric cancer and normal gastric mucosa in GSE79973 dataset were analyzed using GEO2R online tool. GO analysis and KEGG pathway enrichment analysis of the DEGs in DAVID database were performed. The protein interaction network was constructed using STRING database, and the key genes (Hub genes) were screened and their functional modules were analyzed using Cytoscape software. The GEPIA database was used to validate the Hub genes, and the Target Scan database was used to predict the microRNAs that regulate the target genes; OncomiR was used to analyze the expressions of the microRNAs in gastric cancer tissues and their relationship with the survival outcomes of the patients.@*RESULTS@#A total of 181 DEGs were identified in gastric cancer, and 10 hub genes were screened by the protein- protein interaction network. Functional analysis showed that these DEGs were involved mainly in protein digestion and absorption, PI3K-Akt signaling pathway, ECM-receptor interaction and platelet activation signal pathway. GEPIA database validation showed that COL1A1 was highly expressed in gastric cancer tissues and was associated with a poor prognosis of patients with gastric cancer. MiR-129-5p was found to bind to the 3'UTR of COL1A1 mRNA, and compared with that in normal tissues, miR-129-5p expression was obviously down-regulated in gastric cancer tissues, and was correlated with the prognosis of the patients.@*CONCLUSIONS@#COL1A1 under regulation by MiR-129-5p is a potential therapeutic target for gastric cancer.

Cloning, space-time expression and bioinformatics analysis of ACS3 gene from different species of Lonicera macranthoides / 中草药

Objective To clone ACS3 gene from wild type and ‘Xianglei’ cultivar of Lonicera macranthoides, respectively, followed by bioinformatics analysis and detection of spatio-temporal expression pattern. Methods Unigene sequence which is highly homologous with ACS protein from the transcriptome database of L. macranthoides was screened, the primers were designed based on it to amplify the full length of the Unigene by qRT-PCR and RACE techniques; Bioinformatics tools were used to analyze and identify the physicochemical property, conserved domain and gene homology of ACS3 proteins; Finally, qRT-PCR technique was used to detect the gene expression patterns of different species of L. macranthoides. Results Lm-ACS3 (GenBank: MH724196) and Lm-XL-ACS3 (GenBank: MH724197) were isolated from wild type and ‘Xianglei’ cultivar of L. macranthoides, respectively. The length of open reading frame (ORF) were all 1 452 bp, encoding 483 amino acids, containing the conserved Aminotran_1_2 structural domain, which were highly similar to the ACC synthase of other plants; And qRT-PCR results showsed that the expression quantity of ACS3 gene in wild L. macranthoides changed significantly at different blossoming stages, the overall trend was upward from flowering stage 3, while the expression difference between the flowering stages was relatively small in “Xianglei” cultivar. Conclusion Lm-ACS3 and Lm-XL-ACS3 gene were separately obtained from L. macranthoides and L. macranthoides ‘Xianglei’ cultivar, the expression patterns of ACS3 in this two varieties were different; It’s speculated that ACS3 gene might be a possible functional gene that causing different phenotypes of two strains of L. macranthoides, it provides theoretical basis for further verifying the biological function of ACS3 gene in regulating flower’s bud duration and phenotype.

Expression profile analysis of long noncoding RNAs and mRNAs in the human colon cancer cells regulated by VTCN1 / 上海交通大学学报（医学版）

Objective • To investigate the effect of V-set domain containing T cell activation inhibitor 1 (VTCN1) on long noncoding RNAs (lncRNAs) and mRNAs expression in colon cancer cells. Methods • VTCN1 was overexpressed by lentivirus plasmid in colon cancer cell line SW1116. RNA was extracted and sequenced. The differentially expressed lncRNAs and mRNAs were compared with the negative control group. The accuracy of RNA sequencing was verified by real-time quantitative PCR (qRT-PCR) using three differentially expressed lncRNAs and two mRNAs. BLAST2GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze and predict the functions of these differentially expressed lncRNAs and mRNAs. The online platform GEPIA18 (Gene Expression Profiling Interactive Analysis) was used to analyze the correlation between differentially expressed lncRNAs and survival of patients with colorectal cancer. Results • A total of 167 differential genes, i.e., 39 lncRNAs and 128 mRNAs, were identified by RNA sequencing in VTCN1 overexpressed SW1116 cells. The results of qRT-PCR were consistent with those of RNA sequencing. Bioinformatics analysis showed that these different genes regulated by VTCN1 may be involved in endoplasmic reticulum protein processing and RNA monitoring signaling pathways. In addition, three lncRNAs (DNA JC9-AS1, HCG27, and RP11-339B21.13), which were significantly up-regulated in colorectal cancer cells overexpressing VTCN1, were also independent predictors of overall survival of colorectal cancer patients. Conclusion • In colon cancer cells, VTCN1 regulates several downstream lncRNAs and mRNAs, which may be involved in endoplasmic reticulum protein processing and mRNA monitoring signaling pathways.

Cloning of BpTRX gene and promoter and expression pattern analysis under H2S treatment in Betula platyphylla / 中草药

Objective To study the full-length, promoter sequences and its coding structure and properties of thioredoxin (Trx) gene of Betula platyphylla (BpTRX), and reveal the expression pattern of BpTRX under H2S treatment. Methods The BpTRX gene was cloned by PCR, and the promoter region sequences of BpTRX was obtained by using chromosome walking technique. The BpTRX gene, the promoter and its encoded protein were analyzed by bioinformatics software. The phylogenetic tree of BpTRX was constructed. The expression patterns of BpTRX gene under H2S exogenous stress were analyzed by quantitative real-time PCR. Results The full-length of BpTRX gene is 351 bp, encoding 117 amino acids. The BpTRX gene was closely related to the TRX-H protein of castor bean, soybean, alfalfa and grape. The obtained BpTRX promoter region sequences are 873 bp, which contains the essential elements of transcription and a large number of stress response and hormone response elements. Conclusion The full length and partial promoter sequences of the BpTRX gene were obtained. The response trend of BpTRX gene to H2S treatment was in the form of bimodal, which was first rises, then decreases, then rises and then declines.