Construction of endogenous RNA regulatory network for colorectal cancer based on bioinformatics / 中南大学学报(医学版)

OBJECTIVES@#The high morbidity and mortality of colorectal cancer (CRC) have posed great threats to human health. Circular RNA (circRNA) and microRNA (miRNA), acting as competing endogenous RNAs (ceRNAs), have been found to play vital roles in carcinogenesis. This paper aims to construct a circRNA/miRNA/mRNA regulatory network so as to explore the molecular mechanism of CRC.@*METHODS@#The sequencing data of circRNA from CRC were obtained from Gene Expression Omnibus (GEO). The differential circRNA was screened and its structure was identified by Cancer-specific CircRNA Database (CSCD); the sequencing data of miRNA and messenger RNA (mRNAs) were downloaded from The Cancer Genome Atlas (TCGA) database and the differentially expressed genes were screened; the corresponding miRNA of differential circRNAs were predicted by CircInteractome database; DIANA, Miranda, PicTar, and TargetScan databases were used to predict the target genes of different miRNAs; the target genes from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched by R language; String database combined with Cytoscape 3.7.2 software was used to construct protein-protein interaction (PPI) network and hub genes were screened; the expressions of mRNAs in the Top10 hub genes were verified in CRC. The network diagrams of circRNAs/miRNAs/mRNAs and circRNAs/miRNAs/Top10 hub mRNAs were constructed by Cytoscape3.7.2. Real-time PCR was used to examine the expression levels of hsa_circRNA_0065173, hsa-mir-450b, hsa-mir-582, adenylate cyclase 5 (ADCY5), muscarinic acetylcholine receptor M2 (CHRM2), cannabinoid receptor 1 (CNR1), and lysophosphatidic acid receptor 1 (LPAR1) in the CRC tissues and the adjacent normal tissues.@*RESULTS@#A total of 14 differential circRNAs were identified, and 8 were found in CSCD; 34 miRNAs targeted by circRNAs were obtained. The PPI network was constructed, and the Top10 hub genes were identified, which were CHRM2, melanin concentrating hormone receptor 2 (MCHR2), G-protein gamma 3 subunit (GNG3), neuropeptide Y receptor Y1 (NPY1R), CNR1, LPAR1, ADCY5, adenylate cyclase 2 (ADCY2), gamma 7 (GNG7) and chemokine 12 (CXCL12), respectively. The expressions of Top 10 hub genes were also verified, and the results showed that the Top 10 hub genes were down-regulated in CRC; the constructed network diagram showed that hsa_circRNA_0065173 may regulate ADCY5, CHRM2, and Hsa-mir-450b by modulating hsa-mir-450b and hsa-mir-582. CNR1 and LPAR1 genes might serve as potentially relevant targets for the treatment of CRC. Real-time PCR results showed that the expression levels of hsa_circRNA_0065173, ADCY5, CHRM2, CNR1 and LPAR1 in the CRC tissues were significantly reduced compared with the adjacent normal tissues (all P<0.05); the expression levels of hsa-mir-450b and hsa-miR-582 were significantly increased (both P<0.05).@*CONCLUSIONS@#In this study, a potential circRNAs/miRNAs/mRNAs network is successfully constructed, which provides a new insight for CRC development mechanism through ceRNA mediated by circRNAs.

Screening and preliminaryfunctional analysis of differentially expressed lncRNAs and mRNAs in vascular endothelial cells treated by tritiated water / 中华放射医学与防护杂志

Objective:To investigate the expression changes of lncRNAs and mRNAs in human umbilical vein endothelial cells(HUVEC) treated by tritiated water.Methods:HUVEC cells were divided into two groups, the control group cultured in DMEM medium, and the tritiated water exposure group cultured in a medium containing tritiated water with a final concentraion of 3.7×10 3 Bq/ml. After culture for 48 h, cells were collected for RNA extract.The differentially expressed lncRNAs and mRNAs were screened by high-through put chip technology and then analyzed. Results:Compared with the control group, 1 717 lncRNAs were significantly up-regulated and 3 994 lncRNAs significantly down-regulated, and 4 562 mRNAs were significantly up-regulated and 1 433 mRNAs down-regulated. Through co-expression analysis of differential mRNAs and lncRNAs, some key genes including SQSTM1, CXCL8, ITPR1, GADD45A, NF-kB1 and VDAC1 were obtained.Conclusions:Tritiated water exposure can induce multiple changes of mRNAs and lncRNAs in vascular endothelial cells, which may lead to toxic effects through signaling pathways including some key genes such as SQSTM1, CXCL8, and ITPR1.

Effects of tumor biobank storage on polysome stability

Background: Human biological material has become an important resource for biomedical research. Tumor Biobanks are facilities that collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. mRNA-translation has been demonstrated to modulate protein levels and is considered a fundamental post-transcriptional mechanism of gene expression regulation. Thus, determining translation efficiencies of individual mRNAs in human tumors may add another layer of information that contributes to the understanding of tumorigenic pathways. To analyze the RNAs actively engaged in translation, RNAs associated with ribosomes (polysomes) are isolated, identified and compared to total RNA. However, the application of this technique in human tumors depends on the stability of the polysomal structure under Biobank storage conditions that usually consists of ultra-low temperature. Since the effect of freezing on the stability of the polysomal structure in stored tumor samples is not known, it is essential to evaluate this factor in the frozen samples, validating the use of biobank samples in studies of translational efficiency. Methods: Xenograft tumors were divided in two parts, half was subject to immediate processing, and half was frozen for posterior analysis. Both parts were subject to polysomal separation, RNA extraction and identification through RNAseq. Results: It was possible to successfully extract and identify total and polysomal RNA from both fresh and frozen tumoral tissue. The quantification of the polysome profile indicated no difference in the translational efficiency estimated in fresh versus frozen tissue. Gene expression data from the fresh versus frozen tissues were compared and the correlation between the polysome associated fresh x frozen (R = 0,89) and total fresh x frozen (0,90) mRNAs was calculated. No difference was identified between the two conditions. Conclusions: We demonstrated that tissue freezing does not affect the polysomal structure, consequently validating the viability of the use of biobank stored tissue for polysome associated RNA analysis (AU)

The Application of mRNA-based gene transfer in mesenchymal stem cell-mediated migration and cytotox-icity of DBTRG glioma cells / 实用医学杂志

Objective To investigate the mRNA-based anticancer gene transfer in MSCs-mediated cytotox-icity of glioma cells. Methods TRAIL mRNA and PTEN mRNA were synthesized in vitro. Immunoblotting assay was used to detect the expression of TRAIL and PTEN in the transfected MSCs.Transwell co-culture was perform to detect the migration ability of MSCs after gene transfection. The bioluminescence,live/dead staining and real time cell analyzer were used to analyze the viability of DBTRG cells. Results Compared with non-transfected MSCs, an enhanced migration rate was observed in MSCs with two kind of mRNA transfection.TRAIL-and PTEN-mRNA-induced cytotoxicity in DBTRG glioma cell was correlated with the ratio of the conditioned medium of the transfect-ed MSCs. A synergistic action was observed in TRAIL and PTEN in the transwell co-culture model. Conclusion The present study reveals the effect of synthesized mRNA-based gene transfer on mesenchymal stem cell-mediated cytotoxicity of glioma cells(DBTRG).

Candidate genes and pathogenesis investigation for sepsis-related acute respiratory distress syndrome based on gene expression profile

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years, the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment. This study aimed to predict some genes or pathways associated with sepsis-related ARDS based on a public microarray dataset and to further explore the molecular mechanism of ARDS. RESULTS: A total of 122 up-regulated DEGs and 91 down-regulated differentially expressed genes (DEGs) were obtained. The up- and down-regulated DEGs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. Protein-protein interaction network of ARDS analysis revealed 20 hub genes including cyclin B1 (CCNB1), cyclin B2 (CCNB2) and topoisomerase II alpha (TOP2A). A total of seven transcription factors including forkhead box protein M1 (FOXM1) and 30 target genes were revealed in the transcription factor-target gene regulation network. Furthermore, co-cited genes including CCNB2-CCNB1 were revealed in literature mining for the relations ARDS related genes. CONCLUSIONS: Pathways like mitotic cell cycle were closed related with the development of ARDS. Genes including CCNB1, CCNB2 and TOP2A, as well as transcription factors like FOXM1 might be used as the novel gene therapy targets for sepsis related ARDS

A retrotransposição de mRNAs como fator de variabilidade genética no genoma humano e de outros primatas / The retrotransposition of mRNAs as a factor of genetic variability in the human and other primates genomes

Duplicação genica é uma das principais forças levando a evolução dos genomas eucarioto. O impacto de duplicações gênicas/genômicas vem sendo investigado a muito tempo em humanos e outros primatas. Um segundo mecanismo de duplicação gênica, a retrotransposição baseada em RNA maduros, vem sendo menos estudada devido ao seu potencial menor de gerar cópias funcionais. No entanto, recentemente, publicações descreveram retrocópias funcionais em humanos, roedores e mosca de fruta. Nesta tese, para investigar sobre retrocópias causando variabilidade genética no genoma de primatas, nós desenvolvemos a implementamos os métodos para detectar estas inserções. Utilizando nove genomas e transcriptomas publicamente disponíveis (sete primatas e dois roedores) nós confirmamos um número similar, porém, com origem independente, de retrocópias em primatas e roedores. Nós também encontramos um enriquecimento de retrocópias no genoma de Platyrrhini, possivelmente explicado pela expansão de L1PA7 e L1P3 nestes genomas. Posteriormente, nós analisamos a ortologia de retrocópias no genoma de primatas e encontramos 127 eventos específicos à linhagem humana. Nós também exploramos dados do projeto 1000 Genomes para detectar retrocópias polimórficas (retroCNVs germinativos) e encontramos 17 eventos, presentes no genoma referência humano, mas ausentes em mais de um indivíduo. Similarmente, nós investigamos novas retroduplicações de mRNAs no genoma humano, detectando 21 eventos ausentes do genoma referência. Finalmente, investigamos a existência de retroCNVs somáticos e descrevemos sete possíveis retrocópias somáticas. Apesar de sua possível insignificância, nós encontramos que algumas retrocópias compartilhadas entre todos os primatas, espécie específicas, e polimórficas podem ser expressas per se ou como transcritos quiméricos com genes hospedeiros. Sobretudo, nós encontramos que retrocópias são um fator importante da variabilidade genética inter-espécie, intra-espécie e intra-indivíduo e podem estar influenciando a evolução de mamíferos ao criar reservatórios de duplicações potencialmente funcionais

Gene duplication is a major driving force of evolution in eukaryotic genome. The impact of gene/genomic duplication has long been investigated in human and other primates. A second mechanism of gene duplication, retrotransposition, which is based on mature RNA, has been traditionally less studied due to their lower potential to generate functional copies. Recently, however, publications described functional retrocopies in humans, murines and drosophila. Here, to gain insights of the genetic variability arising from retrocopies on primate genomes, we developed and implemented the methods to detect these insertions. Using nine publicly available reference genomes and transcriptomes (seven primates and two rodents) we described a similar number independently arisen retrocopies in primates and rodents. We also found an enrichment of retrocopies in Platyrhinni genomes, putatively explained by the expansion of L1PA7 and L1P3 in these genomes. Next, we evaluated the orthology of retrocopies in primate genomes and found 127 events specific to human lineage. We also explored 1000 Genomes Project data to detect polymorphic events (germinative retroCNVs) on human populations and found 17 events, present on the reference genome, absent in more than one individual. Conversely, we also investigated new insertions of mRNA retroduplications in the human genome, detecting 21 events absent to the human reference genome. Finally, we evaluated the existence of somatic retroCNVs and described seven putative somatic retrocopies. Despite their putative insignificance, we found that some of these shared, specie-specific and polymorphic events may be expressed per se and as chimeric transcripts within host genes. Taken together, we found that retrocopies are a great factor of genetic variation interspecie, intraspecie e intraindividual and may be affecting mammal evolution by creating reservoirs of potentially functional duplications

Effect of immobilization stress on the expression of TH, BDH and CRH gene in rat brain

PURPOSE: Catecholamines are the neuro-transmitters in the sympathetic nervous system (SNS) and are activated by stress stimulus. Tyrosine hydroxylase (TH) and Dopamine-beta-Hydroxylase (DBH) are very important enzymes in the catecholamine synthesis. Corticotropin releasing hormone (CRH) is released in the process of reacting to stresses. The aim of this study is to find out what effects immobilization stresses have on the expression of TH, BDH and CRH mRNA in a rat's brains. METHODS: We compare expression levels in rat's brains of TH, DBH and CRH mRNA induced by immobilization stresses between the test group and controled group. The expression levels of TH, DBH and CRH mRNA are measured by RT-PCR and the Western Blotting Analysis (WBA). RESULTS: In brains and adrenal glands of the immobilization stress group, the expression levels of TH and DBH mRNAs are significantly two to three times higher (P<0.01), and CRH mRNAs are approximately one and a half times higher (P<0.05) than those of controlled group. CONCLUSION: This study suggest that the expression levels of TH, DBH and CRH mRNAs are activated by stress stimulus in a rat's brains and adrenal glands.

Anti-cancer effect of Cyclosporin A on oral squamous cell carcinoma cell line

Squamous cell carcinoma is the most prevalent oral cancer, which is characterized by its low survival rate, high malignancy, mortality with facial defects, and poor prognosis. Exact cause and pathogenesis of the squamous cell carcinoma is still unknown. Various routes including smoking, radiation, and viral infections predispose its genesis, and recent studies revealed that genetic defects which fail to prevent cancer proliferation play a role. Generally, a cancer develops from the decreased rate of apoptosis which is an active and voluntary cell death, and from the altered cell cycles. Anticancer effect can be obtained by recovering the apoptotic process, and by suppressing the cell cycles. Among the apoptosis related factors, bcl-2, caspase-9, and VDAC (voltage-dependent anion channel)are produced in mitochondria of the cell. Cyclosporin-A is known to induce apoptosis through its activation with VDAC. This study was to reveal the anticancer effect of Cyclosporin A to the oral squamous cell carcinoma. The inverted microscope was used to find alterations in the tissue, and sensitivity test to the anticancer cells was performed with MTT (Tetrazolium-based colorimetric) assay. Following cell line culture of primary and metastastic oral squamous cell carcinoma, electrophoresis was performed with extracted total RNA. Finally, semi-quantitative study was carried out through RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The results of this study are as follows: 1. The inverted microscopic observation revealed a poorly defined cytoplasm at 2000ng.3000ng/ml, indistinct nucleus, and apoptosis. 2. The Growth of cancer cells was decreased at 1000ng/ml of cyclosporin-A. No cancer cell growth was observed at over 2000ng/ml concentration of cyclosporin-A, and at one week, growth of cancer cells was ceased. 3. The MTT assays were decreased as cyclosporin-A concentration was increased. This means that the activation of succinyl dehydrogenase in mitochondria was decreased following administration of cyclosporin A. 4. A result of RT-PCR showed that amount of mRNA of VDAC-2 was decreased half times at a cyclosporine-A concentration of 2000ng/ml. In bcl-2, amount of mRNA was significantly decreased 1/5 times at 2000ng/ml. caspase-9, however, showed slight increase compared to the control group. From the results obtained in this study, administration of cyclosporin-A to the cell lines of oral squamous cell carcinoma induced alterations in morphology and growth of the cells as its concentration increased. Since apoptosis related factors such as VDAS-2, bcl-2, and caspase-9 also showed distinct alterations on their mRNAs, further research on cyclosporin A as an anti-cancer agent will be feasible.