The myostatin (MSTN) is a member of transforming growth factor-? superfamily which inhibits muscle growth. In this study, the genomic DNA sequence of MSTN gene was cloned from Takifugu rubripes (T. rubripes). Two polymorphisms of the MSTN gene were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods in 296 T. rubripes. One A748G locates in exon 2 and the other, C1197T, in intron 2. Analysis showed that the A748G mutation caused an amino acid change from Thr to Ala (Ala166Glu). These two SNPs showed a low degree of linkage disequilibrium and four haplotypes were identified. The most frequent haplotype was AC, which occurred at a frequency of 44.3%. Association analyses between these two SNPs and growth traits showed that the individuals with genotype CT and TT of the mutation C1197T had significantly higher body mass, body length and body height than those with genotype CC (P < 0.05). These results show that MSTN gene can be utilized as a candidate gene for molecular marker-assisted breeding of T. rubripes.
Melanotransferrin (MTf), a member of the transferrin families, plays an important role in immune response. But the research about MTf in sea cucumber is limited till now. In this study, the Melanotransferrin (Aj-MTf) gene was firstly cloned and characterized from the sea cucumber Apostichoupus japonicus by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The full-length cDNA of Aj-MTf is 2,840 bp in length and contains a 2,184 bp open reading frame that encodes a polypeptide of 727 amino acids. An iron-responsive element-like structure is located at the 5'-UTR of Aj-MTf cDNA. Sequence analysis shows that the Aj-MTf contains two conserved domains, and the binding-iron (III) sites, including eight amino acid residues (D81,Y109,Y215,H283,D425,Y454,Y565 and H634) and three N-linked glycosylation sites (N121V122S123,N173A174S175 and N673S674T675). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses suggested that the Aj-MTf expressions in the coelomic fluid, body cavity wall and respiratory trees were significantly changed from 4 to 24 h post lipopolysaccharide (LPS) injection. The mRNA levels of Aj-MTf in coelomic fluid was significantly up-regulated at 12 and 24 h in treatment group, and Aj-MTf shared a similar expression pattern with C-type lectin in coelomic fluid, while both genes appears to gradually increase after 4 h of LPS injection. These results indicate that the Aj-MTf plays a pivotal role in immune responses to the LPS challenge in sea cucumber, and provide new information that it is complementary to the sea cucumber immune genes and initiate new researches concerning the genetic basis of the holothurian immune response.
Single nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies in many species. High-throughput sequencing of RNA was developed primarily to analyze global gene expression, while it is an efficient way to discover SNPs from the expressed genes. In this study, we conducted transcriptome sequencing of the gill samples of Takifugu rubripes analyzed by using Illumina HiSeq 2000 platform to identify gene-associated SNPs from the transcriptome of T. rubripes gill. A total of 27,085,235 unique-mapped-reads from 55,061,524 raw data reads were generated. A total of 56,972 putative SNPs were discovered, which were located in 11,327 genes. 35,839 SNPs were transitions (Ts), 21,074 SNPs were transversions (Tv) and 88.1% of 56,972 SNPs were assigned to the 22 chromosomes. The average minor allele frequency (MAF) of the SNPs was 0.26. GO and KEGG pathway analyses were conducted to analyze the genes containing SNPs. Validation of selected SNPs revealed that 63.4% of SNPs (34/52) were true SNPs. RNA-Seq is a cost-effective way to discover gene-associated SNPs. In this study, a large number of SNPs were identified and these data will be useful resources for population genetic study, evolution analysis, resource assessment, genetic linkage analysis and genome-wide association studies. The results of our study can also offer some useful information as molecular makers to help select and cultivate T. rubripes.
MicroRNAs (miRNAs) are a class of endogenous non-coding small RNA with average length of 22 nucleotides, participating in the post-transcriptional regulation of gene expression. In this study, we report the identification and characterization of miRNAs in the tube foot of sea cucumber (Apostichopus japonicus) by next generation sequencing with Illumina HiSeq 2000 platform. Through the bioinformatic analysis, we identified 260 conserved miRNAs and six novel miRNAs from the tube foot small RNA transcriptome. Quantitative realtime PCR (qRT-PCR) was performed to characterize the specific expression in the tube foot. The results indicated that four miRNAs, including miR-29a, miR-29b, miR-2005 and miR-278-3p, were significantly up-regulated in the tube foot. The target genes of the four specifically expressed miRNAs were predicted in silico and validated by performing qRT-PCR. Gene ontology (GO) and KEGG pathway analyses with the target genes of these four miRNAs were conducted to further understand the regulatory function in the tube foot. This is the first study to profile the miRNA transcriptome of the tube foot in sea cucumber. This work will provide valuable genomic resources to understand the mechanisms of gene regulation in the tube foot, and will be useful to assist the molecular breeding in sea cucumber.
Single nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies in many species. High-throughput sequencing of RNA was developed primarily to analyze global gene expression, while it is an efficient way to discover SNPs from the expressed genes. In this study, we conducted transcriptome sequencing of the swimbladder of Takifugu rubripes using Illumina HiSeq2000 platform to identify gene-associated SNPs in the swimbladder. A total of 30,312,181 unique-mapped-reads were obtained from 44,736,850 raw reads. A total of 62,270 putative SNPs were discovered, which were located in 11,306 expressed genes and 2,246 scaffolds. The average minor allele frequency (MAF) of the SNPs was 0.26. GO and KEGG pathway analysis were conducted to analyze the genes containing SNPs. Validation of selected SNPs revealed that 54% of SNPs (26/48) were true SNPs. The results suggest that RNA-Seq is an efficient and cost-effective approach to discover gene-associated SNPs. In this study, a large number of SNPs were identified and these data will be useful resources for population genetic study, evolution analysis, resource assessment, genetic linkage analysis and genome-wide association studies.
The fish gill, as one of the mucosal barriers, plays an important role in mucosal immune response. The fish swimbladder functions for regulating buoyancy. The fish swimbladder has long been postulated as a homologous organ of the tetrapod lung, but the molecular evidence is scarce. In order to provide new information that is complementary to gill immune genes, initiate new research directions concerning the genetic basis of the gill immune response and understand the molecular function of swimbladder as well as its relationship with lungs, transcriptome analysis of the fugu Takifugu rubripes gill and swimbladder was carried out by RNA-Seq. Approximately 55,061,524 and 44,736,850 raw sequence reads from gill and swimbladder were generated, respectively. Gene ontology (GO) and KEGG pathway analysis revealed diverse biological functions and processes. Transcriptome comparison between gill and swimbladder resulted in 3,790 differentially expressed genes, of which 1,520 were up-regulated in the swimbladder while 2,270 were down-regulated. In addition, 406 up regulated isoforms and 296 down regulated isoforms were observed in swimbladder in comparison to gill. By the gene enrichment analysis, the three immune-related pathways and 32 immune-related genes in gill were identified. In swimbladder, five pathways including 43 swimbladder-enriched genes were identified. This work should set the foundation for studying immune-related genes for the mucosal immunity and provide genomic resources to study the relatedness of the fish swimbladder and mammalian lung.
The hepatitis B virus (HBV) X protein (HBx) plays a key role in the molecular pathogenesis of HBV-related hepatocellular carcinoma (HCC). However, its critical gene targets remain largely unknown. RASSF1A gene (Ras-association domain family 1A, RASSF1A), a tumor-suppressor gene, is frequently found to be hypermethylated and downregulated in HCC. In the present study, we investigated whether HBx is involved in the hypermethylation and downregulation of RASSF1A and we examined the potential regulation mechanisms. RT-PCR analysis was used to determine RASSF1A and HBx expression in 9 liver cell lines and the results showed that RASSF1A expression was relatively low in HBx-positive cells. Notably, RASSF1A was downregulated in HepG2.2.15 cells, as compared to HepG2 cells. Further analysis revealed that HBx transfection suppressed RASSF1A expression and HBx knockdown induced its expression. Enforced HBx suppressed RASSF1A and meanwhile induced DNMT1 and DNMT3B expression. In addition, RASSF1A is negatively regulated by DNMT1. ChIP analysis using an antibody against DNMT1 revealed that HBx enhanced the binding of DNMT1 to the RASSF1A promoter but the inhibition of RASSF1A by HBx is DNA methylation-independent as detected by methylation-specific PCR (MSP). Further studies using MSP and bisulfite genomic sequencing (BGS) revealed that no significant methylation changes were observed for regional methylation levels of RASSF1A in DNMT1 knockdown cells, although methylation levels of specific CpG sites at the predicted binding sites for the Sp1 and USF transcription factors were reduced. Additionally, RASSF1A was downregulated in HBV-associated HCC (HBV-HCC) as detected by RT-PCR and immunohistochemistry suggesting RASSF1A expression may be related to HBx in HCC and the clinical relevance of our observations. Collectively, our data showed that HBx suppressed RASSF1A expression via DNMT1 and offered a new mechanism of RASSF1A inactive in HCC in addition to the widely known DNA methylation, enriching the epigenetic mechanism by which HBx contributes to the pathogenesis of HBV-HCC.
Members of human aldo-keto reductase (AKR) superfamily have been reported to be involved in cancer progression, whereas the final conclusion is not generally accepted. Herein, we propose a quantitative method to measure human AKR proteins in cells using mTRAQ-based multiple reaction monitoring (MRM). AKR peptides with multiple transitions were carefully selected upon tryptic digestion of the recombinant AKR proteins, while AKR proteins were identified by SDS-PAGE fractionation coupled with LC-MS/MS. Utilizing mTRAQ triplex labeling to produce the derivative peptides, calibration curves were generated using the mixed lysate as background, and no significantly different quantification of AKRs was elicited from the two sets of calibration curves under the mixed and single lysate as background. We employed this approach to quantitatively determine the 6 AKR proteins, AKR1A1, AKR1B1, AKR1B10, AKR1C1/C2, AKR1C3, and AKR1C4, in 7 different cancer cell lines and for the first time to obtain the absolute quantities of all the AKR proteins in each cell. The cluster plot revealed that AKR1A and AKR1B were widely distributed in most cancer cells with relatively stable abundances, whereas AKR1Cs were unevenly detected among these cells with diverse dynamic abundances. The AKR quantitative distribution in different cancer cells, therefore, may assist further exploration toward how the AKR proteins are involved in tumorigenesis.
Embryogenesis is the initial step in a plants life, and the molecular changes that occur during embryonic development are largely unknown. To explore the relevant molecular events, we used the isobaric tags for relative and absolute quantification (iTRAQ) coupled with the shotgun proteomics technique (iTRAQ/Shotgun) to study the proteomic changes of rice embryos during embryogenesis. For the first time, a total of 2 165 unique proteins were identified in rice embryos, and the abundances of 867 proteins were actively changed based on the statistical evaluation of the quantitative MS/MS signals. The quantitative data were then confirmed using multiple reactions monitoring (MRM) and were also supported by our previous study based on two-dimensional gel electrophoresis (2 DE). Using the proteome at 6 days after pollination (DAP) as a reference, cluster analysis of these differential proteins throughout rice embryogenesis revealed that 25% were up-regulated and 75% were down-regulated. Gene Ontology (GO) analysis implicated that most of the up-regulated proteins were functionally categorized as stress responsive, mainly including heat shock-, lipid transfer-, and reactive oxygen species-related proteins. The stress-responsive proteins were thus postulated to play an important role during seed maturation.
A normalized cDNA library was constructed from the adductor muscle of M. yessoensis and acquired 4595 high quality expressed sequence tags (ESTs). After clustering and assembly of the ESTs, 3061 unigenes containing 654 contigs and 2407 singletons were identified. The contig length ranged from 266 bp to 2364 bp and the average length of these contigs was 544 bp. Blastx nonredundant protein database analysis showed that 1522 unigenes had significant homology to known genes (E value ? 10??). By comparing to Clusters of Orthologous Groups (COG) categories, 460 unigenes were annotated (E value ?10(-10)). Using Kyoto Encyclopedia of Genes and Genomes (KEGG), 345 of 3061 unigenes were assigned into 103 pathways (E value ? 10??). For InterProScan searches, 1237 unigenes were annotated containing 727 different types of protein domains. 941 of the 1237 unigenes were annotated for Gene Ontology (GO) classification using Uniprot2GO associations in any category (biological, cellular, and molecular). By sequences comparability and analysis of Blastx NCBI nonredundant protein database and KEGG, 66 unigenes were identified that may be involved in genetic information processing based on the known knowledge. The study provides a material basis as useful information for the genomic analysis of shellfish.
E-cadherin is a key cell adhesion molecule implicated in tumor suppression that is frequently altered in hepatocellular carcinoma (HCC), particularly in hepatitis B virus-related tumors. Here, we report that the epigenetic drugs 5-azacytidine and trichostatin A up-regulated E-cadherin expression in HCC cells. The depletion of DNMT1 restored E-cadherin expression via demethylation, whereas the depletion of DNMT3A or DNMT3B did not. Activated E-cadherin suppressed HCC cell colony formation. However, E-cadherin expression was repressed by HBx transfection due to the DNA methylation induced by the elevation of DNMT1 in the HCC cell lines. The present study indicates that E-cadherin expression is regulated by epigenetic agents in HCC cells, which suggests a schema for restoring E-cadherin by targeting its epigenetic mechanism.
Promoter hypermethylation mediated by DNA methyltransferases (DNMTs) is the main reason for epigenetic inactivation of tumor suppressor genes (TSGs). Previous studies showed that DNMT1 and DNMT3B play an important role in CpG island methylation in tumorigenesis. Little is known about the role of DNMT3A in this process, especially in hepatocellular carcinoma (HCC). In the present study, increased DNMT3A expression in 3 out of 6 HCC cell lines and 16/25 (64%) HCC tissues implied that DNMT3A is involved in hepatocellular carcinogenesis. Depletion of DNMT3A in HCC cell line SMMC-7721 inhibited cell proliferation and decreased the colony formation (about 65%). Microarray data revealed that 153 genes were upregulated in DNMT3A knockdown cells and that almost 71% (109/153) of them contain CpG islands in their 5 region. 13 of them including PTEN, a crucial tumor suppressor gene in HCC, are genes involved in cell cycle and cell proliferation. Demethylation of PTEN promoter was observed in DNMT3A-depleted cells implying that DNMT3A silenced PTEN via DNA methylation. These results provide insights into the mechanisms of DNMT3A to regulate TSGs by an epigenetic approach in HCC.
The aim of this study was to detect the expression pattern of DNA methyltransferase 3B (DNMT3B) variants in primary gastric cancer (GC) and to explore the clinical significance of DNMT3B variants in gastric carcinogenesis. Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual DNMT3B variants according to their splicing patterns. Expression levels of DNMT3B variants were assessed by quantitative real-time RT-PCR in gastric cancer tissue, normal gastric mucosae and GC cell lines. The relationship between the expression patterns of the DNMT3B variants and corresponding clinical information was analyzed by observing the expression levels of different variants in the tumors. These results demonstrate that DNMT3B overexpression is related to late phase invasion (P=0.029) and intestinal type (P=0.012) in GC. DNMT3B3 expression was higher in normal tissue, compared to tumor tissue (P=0.033). In contrast, only 18, 32 and 35% of the patient tumors overexpressed DNMT3B1, DNMT3B4 and DNMT3B5, respectively. While taking into account environmental factors (H. pylori, Epstein-Barr virus infection), H. pylori infection elevated DNMT3B1 and DNMT3B3 variants in tumors, while increasing DNMT3B4 in both tumor and non-cancerous tissues. Our findings indicated that the expression of DNMT3B3 is the major splice variant in normal gastric mucosae and may be affected by H. pylori infection. Elevated DNMT3B variants may influence the progression of gastric cancer and may possibly be a powerful indicator for the disease.
DNA-methyltransferase (DNMT)-3A plays an important role in the development of embryogenesis and the generation of aberrant methylation in carcinogenesis. The aim of this study was to investigate the role of a DNMT3A promoter genetic variant on its transcriptional activity and to evaluate the association between DNMT3A gene polymorphism and the susceptibility to gastric cancer (GC) and oesophagus carcinoma (EC) in the Chinese population.
Myostatin (MSTN) is a member of the transforming growth factor-beta superfamily which negatively regulates growth of muscle tissue. In this study, 103 cultivated Chlamys farreri individuals were screened for polymorphisms in the MSTN gene using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods. Two mutations were found: A/G at position 327 in exon 2, which caused an amino acid change from Thr to Ala (Thr305Ala), and C/T at position 289 in exon 3, which caused an amino acid change from Cys to Arg (Cys422Arg). One way ANOVA of the SNPs and growth traits showed that genotype GG of primer M5 had significantly higher body mass, soft-tissue mass, adductor muscle mass, shell length, shell height, absolute growth rate of shell height and body mass than those of genotype AG and AA (P<0.05). Genotype frequencies of genotype AA, AG and GG were 68.94%, 27.18% and 3.88%, respectively. The results present evidence that the C. farreri MSTN gene may be selected as a candidate gene for these growth traits.
In current study, the SpRunt-1 gene was screened for the polymorphisms using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods in Strongylocentrotus intermedius (S. intermedius). Three polymorphisms were found with two site mutations, G833A and C1505T mutation, and an insertion mutation (GT) between 931 nt and 932 nt. These two site mutations are synonymous mutations and the insertion mutation is frame shift mutation. One way ANOVA analysis of correlation between the single nucleotide polymorphisms (SNPs) and growth traits showed that the gonad weight of AA genotype (G at 833 nt) was significantly higher than that of BB genotype (A at 833 nt) (P = 0.029). The body weight of the CC genotype (C at 1505 nt) was significantly higher than that of DD genotype (T at 1505 nt) (P < 0.01) and the shell height of CD genotype was significantly higher than that of DD genotype (P = 0.032). These results provided the evidence that S. intermedius SpRunt-1 gene could be selected as a candidate gene for the growth traits.
Traditional ochratoxin A(OTA) competitive antigen are high toxicity, high price and difficult preparation. Non-toxic and easy prepared OTA competitive antigen substitutes were expressed by recombinant filamentous phage which have OTA mimotope displayed on its p VI surface.
DNA-methyltransferase (DNMT)-3A plays a crucial role in embryonic development and aberrant DNA methylation in carcinogenesis. Polymorphisms of the DNMT3A gene may influence its enzymatic activity and its contribution to susceptibility to cancer. This study evaluated the association of DNMT3A rs36012910 A>G with susceptibility to gastric cancer (GC) in a Chinese population. Genomic DNA was extracted from samples taken from 340 patients with GC and 251 healthy control subjects. The genotype frequency of DNMT3A rs36012910 A>G in all subjects was detected by polymerase chain reaction-restriction fragment length polymorphism and confirmed by sequencing. Stratification analyses were used to study subgroups by age and gender and to evaluate the association of rs36012910 A>G polymorphism with genetic susceptibility to GC. All patients and control individuals were successfully genotyped for the DNMT3A rs36012910 A>G polymorphism. The frequency of DNMT3A rs36012910 allele G is 3.39 % in healthy individuals and 7.78 % in GC patients, respectively. The rs36012910 AG genotype was significantly more common in the GC group than in the controls, although the rs36012910 GG genotype was only one case in GC patients. Further stratification indicated that AG+GG genotypes were associated with susceptibility to GC in males older than 60, but this polymorphism has no significant association with GC susceptibility in females. Male individuals who carried AG+GG genotypes had a 2.362-fold increased risk of GC compared to those who carried the AA genotype. The rs36012910 allele G was associated with an increased risk of GC compared to the rs36012910 allele A. This is the first report to investigate the distribution and evaluate the association of a rare SNP in DNMT3A with genetic susceptibility to GC. DNMT3A rs36012910 A>G might become a potential biomarker for use in GC prediction, although further studies in larger groups and different populations are needed for confirmation.
RAS protein activator like 1 (RASAL1) is a member of the RAS GTPase-activating protein (GAP) family, and it is an important molecule in the regulation of RAS activation. In the present study, we investigated the role of RASAL1 in gastric carcinogenesis. Decreased expression pattern of RASAL1 in gastric cancer tissues and cell lines was found in protein and RNA levels, although there was no statistically significant relationship between RASAL1 and clinicopathological features. Restored expression of RASAL1 induced by DNA methylation inhibitor 5-aza-2-deoxycytidine (5-AZA) and HDAC inhibitor trichostatin A (TSA) implied that RASAL1 expression is regulated by epigenetic mechanisms. The biological role of RASAL1 in gastric carcinogenesis was determined by in vitro tumorigenicity assays. Overexpression of RASAL1 showed suppression of cell proliferation due to cell apoptosis. Subsequently, enforced expression of RASAL1 repressed significantly the gastric cancer cell transformation ability. These findings demonstrated that decreased RASAL1 expression is a characteristic of gastric cancer and regulated by epigenetic mechanisms. RASAL1 may be a functional tumor suppressor involved in gastric cancer. This study provides novel insights into the biological role of RASAL1 in gastric carcinogenesis.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.