Project description:To gain insights into the mechanisms of Eupolyphaga sinensis Walker (ESW) and taspine derivate on inhibition to HCC, we have employed whole genome microarray expression profiling as a discovery platform to identify different genes between ESW-treated sample and control. At the same time, the differences were investigated between taspine derivate and the same control. SMMC-7721 cells were cultivated in the absence or presence of 0.1mg/mL ESWE and Taspine derivate of 2.5x10-5mol/L for 48 h, followed by the Agilent Whole Human Genome Oligo Microarray. The PLG, PKCM-NM-2 and IL3RA genes confirmation of Microarray analysis was confirmed by Real-time PCR. SMMC-7721 cells were cultivated in the absence or presence of 0.1mg/mL Eupolyphaga sinensis Walker extract (ESWE) and Taspine derivate of 2.5x10-5mol/L for 48 h, followed by the Agilent Whole Human Genome Oligo Microarray
Project description:To gain insights into the mechanisms of Eupolyphaga sinensis Walker (ESW) and taspine derivate on inhibition to HCC, we have employed whole genome microarray expression profiling as a discovery platform to identify different genes between ESW-treated sample and control. At the same time, the differences were investigated between taspine derivate and the same control. SMMC-7721 cells were cultivated in the absence or presence of 0.1mg/mL ESWE and Taspine derivate of 2.5x10-5mol/L for 48 h, followed by the Agilent Whole Human Genome Oligo Microarray. The PLG, PKCβ and IL3RA genes confirmation of Microarray analysis was confirmed by Real-time PCR.
Project description:NLRC5 is a member of the NLR family of proteins. The observation that NLRC5 is found in the nucleus prompted us to perform a gene array to identify putative target genes of NLRC5. We generated Jurkat T cell lines that stably express either the wild-type or mutant forms of NLRC5 harboring mutations in the nucleotide binding domain (NBD): Walker A (deficient in nucleotide binding), Walker B (deficient in nucleotide hydrolysis), and the combined Walker AB, carrying both mutations. Site-directed mutagenesis was used to create the NLRC5 NBD mutants: Walker A (K234A), Walker B (E311Q), and Walker AB (both mutations).
Project description:NLRC5 is a member of the NLR family of proteins. The observation that NLRC5 is found in the nucleus prompted us to perform a gene array to identify putative target genes of NLRC5. We generated Jurkat T cell lines that stably express either the wild-type or mutant forms of NLRC5 harboring mutations in the nucleotide binding domain (NBD): Walker A (deficient in nucleotide binding), Walker B (deficient in nucleotide hydrolysis), and the combined Walker AB, carrying both mutations. Site-directed mutagenesis was used to create the NLRC5 NBD mutants: Walker A (K234A), Walker B (E311Q), and Walker AB (both mutations). We compared the expression profiles of Jurkat T cells expressing GFP, or the GFP-tagged wild-type, Walker A, Walker B, or Walker AB forms of NLRC5. GFP and the Walker A and Walker AB forms of NLRC5 were considered functionally inactive, while the wild-type and Walker B forms of NLRC5 were considered functionally active. These groups were used as biological replicates to assess the effect of NLRC5 activity on gene expression.
Project description:Clonorchis sinensis is a zoonotic parasite causing clonorchiasis associated with human diseases such as biliary calculi, cholecystitis, liver cirrhosis, and is classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs) are non-coding, regulating small RNA molecules essential for the complex life cycle of parasites and involved in parasitic infections. To identify and characterize miRNAs expressed in adult C. sinensis residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing, bioinformatic predictions with stem-loop real-time PCR analysis. Here we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belong to 284 families. There is strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil is the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and the end of conserved miRNAs. There is no significant “seed region” at the first and ninth positions commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis are still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There are two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping a stringent conserved seed region with high active innovation in other place of miRNA mainly in the middle and the end, which are perfect for the parasite to perform its complex life style and for host changes. The present study represents the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as the miRNA studies of other related species such as Opisthorchis felineus and O. viverrini of human and animal health significance.
Project description:Clonorchis sinensis is a zoonotic parasite causing clonorchiasis associated with human diseases such as biliary calculi, cholecystitis, liver cirrhosis, and is classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs) are non-coding, regulating small RNA molecules essential for the complex life cycle of parasites and involved in parasitic infections. To identify and characterize miRNAs expressed in adult C. sinensis residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing, bioinformatic predictions with stem-loop real-time PCR analysis. Here we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belong to 284 families. There is strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil is the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and the end of conserved miRNAs. There is no significant M-bM-^@M-^\seed regionM-bM-^@M-^] at the first and ninth positions commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis are still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There are two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping a stringent conserved seed region with high active innovation in other place of miRNA mainly in the middle and the end, which are perfect for the parasite to perform its complex life style and for host changes. The present study represents the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as the miRNA studies of other related species such as Opisthorchis felineus and O. viverrini of human and animal health significance. Analysis of miRNA profile in parasite of C. sinensis