Project description:Antrodia cinnamomea (Ac), a traditional medicine and an endemic fungus in Taiwan, has been used in cancer research. Recent research has revealed decreased cell proliferation after treatment of Ac on tumor. In this study, we profiled the 2 hours and 4 hours genome-wide miRNA and mRNA transcriptome by next-generation sequencing techniques to report the early apoptotic effect on Ac fruiting body extract treated human hepatocarcinoma cells, SK-HEP-1, instead of prolonged treatment. Results showed that miRNAs were globally downregulated during the first 2-4 hours in, and solely in, AcFBE-treated SK cells. The inhibition of miRNAs imposed no discrimination against any particular miRNA species, but oncogenic miR-21, miR-191 and two oncogenic clusters miR-17-92 and miR-106b-25 were among the most significantly inhibited miRNAs. In addition to miRNA expression, mRNA transcriptome data indicated the association of apoptosis mechanism with AcFBE treatment. Western blotting indicated a decrease in key proteins Drosha and Dicer required for miRNA biogenesis, and an increase of XRN2 involved in miRNA degradation. Our results suggest that miRNAs appeared to be the prime targets of Ac in disrupting multiple miRNA regulatory pathways and global disruption of miRNA transcriptome resulting in activation of extrinsic and intrinsic (mitochondrial) pathways. Human liver SK-Hep-1 cells with or without Antrodia cinnamomea treatment at 2 hours and 4 hours were sequenced by SOLiD 3 and SOLiD 5500xl to obtain miRNA profiles; mRNA profiles also were profiled by SOLID 3. Mouse liver BNL CL.2 cells with or without Antrodia cinnamomea treatment at 2 hours and 4 hours were sequenced by SOLiD 3 to obtain miRNA profiles.
Project description:Combined genome and transcriptome sequencing to identify plant cell wall degrading enzymes in the thermophilic fungus Malbranchea cinnamomea
Project description:Antrodia cinnamomea (Ac), a traditional medicine and an endemic fungus in Taiwan, has been used in cancer research. Recent research has revealed decreased cell proliferation after treatment of Ac on tumor. In this study, we profiled the 2 hours and 4 hours genome-wide miRNA and mRNA transcriptome by next-generation sequencing techniques to report the early apoptotic effect on Ac fruiting body extract treated human hepatocarcinoma cells, SK-HEP-1, instead of prolonged treatment. Results showed that miRNAs were globally downregulated during the first 2-4 hours in, and solely in, AcFBE-treated SK cells. The inhibition of miRNAs imposed no discrimination against any particular miRNA species, but oncogenic miR-21, miR-191 and two oncogenic clusters miR-17-92 and miR-106b-25 were among the most significantly inhibited miRNAs. In addition to miRNA expression, mRNA transcriptome data indicated the association of apoptosis mechanism with AcFBE treatment. Western blotting indicated a decrease in key proteins Drosha and Dicer required for miRNA biogenesis, and an increase of XRN2 involved in miRNA degradation. Our results suggest that miRNAs appeared to be the prime targets of Ac in disrupting multiple miRNA regulatory pathways and global disruption of miRNA transcriptome resulting in activation of extrinsic and intrinsic (mitochondrial) pathways.
Project description:The thermophilic fungus Malbranchea cinnamomea belongs to the order of Onygenales and is a promising source of thermostable, industrially relevant biocatalysts, as it can grow at temperatures of more than 50°C and is able to utilise many different types of plant biomass. Enzymes from M. cinnamomea that have been characterised so far include an α-amylase, an α-glucosidase, xylanases and an alkaline β-1,3-1,4-glucanase (lichenase), all of which have been reported to have temperature optima between 50°C and 80°C. With this study, we complement the knowledge of the enzymatic repertoire of M. cinnamomea with a transcriptomic analysis of strain FCH 10.5 to provide a more comprehensive view of its lignocellulolytic enzyme system. Genes differentially expressed during growth on two different polymeric substrates, beechwood xylan and wheat bran, point to differences in the fungal response to the deconstruction of a hardwood hemicellulose (beechwood xylan) and a cereal hemicellulose (wheat bran). The data presented here will form the basis for a systematic exploration of the full potential of this fungus as a source of thermostable enzymes. We sequenced the genome of M. cinnamomea FCH 10.5, which was isolated from the compost of a waste treatment plant in Hanoi, Vietnam (PMC5604768, https://www.ncbi.nlm.nih.gov/nuccore/FQSS02000000). For RNAseq, the fungus was grown on three different carbon sources (glucose, wheat bran, beechwood xylan) at 50°C. Mycelium was harvested after 4h and 48h and RNA was extracted. For RNAseq analysis, the RNA of 4h and 48h samples was mixed 1:1, to get information about both early- and late-response genes during growth on the different carbon sources. Two independent duplicate experiments were done for each substrate. Total RNA was extracted using TRIzol (Invitrogen) and chloroform, and further purified with the RNeasy Plant RNA Kit with on-column DNAse digestion (QIAGEN). The quality of the purified RNA was verified by agarose gel electrophoresis, Nanodrop (Thermo Scientific) and Qubit (Life Technologies). The NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used to process the samples according to the manufacturer’s instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads and used to synthesise cDNA. The cDNA was ligated with sequencing adapters and PCR amplified. The quality and yield after sample preparation were determined with the Fragment Analyzer (Advanced Analytical). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Standard Illumina primers for Illumina cBot and HiSeq 2500, and the HiSeq control software HCS v2.2.58 were used according to manufacturer’s protocols for clustering and DNA sequencing with a concentration of 16.0 pM. The Illumina data analysis pipelines RTA v1.18.64 and Bcl2fastq v2.17 were used for image analysis, base calling, and quality check. Sequencing was performed on an Illumina HiSeq 2500 sequencer. The assembled genome from the DNA sequencing was used as a reference to map the reads using the packages Tophat (v2.0.14. Linux_x86_64) and Bowtie (v2-2.1.0) with a default mismatch rate of 2%. The frequency with which a read was mapped on a transcript was determined based on the mapped locations from the alignment. To normalise for transcript length, fpkm (fragments per kilobase of transcript per million mapped reads) were calculated. For differential expression analysis, the read counts were loaded into the DESeq package v 1.10.1. Genes were considered differentially expressed if they showed a log2 fold change ≥ 1 and the adjusted p-value was < 0.05.