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MRNA expression profiling in MDA-MB-231 and HepG2 cells with 2 µM of ACP52C peptide treatment for 0, 12, 24, and 48 hours

ABSTRACT: The goal of this study is to compare mRNA expression profiles of the ACP52C-treated cells in time dependent manner. All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For the drug treatment, MDA-MB-231 and HepG2 cells were incubated in the presence of 2 μM ACP52C for 0, 12, 24, and 48 hours. Total cellular RNAs were extracted using Qiazol reagent. The RNA quality was examined by spectrophotometry, agarose gel electrophoresis (calculating the 18S and 28S rRNA ratio) and an Agilent Technologies 2100 Bioanalyzer (ensuring a RIN value greater than 7). The library was prepared using the RNA seq3’ mRNA-Seq Library Prep Kit FWD. The constructed libraries were subjected to 150-bp two-end sequencing using an Illumina NextSeq 500 sequencer at Insilicogen. All procedures were performed following the manufacturer’s instructions. Tophat 2 and cufflinks were used for reads mapping and expected read counts, respectively. DEGs were analyzed using an edgeR program and functional annotation of genes were achieved using a DAVID program.

ORGANISM(S): Homo sapiens

PROVIDER: GSE175393 | GEO | 2021/05/25

REPOSITORIES: GEO

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Project description:Purpose: Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Therefore, we aimed to investigate the mechanism of IFN-I regulation upon West Nile virus infection using TRIM6 knockout (TRIM6-KO) human airway epithelial cells (A549). Methods: RNAs were extracted from wild-type (WT) and TRIM6KO A549 cells infected with WNV or mock-treated. RNA quality was assessed by visualization of 18S and 28S RNA bands using an Agilent BioAnalyzer 2100 (Agilent Technologies, CA); the electropherograms were used to calculate the 28S/18S ratio and the RNA Integrity Number. Poly-A+ RNA was enriched from total RNA (1 μg) using oligo dT-attached magnetic beads. First and second strand synthesis, adapter ligation, and amplification of the library were performed using the Illumina TruSeq RNA Sample Preparation kit as recommended by the manufacturer (Illumina, Inc). Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantification of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1500 using protocols defined by the manufacturer. The alignment of NGS sequence reads was performed using the Spliced Transcript Alignment to a Reference (STAR) program, version 2.5.1b, using default parameters. We used the human hg38 assembly as a reference with the UCSC gene annotation file. The –quantMode GeneCounts option of STAR provided read counts per gene, which were input into the DESeq2 (version 1.12.1) differential expression analysis program to determine expression levels and differentially expressed genes. Processed data are presented as reads per kilobase transcript per million (RPKM). Results: Using next generation sequencing, we identified VAMP8 as a factor involved in this TRIM6-mediated antiviral response. In support, VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFNβ treatment. Conclusions: Our results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system

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MRNA expression profiling in MDA-MB-231 and HepG2 cells with 2 µM of ACP52C peptide treatment for 0, 12, 24, and 48 hours

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