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The gene expression signatures for Huh7 cell lines with HCC-associated HBV variants in small S proteins transfection

ABSTRACT: To further investigating the novel NGS-defined HCC-associated SNVs in small S region involve hepatocarcinogenesis pathways, the plasmid pEGFP-N1-HBVS-genoB was used as the template for mutagenesis to create mutations T216C, A273G, and double mutant T216C/A273G at small surface (S) region, with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid pEGFP-N1-HBVS-genoC was used as the template for mutagenesis to create mutations A293G, C446G, A456G, A293G/C446G, C446G/A456G, A293G/A456G, and triple mutant A293G/C446G/A456G at small S region. Wild-type or mutant envelope expression vectors were transfected into Huh7 cells by using lipofectamine 3000 (Thermo Fisher Scientific) according to the manual of the manufacturer. All mutants were confirmed by DNA sequencing. all the novel NGS-defined HCC-associated SNVs in small S region could involve hepatocarcinogenesis pathways with inducing cell apoptosis, regulating cell death and survival, affecting DNA replication, recombination, and repair.

ORGANISM(S): Homo sapiens

PROVIDER: GSE87804 | GEO | 2019/10/03

REPOSITORIES: GEO

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Project description:Polymorphisms in pre-miRNAs may affect its expression, then having effect on its target mRNAs and be associated with cancer susceptibility. In the current study, we evaluated the association of a 3-base pair (3-bp) indel polymorphism (rs57408770) in pre-miR-3131 with hepatocellular carcinoma (HCC) susceptibility in a Chinese population. Logistic regression analysis showed that the insertion allele of rs57408770 was significantly associated with an increased risk for HCC occurrence in both case-control studies. To further investigated the molecular mechanism underlying the correlation between rs57408770 and the risk of HCC, overexpression of pre-miR-3131 in HCC cell line was performed. Human HCC cell lines Sk-Hep-1 was obtained from Shanghai Cell Bank of Chinese Academy of Sciences on 9 December 2013.The fragment of 366bp including the insert allele of rs57408770 in pre-miR-3131 was directly synthesized and cloned into BamHI and EcoRI sites of pEGFP-N1 yielding the wild-type vector (pEGFP-N1-miR3131-WT). The mutant-type vector (pEGFP-N1-miR3131-MT) including the delete allele of rs57408770 was generated using QuikChange Lightening Site-Directed Mutagenesis Kit. The resulting constructs were verified by direct sequencing. For overexpression experiments, cells were cultured in 6-well plates at a density of 200 000 cells per well the day before transfection. 2.5Î¼g plasmids of empty vector pEGFP-N1, pEGFP-N1-miR3131-WT or pEGFP-N1-miR3131-MT were transfected into the growing cells (about 80%-90% confluent) using Lipofectamine2000. After 24 hours of the transfection, the cells were collected for subsequent experiments.And human genome-wide gene expression profile assay was used to screen the targets of miR-3131. For overexpression experiments, cells were cultured in 6-well plates at a density of 200 000 cells per well the day before transfection. 2.5Î¼g plasmids of empty vector pEGFP-N1, pEGFP-N1-miR3131-WT or pEGFP-N1-miR3131-MT were transfected into the growing cells (about 80%-90% confluent) using Lipofectamine2000 (Invitrogen). After 24 hours of the transfection, the cells were collected for subsequent experiments. There are 3 samples in this experiment.

Project description:Abstract：DNA transfection is widely used in biomedical research. However, transfection efficiency is often the bottleneck of research and gene therapy practices. To explore the mechanism regulating transgene expression, we investigated the possible involvement of the cGAS-STING signaling pathway, which induces type-I interferons in response to cytosolic DNA. We found deletion of cGAS enhances the expression of transfected genes. This enhancement is inversely correlated with the expression of type I interferons and interferon stimulated genes (ISGs). We provide evidence that the interferon responses can suppress expression of transfected genes at the mRNA level. Mechanistically, DNA transfection activates cGAS-STING pathway and induces the expression of the OAS family proteins, leading to the activation of RNaseL and degradation of mRNA derived from transgenes. As a result, administration of chemical inhibitors that block cGAS-mediated signaling cascades improves the expression levels of exogenous genes in multiple cell lines and in primary cells, including T cells. These data suggest that targeting the cGAS-STING pathway can improve transgene expression and this strategy may be applied to gene therapy. Goal：Analysis of mouse fibroblast cell line L929 transfected with pEGFP-N1 plasmid in the presence of DMSO or TBK1 inhibiter (BX795) . Results provide insight into molecular mechanisms underlying a inhibitor effect of BX795 on DNA-induced cGAS signaling which improves expression of transfected genes. Methods： L929 cells were pretreated with TBK1 inhibitor BX795 or mock treated with DMSO for 1 hour, then transfected with pEGFP-N1 plasmid. There are four groups in this experiment. S1, L929 cells mock-treated; S2, L929 cells transfected with pEGFP-N1 plasmid; S3, L929 treated with 0.5 uM BX795; S4, L929 treated with 0.5 uM BX795 and transfected with pEGFP-N1 plasmid. Each group has three replicates. There are total 12 samples in this experiment. Six hours later, total RNA of 12 samples was extracted using Trizol reagent from cells according to the manufacturer’s instruction. cDNA library construction and sequencing were performed by BGI Genomics (BGI-SHENZHEN, China) using BGISEQ-500 platform. High-quality reads were aligned to the Mus_musculus reference genome (Mus musculus GRCm38.p5) using Bowtie2 (version:2.2.5, parameters: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200). The expression levels for each of the genes were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM) (version:1.2.12, default parameters). For the identification of differentially expressed genes (DEGs), we used PossionDis, which is developed by BGI Genomics, implementing the passion distribution method. We analyzed up-expression genes in S2 other than S1, S3 and S4.

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The gene expression signatures for Huh7 cell lines with HCC-associated HBV variants in small S proteins transfection

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