Project description:The isoform delta of class I Phosphoinositide 3-kinase (PI3Kδ) is expressed primarily in hematopoietic cells, and its inhibitor, idelalisib, is approved for the treatment of leukemia and lymphoma. PI3K orients the apicobasal axis and regulates lumen formation through assembly of the extracellular matrix. PI3Kδ overexpression in hepatocellular carcinoma (HCC) cells (Huh7) leads to profound changes in cell morphogenesis and is associated to the up-regulation of stem cell markers. Transcriptomic analysis performed in triplicate on mRNA of Huh7 cells transfected with PI3Kδ construct (PI3KCD) revealed down regulation of transcripts of hepatocyte differentiation genes and up regulation of transcripts that allow prediction of ICC and mesenchymal stem cells.
Project description:To study the role of Dengue virus serotype 4 NS1 in modulation of host cell transcriptome we performed gene expression profiling using data obtained from RNA- sequencing of total RNA from 3 different samples: Huh7 cells (no transfection), Huh7 cells transfected with pTracer-SV40 empty vector and Huh7 cells transfected with pTracer-SV40 encoding dengue virus serotype 4 NS1 (pDENV4 NS1).
Project description:RNA chemical modifications have been found to play important biological functions. Among which, the 5-methylcytosine (m5C) modification has been reported to participate in viral replication through affecting RNA processing, such as export, decay, translation and so on. In this study, we performed bisulfite sequencing (BS-seq) on HBV 1.1-mer-transfected huh7 cells to identify the m5C sites in HBV mRNA and their function in virus replication was verified. To investigate the mechanism by which m5C methyltransferase NSUN2 suppresses HBV replication, altered global m5C levels in host genes in HBV 1.1-mer-transfected cells were examined by BS-seq. We found that the m5C modification of genes associated with antiviral immunity changed significantly after viral infection. Our study provide new molecular insights into the mechanism of HBV-mediated IFN inhibition
Project description:Studies have shown that miR-361-3p is associated the progression of HCC cells and has potential to be used as a biomarker for cancer therapy. However, the mechanism of how miR-361-3p regulates the progression of HCC cells is not clear. To explore this mechanism, we transfected miR-361-3p mimics into Huh 7 cells , and verified the transfection efficiency with qRT-PCR. Then, we performed transcriptomic analysis using data obtained from RNA-seq of 3 pairs of these transfected cells.
Project description:The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 long-time up-regulation in hepatocytes that can be further used to decipher the mechanism of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, bulk RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. Hepatocytes from human hepatocarcinoma (Huh7 line, Cell Lines Service GmbH, Germany) were cultured in RPMI-1640 supplemented with FBS (10%, v/v), penicillin (100U/mL), and streptomycin (0.1mg/mL). Huh7 cells were seeded into 12 well plates at a density of 70.000 cells/well in complete RPMI media, without antibiotics. At 70-80% confluency, the hepatocytes were transfected using the CRISPR/(d)Cas9 activation plasmids for APOA1, PON1, or Control plasmids (CP) containing resistance genes for Blasticidin, Hygromycin B and Puromycin. 1 µg DNA plasmid/mL and 2.5µL/mL transfection reagent for each transfection condition were used according to the manufacturer’s instructions (SantaCruz Biotech., USA). After 48h, the cells’ media was changed and the cells were left to recover for another 48h. After transfection, the culture media was replaced with RPMI supplemented with 10% FBS, containing selection antibiotics, Hygromycin B (50 µg/mL), Blasticidin S HCl (2 µg/mL), and Puromycin dihydrochloride (1 µg/mL). The culture media with selection antibiotics was changed every 2 days for a period of 12 days, which was sufficient for the selection antibiotics to induce the death of the un-transfected cells. After selection, the medium was replaced with antibiotics-free RPMI and the transfected and selected Huh7 were allowed to grow until confluency. Total RNA was extracted from cells using Trizol (Invitrogen, USA) based on manufacturer’s instructions. To assess RNA purity and integrity, samples were analyzed via 1% agarose gel electrophoresis, and a NanoDrop spectrophotometer was used to confirm sample purity and quantity. Bulk long-RNA sequencing (longRNA-seq) of total RNA isolated from hepatocytes was done by Novogene, UK. The analysis included an additional RNA sample quality control, directional library preparation (rRNA removal), and long-RNA sequencing on NovaSeq X Plus Series (PE150, 12G raw data per sample).
Project description:This study investigates the transcriptomic changes following QRICH1 knockdown in Huh7 hepatocellular carcinoma cells using RNA sequencing.
Project description:To understand the roles of JMJD5 in liver cells, we performed DNA microarray analysis by using JMJD5KO Huh7 cells. We noticed that several transcriptional factors involved in differentiation to hepatocytes were down-regulated in JMJD5KO Huh7 cells compared to parent Huh7 cells..