Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary rat mesenchymal precursor (MM) cells. Regulation of cell cycle progression and apoptosis by KSHV-encoded microRNAs is required for KSHV-induced cellular transformation and tumorigenesis. To determine the roles of KSHV miRs in growth deregulation, we infected MM cells with the KSHV miR cluster deletion mutant and monitored infection by examining green fluorescence protein expression from a cassette inserted in the KSHV genome.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary rat mesenchymal precursor (MM) cells. Regulation of cell cycle progression and apoptosis by KSHV-encoded microRNAs is required for KSHV-induced cellular transformation and tumorigenesis.
Project description:Knee osteoarthritis (KOA), as a degenerative multifactorial disease, affects the quality of life and mental health of patients, and also brings a huge socioeconomic burden. Treating synovitis have shown promise as anti-inflammatory therapeutics in mitigating OA symptoms and disease progression. Here, by analysing synovial single-cell sequencing (scRNA-seq) data from KOA, we found that synovial fibroblasts (FLS) in OA synovium showed a distinct pro-inflammatory phenotype. We collected synovial tissue from patients with clinical OA as well as from healthy donors, and histological examination was consistent with findings in scRNA-seq. Inspired by recent cross-tissue fibroblast lineage studies, we identified by sequencing that healthy FLS in synovial tissues share transcriptome-level similarities with dermal fibroblasts (DFb). Subsequently, we revealed the local as well as systemic distribution of intra-articular injected DFbs by constructing/extracting two types of rat fibroblasts (luciferase DFbs as well as GFP DFbs). The results demonstrate that DFbs can be locally retained in the synovium for up to three weeks following targeted engrafting on it. And intra-articular injection does not result in DFbs migration to vital organs or the occurrence of histological changes in these organs. A rat model of KOA was constructed by anterior cruciate ligament transection (ACLT) in order to study the therapeutic effect of DFbs on KOA. After injection, the rats showed improvement in painful gait. In addition, histological as well as imaging results showed reduced synovitis and improvement in articular cartilage. Finally we verified the protective effect of DFbs on cytokine-stimulated chondrocytes in a co-culture system.
Project description:RNA alternative splicing is a fundamental cellular process implicated in cancer development. Kaposi’s sarcoma-associated herpesvirus (KSHV), the etiological agent of multiple human malignancies, including Kaposi’s sarcoma (KS), remains a significant concern, particularly in AIDS patients. A CRISPR-Cas9 screening of matched primary rat mesenchymal stem cells (MM) and KSHV-transformed MM cells (KMM) identified key splicing factors involved in KSHV-induced cellular transformation. To elucidate the mechanisms by which KSHV-driven splicing reprogramming mediates cellular transformation, we performed transcriptomic sequencing, identifying 131 differential alternative splicing transcripts, with exon skipping as the predominant event. Notably, these transcripts were enriched in vascular permeability, multiple metabolic pathways and ERK1/2 signaling cascades, which play key roles in KSHV-induced oncogenesis. Further analyses of cells infected with KSHV mutants lacking latent genes including vFLIP, vCyclin and viral miRNAs, as well as cells overexpressing LANA, revealed their involvement in alternative splicing regulation. Among the identified splicing factors, FAM50A, a component of the spliceosome complex C, was found to be crucial for KSHV-mediated transformation. FAM50A knockout resulted in distinct splicing profiles in both MM and KMM cells, and significantly inhibited KSHV-driven proliferation, cellular transformation and tumorigenesis. Mechanistically, FAM50A knockout altered SHP2 splicing, promoting an isoform with enhanced enzymatic activity that led to reduced STAT3 Y705 phosphorylation in KMM cells. These findings reveal a novel paradigm in which KSHV hijacks host splicing machinery, specifically FAM50A-mediated SHP2 splicing, to sustain STAT3 activation and drive oncogenic transformation.
Project description:In order to establish a rat embryonic stem cell transcriptome, mRNA from rESC cell line DAc8, the first male germline competent rat ESC line to be described and the first to be used to generate a knockout rat model was characterized using RNA sequencing (RNA-seq) analysis.