Project description:Set domain-containing 2 (SETD2) is the most frequently mutated gene among all the histone methyltransferases (HMTs) in Clear cell renal cell carcinoma (ccRCC). Loss of function of SETD2 is significantly associated with poor prognosis in patients with ccRCC. A better understanding of the roles of SETD2 played in ccRCC can greatly improve the prognosis and quality of life of patients with kidney cancer. Clear cell renal carcinoma cell A498 were treated with si-SETD2 and si-NC, and the exosomes were extracted.
Project description:We have reported that microRNAs are present in human, bovine, and rat milk whey. Milk whey miRNAs were resistant to acidic condition and to RNase. Thus, milk miRNAs were thought to be present packaged into membrane vesicles like exosome. However, body fluid miRNAs have been reported that there are in different forms. To clarify which miRNAs species are exist in exosome and which species are exist in another form, we used bovine raw milk and purified total RNA from exosome fraction and ultracentrifugated supernatant fraction, and analyzed by miRNA microarray.
Project description:We have reported that microRNAs are present in human, bovine, and rat milk whey. Milk whey miRNAs were resistant to acidic condition and to RNase. Thus, milk miRNAs were thought to be present packaged into membrane vesicles like exosome. However, body fluid miRNAs have been reported that there are in different forms. To clarify which miRNAs species are exist in exosome and which species are exist in another form, we used bovine raw milk and purified total RNA from exosome fraction and ultracentrifugated supernatant fraction, and analyzed by miRNA microarray.
Project description:Each condition (cellular interaction with bacteria or with bacterial supernatant) was hybridized on 6 slides (3 slides, corresponding to different interactions and RNA extractions, were swapped).
Project description:Each condition (cellular interaction with bacteria or with bacterial supernatant) was hybridized on 6 slides (3 slides, corresponding to different interactions and RNA extractions, were swapped). Keywords: parallel sample
Project description:Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation, in which the contrasting effects of pathogens and commensals on host tissues are clearly displayed. While virulent Escherichia coli cause severe, potentially life-threatening disease by breaking the inertia of the mucosal barrier and infecting the kidneys, the most common outcome of bacteriuria is an asymptomatic carrier state resembling commensalism at other mucosal sites. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease associated responses in the host. To address this question, we examined the effects of asymptomatic bacterial carriage on host gene expression. The asymptomatic strain E. coli 83972 caused reduction in Pol II phosphorylation in the nuclei of human kidney epithelial A498 cells. To specifically address if Pol II inhibition alters the response to infection, A498 cells were pretreated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). This adenosine analogue has been proposed to specifically and reversibly inhibit Pol II transcription without directly affecting other cellular functions. A498 cultered cells were infected with E. coli 83972 or DRB for 4 hours. The culture medium with DMSO was used as a background control. A498 cells were infected with E. coli 83972 or DRB for 4 h. Isolated RNA was subjected to whole genome transcriptome analysis.
Project description:Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation, in which the contrasting effects of pathogens and commensals on host tissues are clearly displayed. While virulent Escherichia coli cause severe, potentially life-threatening disease by breaking the inertia of the mucosal barrier and infecting the kidneys, the most common outcome of bacteriuria is an asymptomatic carrier state resembling commensalism at other mucosal sites. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease associated responses in the host. To address this question, we examined the effects of asymptomatic bacterial carriage on host gene expression. The asymptomatic strain E. coli 83972 caused reduction in Pol II phosphorylation in the nuclei of human kidney epithelial A498 cells. To specifically address if Pol II inhibition alters the response to infection, A498 cells were pretreated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). This adenosine analogue has been proposed to specifically and reversibly inhibit Pol II transcription without directly affecting other cellular functions. A498 cultered cells were infected with E. coli 83972 or DRB for 4 hours. The culture medium with DMSO was used as a background control.
Project description:A498 cells, established from a clear cell renal cell cancer of a 52 years old male patient, were obtained from ATCC and maintained in DMEM supplemented with 10% fetal calf serum (FCS). This cell line does not express functional VHL. A498 subclones stably transfected with HA-pVHL30 or control plasmid have been described and were maintained in 0.5 mg/ml G418.
Project description:Large fractions of genomes are expressed in most eukaryotic cells, generating a diverse repertoire of protein-coding and non-coding transcripts. With such complexity, and as the number of mRNA molecules per cell are often low, it is clear that errors anywhere in the gene expression process could have profound consequences on cellular functions. The exosome is a conserved RNA degradation machine that plays a fundamental role in monitoring the quality of gene expression in the nucleus. The current view is that the nuclear exosome constantly scrutinizes transcription and contributes to post-transcriptional turnover of faulty mRNAs. Yet, how nuclear RNA surveillance by the exosome is coordinated with transcription is still unknown. Here we show that the exosome can directly target the transcription machinery by terminating transcription events associated with paused and backtracked RNA polymerase II (RNAPII), contrary to the notion that the exosome acts exclusively on RNAs that have been released by RNAPII. Our data support a mechanism by which RNAPII backtracking provides a free RNA 3' end for the core exosome, resulting in transcription termination with the concomitant degradation of the associated transcript. These findings uncover a mechanism of co-transcriptional RNA surveillance whereby termination of transcription by the exosome prevents aberrant read-through RNAs and transcriptional interference at neighboring genes.
Project description:AS a key medium for intercellular communication, exosome plays an important role in the occurrence and development of various forms of cancers, including breast cancer. However, the RNA from breast cancer-derived exosome remains to be explored. The HR+/HER2− breast cancer is the most common subgroup of breast cancer, we collected and primary cultured breast cancer and normal breast epithelial cells from three HR+/HER2− breast cancer patients, isolated the supernatant and extracted RNA from exosomes, and then performed microarray assays.