Cellular glucuronidation

Abstract: Increasing evidence has indicated that circular (circ)RNAs participate in carcinogenesis; however, the specific regulatory mechanisms underlying the effects of circRNAs, microRNAs (miRNAs/miRs) and genes on the development of clear cell renal cell carcinoma (CCRCC) remain unclear. In the present study, RNA microarray data from CCRCC tissues and control samples were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas, in order to identify significantly dysregulated circRNAs, miRNAs and genes. The Cancer-Specific circRNA Database was used to explore the interactions between miRNAs and circRNAs, whereas TargetScan and miRDB were employed to predict the mRNA targets of miRNAs. Functional enrichment and prognostic analyses were conducted in R. The results revealed that 324 circRNAs were downregulated, whereas 218 circRNAs were upregulated in cancer. In addition, a circRNA-miRNA-mRNA interaction network was constructed. Gene Ontology analysis of the upregulated genes revealed that these genes were enriched in biological processes, including ‘flavonoid metabolic process’, ‘cellular glucuronidation’ and ‘T cell activation’. The downregulated genes were mainly enriched in biological processes, such as ‘nephron development’, ‘kidney development’ and ‘renal system development’. The hub genes, including membrane palmitoylated protein 7, aldehyde dehydrogenase 6 family member A1, transcription factor AP-2α, collagen type IV α 4 chain, nuclear receptor subfamily 3 group C member 2, plasminogen, Holliday junction recognition protein, claudin 10, kinesin family member 18B and thyroid hormone receptor β, and the hub miRNAs, including miR-21-3p, miR-155-3p, miR-144-3p, miR-142-5p, miR-875-3p, miR-885-3p, miR-3941, miR-224-3p, miR-584-3p and miR-138-1-3p, were significantly associated with CCRCC survival. In conclusion, these results suggested that the significantly dysregulated circRNAs, miRNAs and genes identified in this study may be considered potential biomarkers of the carcinogenesis of CCRCC and the survival of patients with this disease.

Abstract: Purpose Although human umbilical cord mesenchymal stem cells (hucMSCs) can contribute to the growth of tumors, including pancreatic ductal adenocarcinoma (PDAC), however, little is known about the exact mechanisms by which the exosomes secreted from hucMSCs (hucMSCs-exo) have an oncogenic effect on the physiopathology of PDAC. The effects of hucMSCs on tumor development are attributed to hucMSCs-exo, which deliver unique proteins and miRNAs to cancer cells. Methods HucMSCs and exosomes were isolated and confirmed via transmission electron microscopy, nanoparticle tracking analysis and western blot. The nude mice were inoculated subcutaneously on both flanks with human pancreatic cancer Panc-1 cells (1 × 106), and hucMSCs-exo were directly administered via intratumoral injection once a day for three days each week. Cell proliferation assays were performed using a Cell Counting Kit-8 assay and the cell invasion assay was performed using Transwell assay. The miRNA data were predicted and analyzed by miRanda software. The analysis of the target genes of the miRNAs was proformed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Results Firstly, we observed that hucMSCs-exo promoted Panc-1 and BxPC3 cell growth by increasing proliferation and migration in vitro. Secondly, in a xenograft tumor model, hucMSCs-exo increased the growth of Panc-1 cells. Thirdly, high-throughput sequencing of hucMSCs-exo showed that hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, hsa-miR-21-5p and hsa-miR-92a-3p were highly expressed. For the five identified miRNAs, 1308 target genes were predicted by miRanda software. From the GO and KEGG analyses of the target genes of the identified miRNAs, it was found that the main GO function was the regulation of cellular glucuronidation, and the main KEGG metabolic pathway involved the metabolism of ascorbic acid and aldehyde acid. These processes are related to the occurrence and development of pancreatic cancer. Finally, we observed that miR-100-5p promoted Panc-1 and BxPC3 cell growth in vitro and in vivo. Conclusion Here, by utilizing exosomes secreted from hucMSCs, we systematically investigated the effects of hucMSCs-exo on PDAC growth in vitro and in vivo for the first time. Building on these results, we provided new insights into the role of hucMSCs-exo in the PDAC growth and revealed the attractive communication between hucMSCs and PDAC cells that occurs through MSCs-exosomes-miRNAs.

Abstract: Objective This study aims to identify several RNA transcripts associated with the prognosis of kidney renal clear cell carcinoma (KIRC). Methods The differentially expressed mRNAs, lncRNAs, and miRNAs (DEmRNAs, DElncRNAs, and DEmiRNAs) between KIRC cases and controls were screened based on an RNA-seq dataset from The Cancer Genome Atlas (TCGA) database. Subsequently, miRcode, miRDB, and TargetScan database were used to predict interactions between lncRNAs, miRNAs and target mRNAs. Then, a ceRNA network was built using miRNAs-mRNAs and lncRNAs-miRNAs pairs. Functional analysis of mRNAs in ceRNA was performed. Finally, the survival analysis of RNA transcripts in ceRNA network and correlation analysis for key RNA regulators were carried out. Results There were 1527 DElncRNAs, 54 DEmiRNAs, and 2321 DEmRNAs. A ceRNA network was constructed among 81 lncRNAs, 9 miRNAs, and 197 mRNAs. Functional analysis showed that numerous mRNAs were significantly associated with regulation of cellular glucuronidation. In addition, 35 lncRNAs, 84 mRNAs and two miRNAs were significantly corelated to the survival of patients with KIRC (P < 0.05). Among them, miRNA-21 and miRNA-155 were negatively related to three lncRNAs (LINC00472, SLC25A5.AS1, and TCL6). Seven mRNA targets of miRNA-21 (FASLG, FGF1, TGFBI, ALX1, SLC30A10, ADCY2, and ABAT) and 12 mRNAs targets of miRNA-155 (STXBP5L, SCG2, SPI1, C12orf40, TYRP1, CTHRC1, TDO2, PTPRQ, TRPM8, ERMP1, CD36, and ST9SIA4) also acted as prognostic biomarkers for KIRC patients. Conclusion We screened numerous novel prognosis-related RNA markers for KIRC patients by a ceRNA network analysis, providing deeper understandings of prognostic values of RNA transcripts for KIRC.

Abstract: Active efflux transport of glucuronides out of cells is a critical process in elimination of drugs and food-derived compounds. Wushanicaritin, a natural polyphenol from Epimedium species, has shown many biological activities. However, the transporters responsible for excretion of wushanicaritin glucuronides still remain undefined. Herein, chemical inhibitors (Ko143, MK571, dipyridamole and leukotriene C4) and single stable knocked-down efflux transporters (BCRP, MRP1, MRP3 and MRP4) were used to determine the contributions of efflux transporters to glucuronide efflux and cellular glucuronidation in UGT1A1-overexpressing HeLa cells (HeLa1A1). Knock-down of transporters was performed by stable transfection of short hairpin RNA (shRNA) using lentiviral vectors. The HeLa1A1 cell lysate catalyzed wushanicaritin glucuronidation, generating wushanicaritin-3-O-glucuronide and wushanicaritin-7-O-glucuronide. Ko143 (a dual inhibitor of BCRP, 5-20 μM) caused a marked decrease in excretion rate (maximal 53.4%) and increase of intracellular glucuronides (maximal 86.0%), while MK-571 (an inhibitor of MRPs, 5-20 μM) resulted in a significant reduction in excretion rate (maximal 64.6%) and rise of intracellular glucuronides (maximal 98.0%). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on glucuronide excretion. Furthermore, shRNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of wushanicaritin glucuronides (maximal 33.8% for BCRP; 25.9% for MRP1; 26.7% for MRP3; 39.3% for MRP4). Transporter silencing also led to substantial decreases in efflux clearance (maximal 61.5% for BCRP; 48.7% for MRP1; 35.1% for MRP3; 63.1% for MRP4). In conclusion, chemical inhibition and gene silencing results suggested that BCRP, MRP1, MRP3 and MRP4 were significant contributors to excretion of wushanicaritin glucuronides.