RESUMO
Toll-like receptor 2 (TLR2) is an important sensor for innate immune cells, including neutrophils, for the recognition of pathogen infection. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is a TLR2 ligand. LTA-induced TLR2 signaling pathways are well established in neutrophils. However, experimental studies regarding transcriptional regulation and the molecular mechanisms in primary human neutrophils are limited due to their short lifespan. The promyelocytic leukemia cell line, HL-60, can differentiate into a neutrophil-like phenotype following treatment with dimethyl sulfoxide. The aim of the present study was to investigate whether differentiated HL-60 (dHL-60) cells induced a similar gene expression profile upon LTA treatment as that previously determined for primary human neutrophils. After 4 or 24 h of Staphylococcus aureus LTA treatment, undifferentiated HL-60 (uHL-60) and dHL-60 cells were collected for RNA sequencing. The results demonstrated that hundreds of identical differentially expressed genes (DEGs) were observed in 1 and 10 µg/ml LTA-treated dHL-60 cells following 4 and 24 h of incubation, while almost no DEGs between LTA-treated HL-60 and dHL-60 cells were observed. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG), it was noted that the pathways of shared DEGs between the 1 and 10 µg/ml LTA-treated dHL-60 cells at both time points were significantly enriched in immune and inflammatory response-related pathways, such as cellular response to tumor necrosis factor, interleukin-1, interferon γ, neutrophil chemotaxis, the NF-κB signaling pathway and the Toll-like receptor signaling pathway. In addition, when comparing the effect of 1 and 10 µg/ml LTA treatment on dHL60 cells, it was found that all enriched GO and KEGG pathways were associated with the TLR signaling pathways of neutrophils. The results of the present study provided important information for the implementation of mRNA profiling in LTA-treated dHL-60 cells and may indicate the feasibility of using dHL-60 cells as a research model for TLR2 signaling in human neutrophils.
RESUMO
OBJECTIVE: Ribonuclease P RNA component H1 (RPPH1) is a long non-coding RNA (lncRNA) associated with cancer progression. Higher RPPH1 expression in breast and cervical cancer samples than that in normal tissues were observed through the lncRNASNP2 database; therefore, silencing RPPH1 expression might be a potential strategy for cancer treatments, even though RPPH1 is also an RNA subunit of ribonuclease P involved in processing transfer RNA (tRNA) precursors and the effect of RPPH1 knockdown is not yet fully understood. METHODS: Differentially expressed genes (DEGs) were identified through RNA sequencing in each shRNA-transfected RPPH1 knockdown MDA-MB-231, RPPH1 knockdown HeLa cell, and respective control cells, then the gene ontology enrichment analysis was performed by IPA and MetaCore database according to these DEGs, with further in vitro experiments validating the effect of RPPH1 silencing in MDA-MB-231 and HeLa cells. RESULTS: Hundreds of down-regulated DEGs were identified in RPPH1 knockdown MDA-MB-231 and HeLa cells while bioinformatics analysis revealed that these genes were involved in pathways related to immune response and cancerogenesis. Compared to mock- and vector-transfected cells, the production of mature tRNAs, cell proliferation and migration capacity were inhibited in RPPH1-silenced HeLa and MDA-MB-231 cells. Additionally, RPPH1 knockdown promoted G1 cell cycle arrest mainly through the down-regulation of cyclin D1, although glycolytic pathways were only affected in RPPH1 knockdown HeLa cells but not MDA-MB-231 cells. CONCLUSION: This study demonstrated that knockdown RPPH1 affected tRNA production, cell proliferation and metabolism. Our findings might provide insight into the role of RPPH1 in tumor development.