Investigation of cellular communication and signaling pathways in tumor microenvironment for high TP53-expressing osteosarcoma cells through single-cell RNA sequencing.

Osteosarcoma (OS) stands as the most prevalent primary bone cancer in children and adolescents, and its limited treatment options often result in unsatisfactory outcomes, particularly for metastatic cases. The tumor microenvironment (TME) has been recognized as a crucial determinant in OS progression. However, the intercellular dynamics between high TP53-expressing OS cells and neighboring cell types within the TME are yet to be thoroughly understood. In our study, we harnessed the single-cell RNA sequencing (scRNA-seq) technology in combination with the computational tool-Cellchat, aiming to elucidate the intercellular communication networks present within OS. Through meticulous quantitative inference and subsequent analysis of these networks, we succeeded in identifying significant signaling pathways connecting high TP53-expressing OS cells with proximate cell types, namely Macrophages, Monocytes, Endothelial Cells, and PVLs. This research brings forth a nuanced understanding of the intricate patterns and coordination involved in the TME's intercellular communication signals. These findings not only provide profound insights into the molecular mechanisms underpinning OS but also indicate potential therapeutic targets that could revolutionize treatment strategies.

LncRNA UCA1 accelerates osteosarcoma progression via miR-145 and Wnt/ß-catenin pathway.

Long non-coding (lnc) urothelial cancer associated 1 (UCA1) has been confirmed to participate in osteosarcoma (OS), but its specific mechanism is still under investigation. The study was designed to reveal the interaction between UCA1 and its downstream effector molecules, so as to determine whether there is any interaction of regulating physiological processes in tumor cells. Here, we studied the signaling cascade involving UCA1, miR-145, and HMGA1. The expression of UCA1 and miR-145 levels was interfered to assess their effects on physiological processes of tumor cells. The relationship between UCA1 and miR-145 as well as between HMGA1 and miR-145 was identified by the dual-luciferase reporter (DLR) assay, and the in vivo effect of UCA1 was estimated in nude mouse xenografts. As a result, a negative association was found between UCA1 and miR-145 in OS cells. Both UCA1 knockout and miR-145 over-expression inhibited malignant progression and induced apoptosis in MG-63 and U2OS cells. UCA1 knockout led to an increase in miR-145 and decreases in HMGA1, p-ß-catenin and cyclin D1. In addition, UCA1 upregulation promoted tumor growth in vitro and changed miR-145 and HMGA1 levels in vivo. Moreover, the DLR assay and RNA immunoprecipitation (RIP) showed that UCA1 was likely to regulate HMGA1 levels by sponging miR-145. Overall, the inhibition of UCA1 increases miR-145 levels and decreases HMGA1 levels, thereby exerting an anti-tumor role in OS.

Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme-linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5'- and 3'-ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function.