MiR-183-5p-PNPT1 Axis Enhances Cisplatin-induced Apoptosis in Bladder Cancer Cells

Objective:It has been reported that intrinsic apoptosis is associated with the progression of bladder cancer(BC).Recent evidence suggests that polyribonucleotide nucleotidyltransferase 1(PNPT1)is a pivotal mediator involved in RNA decay and cell apoptosis.However,the regulation and roles of PNPT1 in bladder cancer remain largely unclear.Methods:The upstream miRNA regulators were predicted by in silico analysis.The expression levels of PNPT1 were evaluated by real-time PCR,Western blotting,and immunohistochemistry(IHC),while miR-183-5p levels were evaluated by qPCR in BC cell lines and tissues.In vitro and in vivo assays were performed to investigate the function of miR-183-5p and PNPT1 in apoptotic RNA decay and the tumorigenic capability of bladder cancer cells.Results:PNPT1 expression was decreased in BC tissues and cell lines.Overexpression of PNPT1 significantly promoted cisplatin-induced intrinsic apoptosis of BC cells,whereas depletion of PNPT1 potently alleviated these effects.Moreover,oncogenic miR183-5p directly targeted the 3′UTR of PNPT1 and reversed the tumor suppressive role of PNPT1.Intriguingly,miR-183-5p modulated not only PNPT1 but also Bcl2 modifying factor(BMF)to inhibit the mitochondrial outer membrane permeabilization(MOMP)in BC cells.Conclusion:Our results provide new insight into the mechanisms underlying intrinsic apoptosis in BC,suggesting that the miR-183-5p-PNPT1 regulatory axis regulates the apoptosis of BC cells and might represent a potential therapeutic avenue for the treatment of BC.

Identification of Residue Involved in Nucleotidyltransferase Activity of LinA from Staphylococci by Site-directed Mutagenesis

The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg^(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.