DCTPP1-MYC Positive Feedback Loop Promotes Liver Cancer Proliferation

Background: Liver cancer remains a global health challenge,with 4.6 million new cases of liver cancer each year in China and a median survival time of only 6months for advanced liver cancer.Targeting enzymes related to pyrimidine metabolism is one of the strategies to slow down the progression of liver cancer.Studies have shown that d CTP pyrophosphatase 1(DCTPP1)is involved in the progression of multiple malignancies,but its mechanism of action and therapeutic implications in liver cancer are unclear.Objective: To explore the clinical significance of DCTPP1 in liver cancer,to investigate the role and regulatory mechanism of DCTPP1 in the proliferation process of liver cancer cells,and to provide new ideas for finding potential therapeutic targets for liver cancer.Methods: Immunohistochemistry and protein immunoblotting were used to detect the expression of DCTPP1 in liver cancer tissue microarrays and postoperative liver cancer tissues,combined with Kaplan-Meier plotter database to analyze the effect of high and low DCTPP1 expression on the prognosis of liver cancer patients;RNA interference technology was used to construct DCTPP1-silenced liver cancer cell lines by CCK-8 assay,clone formation assay,Transwell assays,flow cytometry and animal studies to detect the effects of DCTPP1 on proliferation and drug responsiveness of liver cancer cells;protein immunoblotting to detect the effects of DCTPP1 on nucleotide metabolism-related enzymes(PPAT,ADSL,ADA,PDE4 D,CTPS1,TYMS,TK1,NME1)and β-catenin/MYC;GEO dataset GSE14520 to analyze the correlation of DCTPP1 expression with MYC and validate it at the cellular and tissue level by protein immunoblotting;online databases PROMO,CHEA,h TFtarget,Cistrome DB and JASPAR to predict transcription factors of DCTPP1 and potential binding sites of transcription factors by chromatin immunoprecipitation reverse transcription quantitative real-time polymerase chain reaction(Ch IP-q PCR).The expression of pyrimidine metabolism-related enzymes(DCTPP1,CTPS1,TYMS,TK1,NME1)after MYC silencing was detected by RT-q PCR,and the expression of DCTPP1 after MYC silencing was detected by cellular immunofluorescence assay.High performance liquid chromatography combined with mass spectrometry was used to detect the energy metabolite profile of DCTPP1 in Hep G2 cell line after silencing.Results: The expression level of DCTPP1 in liver cancer tissues was significantly higher than that in paraneoplastic tissues(P < 0.05),and the overall survival of liver cancer patients with high expression of DCTPP1 was shorter with a risk ratio of 1.51(P < 0.05);liver cancer cells with DCTPP1 silencing had reduced proliferative capacity in vitro and vivo,clonogenic ability,migration ability and G2/M block in cell cycle,as well as a greater response to the nucleotide analogs capecitabine,No significant differences were observed in purine metabolism-related enzymes(PPAT,ADSL,ADA,PDE4D)and pyrimidine metabolism-related enzymes(CTPS1,TYMS,TK1,NME1)in DCTPP1-silenced liver cancer cells,and the differences were statistically significant(P < 0.05);downregulation of β-catenin and MYC was detected in both DCTPP1-silenced liver cancer cells;m RNA expression levels of DCTPP1 and MYC were positively correlated in 488 samples from the GSE14520 dataset,with a correlation coefficient of 0.376(P < 0.001);the databases PROMO,CHEA and h TFtarget predicted that the DCTPP1 Cistrome DB,JASPAR database predictions and chromatin immunoprecipitation-real-time quantitative fluorescence polymerase chain reaction experiments showed that MYC binds to the promoter region of DCTPP1 by binding "AAACACGTGGCC" sequence in the DCTPP1 promoter region,while DCTPP1 silencing impairs the transcriptional effect of MYC on DCTPP1;MYC silencing causes downregulation of the expression levels of pyrimidine metabolism-related enzymes(DCTPP1,CTPS1,TYMS,TK1,NME1);DCTPP1-silenced Hep G2 intracellular rearrangement of energy metabolites,mainly manifested by down-regulation of argininosuccinic acid,arginine and accumulation of metabolites such as malate,lysine,fumarate,α-ketoglutarate and cis-aconitate.Conclusions: DCTPP1 was highly expressed in liver cancer tissues,and the overall survival rate of liver cancer patients with high expression of DCTPP1 was shorter;DCTPP1 silencing inhibited liver cancer cell proliferation and migration ability,and enhanced drug sensitivity of liver cancer cells to nucleotide analogues;DCTPP1 regulated MYC expression through the Wnt/β-catenin pathway,while MYC in the promoter region of DCTPP1 DCTPP1 is a potential target for antitumor drug development.