| Background:Metabolic reprogramming is the energy basis for the survival of cancer cells.Targeting the active metabolic pathway of tumor can induce metabolic trauma and affect the proliferation ability and genomic stability of tumor cells,it is a hot issue in tumor prevention and treatment.Urea cycle is connected with multiple metabolic pathways such as polyamine metabolism,pyrimidine metabolism and tricarboxylic acid cycle.Many key enzymes are significantly regulated in melanoma cells,which has become an important metabolic support for the adaptive growth of tumors after oncogene mutation.Urea transporter UT-B is an important membrane channel protein for urea transmembrane transport.Previous studies found that overexpression of UT-B can significantly change the urea cycle and polyamine metabolic flow of melanoma B16 cells,suggesting that the down-regulation of UT-B gene expression is also an important component of metabolic reprogramming of melanoma cells.Transcriptome sequencing found that the gene regulation of pyrimidine metabolism,DNA replication,cell cycle and other pathways was obvious.At present,there is no research report on the relationship between UT-B and melanoma cell Dan damage and DNA replication stress.Therefore,this project focuses on the hot issues of metabolic reprogramming and genomic stability regulation,discusses the mechanism of UT-B expression regulation in inhibiting cancer through its impact on the cell cycle and DNA replication stress of melanoma cells,clarifies the role of UT-B in tumor biological function,and explores its possibility as a biomarker for melanoma diagnosis and potential therapeutic target.Objective:After transient transfection of UT-B expression plasmid to increase the expression level of UT-B,observe the changes of proliferation ability,cell cycle and DNA replication stress related genes of melanoma B16 cells,and explore whether overexpression of UT-B induces DNA replication stress of melanoma cells through cycle regulation and metabolic remodeling.Methods:1.MTT assay was used to observe the cell growth activity.2.After PI staining,the cell cycle was detected by flow cytometry;After annexin V staining,apoptosis was observed by flow cytometry;3.Transcriptome gene sequencing was used to analyze the expression changes of gene after transfection with UT-B overexpression,and GO and KEGG were used for gene enrichment analysis to find genes and signal pathways with obvious modulation;4.Quantitative Q-PCR was used to detect the m RNA level of UT-B,observe the transfection efficiency,verify the m RNA level of genes significantly changed by transcriptome sequencing,and analyze the m RNA level of genes related to cell cycle and DNA replication stress;5.DNA replication and synthesis were analyzed by fluorescence microscope after edu staining;6.The morphological changes of nuclei were detected by fluorescence microscope after DAPI staining;7.The levels DNA damage molecule H2 PA was detected by Western blot.Results:1.After transfection of UT-B expression plasmid,the m RNA level of UT-B increased nearly three times.After 48 hours,it can significantly inhibit the growth of B16 cells,block G1 phase and increase the level of apoptosis.2.Transcriptome sequencing showed that 1014 differential genes were up-regulated and 657 genes were down regulated in B16 cells with UT-B overexpression compared with the control group.After go function enrichment analysis and KEGG pathway enrichment analysis,it was found that the differentially expressed m RNA was significantly modulated by genes related to cell cycle,DNA replication,ribosome,pyrimidine synthesis and other cell processes.3.The transcriptome results were verified by qPCR.It was found that the expression of MCM5 gene involved in assembling DNA helicase and regulating the starting point of replication,and the expression of RRM1 gene of ribosome involved in d NTP de novo formation was significantly down regulated,suggesting that DNA replication was affected;The expression level of thymidylate synthase TYMS involved in pyrimidine synthesis was also down-regulated to only 1 / 4 of the control group,while the expression of key enzyme CPS1 in urea cycle was up-regulated,suggesting that the intracellular n flow may mainly enter the urea cycle and affect pyrimidine metabolism.4.WB detection found that the expression level of DNA damage marker H2 AX was significantly up-regulated,and edu staining found that DNA replication in S phase was also significantly reduced,combined with gene changes,suggesting significant DNA replication stress.5.QPCR found that after overexpression of UT-B,the expression levels of chek1,CHEK2,ATR and ccne2,which are important regulatory proteins involved in the repair of damaged DNA in DDR process,were also significantly down regulated and increased with time,suggesting that the DNA repair function of cells was damaged;DAPI staining showed that the morphology of the nucleus was also significantly different from that of the control group.The integrity of the nuclear membrane of some cells was destroyed,and there were micronucleus and binucleate cells,indicating that the cells had chromosome disaster.6.The sensitivity of B16 cells to DDP after transfection with UT-B was observed,and it was found that when combined with 25μM DDP,the therapeutic effect of 50μM DDP could be achieved,resulting in synthetic lethality.Conclusion:1.After overexpression of UT-B,DNA synthesis and replication related genes of melanoma B16 cells were down regulated,the expression of DNA damage related protein H2 AX was increased,and edu labeled DNA replication was reduced,suggesting that the cells had DNA replication stress;2.After overexpression of UT-B,melanoma B16 cells undergo G1 phase arrest,which may affect the formation of DNA replication fork;The down-regulation of pyrimidine metabolism related genes may lead to the shortage of DNA synthesis materials;3.After overexpression of UT-B,the gene expression of g2-s phase regulator y point related to damaged DNA repair in melanoma B16 cells is down regulated,the cells have mitotic disaster,and can synthesize lethal effect with DDP,which provides a new experimental basis for cancer treatment with targeted metabolic reprogramming. |
