| 1.Research backgroundLiver cancer is the fourth leading cause of cancer-related death worldwide,and the 5year overall survival rate for untreated liver cancer is less than 10%.Despite a variety of treatment options,such as liver transplantation,systemic chemotherapy,locoregional therapy and radioembolization or targeted therapy with sorafenib,lenvatinib or other multikinase inhibitors,most patients have a poor prognosis and overall profitability remains low.Elucidating the key factors of hepatocarcinogenesis will help to understand the pathogenesis of liver cancer and provide a basis for the development of new and effective targeted therapies.Metabolic reprogramming is an important hallmark of cancer,and cancer cells alter metabolic pathways to meet their needs for rapid proliferation,invasion and metastasis.Abnormally elevated levels of glucose,glutamine,fatty acids,and amino acids are major drivers of tumor growth,and these substances also serve as substrates for the hexosamine biosynthesis pathway(HBP).HBP ultimately produces uridine diphosphate-Nacetylglucosamine(UDP-GlcNAc),serving as the basis for the biosynthesis of glycoproteins or other glycoconjugates together with other charged nucleotide sugars.These nutrient-driven post-translational modifications,highly dyregulated in cancer,regulate target protein function in various cancer-related processes.A series of studies have shown that HBP promotes the metabolism,growth,survival and proliferation of cancer cells,but the role and molecular mechanism of HBP in liver cancer remain still unclear.Therefore,this thesis focuses on the relationship between HBP and the development of liver cancer and its mechanism of action.2.Research aimsTo clarify the role of HBP in the development of liver cancer,to explore the molecular mechanism of HBP’s role in liver cancer,and to provide a theoretical basis for new and effective molecular targets for liver cancer treatment.3.Materials and Methods(1)A mouse liver cancer model was constructed to study the mechanism of the development of liver cancer.We specifically overexpressed c-MYC and knocked out Trp53 in the liver of C57BL/6 mice by hydrodynamic tail vein injection of plasmid vector to construct a liver cancer model.After the model was successfully constructed,the liver tumors and adjacent tissues were sequenced by RNA-seq technology,and the differential gene expression between cancer and adjacent cancer was analyzed at the transcriptome level.RT-qPCR technology was used to detect the differences in the mRNA levels of key enzymes in HBP between cancer and adjacent non-cancer tissues,and Western Blot was used to detect the differences in protein levels of key enzymes in HBP.(2)The HBP pathway was inhibited by knocking out the HBP rate-limiting enzyme Gfptl to explore the role of HBP in the development of liver cancer induced by overexpression of c-MYC and deletion of Trp53.Firstly,the knockout vector px330-sg-Gfptl targeting HBP rate-limiting enzyme Gfptl was constructed by molecular cloning technology.Then we divided 6-week-old C57BL/6 mice into 2 groups to induce liver cancer by tail vein injection.The control group was injected with plasmids pT3-EF1a-c-MYC-acGFP,pCMV(CAT)T7-SB 100,px330-sg-Trp53,px330-sg-Renilla(control plasmid),while experimental group was injected with plasmids pT3-EF1a-c-MYC-acGFP,pCMV(CAT)T7SB100,px330-sg-Trp53,px330-sg-Gfpt1(knockout plasmid).Liver tissues were harvested 30 days after plasmid injection,and the differences in tumor number,size,and liver-to-body weight ratio were compared between the control and experimental groups.The survival time of mice in the control group and the experimental group was recorded,and the survival analysis was performed to compare the effect of knocking out Gfptl on the occurrence and development of liver tumor and the survival of mice.(3)In order to explore the mechanism by which HBP affects the occurrence and development of liver cancer,the expression levels of two key enzymes downstream of HBP were detected at the mRNA and protein levels by qPCR and Western Blot,respectively.In addition to the key enzymes of the two downstream pathways,??.Firstly,the vector px330sg-Ogt targeting O-linked glycosylation pathway key enzyme N-acetylglucosamine transferase(Ogt)and the vector px330-sg-Nans targeting the key enzyme sialic acid-9phosphate synthase(Nans)in the sialic acid synthesis pathway were constructed by molecular cloning technology.Then we divided 6-week-old C57BL/6 mice into 3 groups to induce liver cancer by tail vein injection,and the control group was injected with plasmids pT3-EF1a-cMYC-acGFP,pCMV(CAT)T7-SB100,px330-sg-Trp53,px330-sg-Renilla(control plasmid);2 experimental groups were injected with plasmids pT3-EF1a-c-M YC-acGFP,pCMV(CAT)T7-SB100,px330-sg-Trp53,px330-sg-Ogt(Ogt gene knockout)and pT3-EF1ac-MYC-acGFP,pCMV(CAT)T7-SB 100,px330-sg-Trp53,px330-sg-Nans(Nans gene knockout),respectively.Liver tissues were harvested 30 days after plasmid injection,and the differences in tumor number,size,and liver-to-body weight ratio were compared between the control and experimental groups.The survival time of mice in the control group and the experimental group was recorded,and statistical analysis of survival was performed.4.Research result(1)After the liver cancer model was successfully induced,RNA-seq was performed on the cancerous and non-cancerous tissues.Fructose-6-phosphate(F6P)metabolic processes,fructose and mannose metabolism,glutamine metabolism,and glutamine family amino acid metabolism processes were found to be enriched in tumors by GOBP and KEGG analysis.The expression of key enzymes in HBP was up-regulated in tumors,and the same results were obtained by qPCR and Western Blot,suggesting that HBP is activated in c-MYC;sgTrp53induced liver cancer.(2)The size and number of tumors in Gfptl-knockout mice were significantly smaller than those in control mice,and the 50-day survival rate was significantly higher than that in control mice.These results suggested that HBP plays an important role in the initiation and development of liver cancer induced by c-MYC;sgTrp53.(3)The expression levels of key enzymes in the two downstream branches of HBP were detected by qPCR and Western Blot,and both Ogt and Nans were significantly up-regulated in tumors at the mRNA level.At the protein level,only the expression of Nans was significantly up-regulated,while Ogt did not change significantly,and the downstream end product of Ogt,O-GlcNAc,did not change significantly.Ogt and Nans were knocked out respectively when inducing liver cancer.Ogt knockout had no obvious effect on the survival time of mice,while Nans knockout significantly prolonged the survival time.The tumor size and number of Nans knockout mice were significantly smaller than those of control mice at 30 days after induction,which was comparable to the effect of Gfptl knockout.These results suggested that the pro-carcinogenic effect of HBP on the initiation and development of liver cancer is achieved through the sialic acid synthesis pathway.5.ConclusionThrough the above research,we have concluded that HBP is activated in c-MYC;sgTrp53-induced liver cancer;HBP plays an important role in the initiation and development of c-MYC;sgTrp53-induced liver cancer,and inhibition of HBP can inhibit the progression of liver cancer;the promoting effect of HBP on the occurrence and development of liver cancer is achieved through the sialic acid synthesis pathway,and inhibition of the sialic acid synthesis pathway can also inhibit the initiation and development of liver cancer.This study provides experimental data for further understanding the mechanism of primary liver cancer,and provides a theoretical basis for novel and effective molecular therepies for liver cancer treatment. |
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