| Background and Purpose:The international situation of gastric cancer(GC)is still severe.In 2020,the incidence of GC ranked the second in all cancer incidence and the mortality ranked the third in all cancer mortality in China.Based on this situation,it is still urgent to explore new specific biomarkers for diagnosis and treatment.Recently,the exploration of the relationship between Long non-coding RNAs(Lnc RNAs)and GC has become a hot topic in academic research.Lnc RNAs play an important role in the occurrence,development and prognosis of tumors,and its role in GC cannot be ignored.Lnc RNAs are expected to become biomolecules for early diagnosis and effective treatment of GC.Our previous study found that Long non-coding RNA Cancer susceptibility candidate 19(Lnc RNA CASC19)was related to the occurrence and development of GC.However,the deep relationship between it and GC needs to be further studied.Exploring the mechanism of CASC19 in GC is expected to find new molecular markers or therapeutic targets for GC diagnosis and treatment.Research methods:(1)The expression levels of CASC19 in different human GC cell lines and normal human gastric mucosal epithelial cell lines were detected by real-time fluorescence quantitative PCR.CASC19 expression was knocked down or overexpressed in GC cells,and then the following experiments were performed: the proliferation of GC cells was detected by colony formation assay;The cell migration ability was detected by scratch test.Transwell assay was used to detect the changes of cell migration and invasion.Glucose and lactate detection kits were used to determine the changes of glucose and lactate metabolism in GC cells.Western blot was used to detect the changes in the levels of aerobic glycolysis-related proteins in GC cells.(2)GC cells from control group and CASC19 knockdown group were injected subcutaneously into nude mice to construct a transplanted tumor model,and the length and short diameter of tumors were measured every other day after tumor formation.At the end of the experiment,the tumors were removed,and the expression levels of aerobic glycolysis related proteins and NPM1 protein in the transplanted tumor tissues were detected by immunohistochemistry.(3)The expression of NPM1 m RNA in GC tissues and adjacent tissues was detected by q RT-PCR.The online database was used to analyze the effect of NPM1 m RNA on prognosis.The expression level of NPM1 in GC and adjacent tissues was detected by tissue microarray,and the relationship between NPM1 expression and clinicopathological factors and prognosis of GC was analyzed.(4)Metabolomics analysis was used to analyze the effect of CASC19 knockdown on metabolites in GC cells.(5)Transcriptome sequencing technology was used to analyze the effect of CASC19 knockdown on gene expression in GC cells.(6)Combined metabolomics and transcriptomics analyses were performed to explore the molecular mechanism of CASC19 affecting GC progression.Research results:(1)CASC19 was highly expressed in GC cell lines compared with normal gastric epithelial cells.Down-regulation of CASC19 expression significantly inhibited the proliferation,invasion,migration,glucose consumption and lactate production of GC cells.In turn,up-regulation of CASC19 expression promoted GC cell proliferation,invasion,migration,glucose consumption and lactate production.After downregulating CASC19 expression in GC cells,the expression of aerobic glycolysis-related proteins decreased.(2)The subcutaneous tumor formation experiment in nude mice showed that the proliferation of GC xenografts was significantly inhibited and the tumor growth was slowed down after down-regulating the expression of CASC19.The results of immunohistochemistry showed that the expression levels of aerobic glycolysis-related enzymes in the transplanted tumor tissues were significantly decreased after downregulating the expression of CASC19,but the expression of NPM1 in the transplanted tumor tissues was higher than that in the control group.(3)Microarray immunohistochemical analysis and q RT-PCR results showed that NPM1 was highly expressed in all adjacent normal tissues and lowly expressed in most GC tissues.Compared with the high NPM1 expression group,the overall survival and disease-free survival of the low NPM1 expression group were worse(P<0.01).The above results are consistent with those from the online database.(4)Metabolomics data showed that the levels of many metabolites were changed after CASC19 knockdown in GC cells.Kyoto Encyclopedia of Genes and Genomes(KEGG)annotation results showed that differential metabolites were enriched in a variety of biological pathways.(5)The transcriptome data showed that the expression of multiple differential genes was changed after down-regulating CASC19 expression in GC cells.GO analysis showed that the enrichment of differential genes was mainly related to cytoplasmic,cytoplasmic mitochondrial translation elongation,mitochondrial translation termination and mitochondrial respiratory chain complex.Differentially expressed genes were enriched in multiple tumor-related pathways.(6)The combined analysis of transcriptome and metabolome revealed that a variety of metabolites,gene expression and pathways related to the occurrence and development of cancer were significantly changed after down-regulating CASC19 expression in GC cells.Especially,the metabolites and pathways related to the transition of oxidative phosphorylation(OXPHOS)to aerobic glycolysis have changed significantly.Conclusion:We systematically demonstrated the function of CASC19 through cell experiments,animal experiments,clinicopathological data studies,transcriptomic analysis,metabolomics analysis,and combined transcriptome and metabolome analysis.We verified the results of the omics studies with the results of cell and animal experiments,showing that CASC19 promotes the progression of GC mainly through the regulation of NPM1 and aerobic glycolysis.The study of CASC19 is expected to find ideal molecular markers for the diagnosis and treatment of gastric cancer. |
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