| Objective: Hepatocellular carcinoma(HCC)is the most common manifestation of primary liver cancer.HCC has high invasive properties and heterogeneity and is associated with postoperative recurrence and metastasis.Ceramide can induce tumor cell apoptosis and has become a hot research topic in the treatment of liver cancer.Sphingomyelin phosphodiesterase 1(SMPD1)catalyzes the production of ceramide from sphingomyelin,but its expression and role in hepatocellular carcinoma are still unclear.Therefore,this study aimed to explore the expression of SMPD1 in HCC and its effects on the proliferation,migration,apoptosis and lipid metabolism of Hep G2 cells,so as to provide the basis for the targeted and precise treatment of HCC.Methods:1.Download The Cancer Genome Atlas(TCGA)data,comparing the expression of SMPD1 in paracancerous tissue and liver cancer tissue at the m RNA level,and analyzing the relationship between the expression level of SMPD1 and the survival and prognosis of liver cancer patients;The Clinical proteomic tumor analysis consortium(CPTAC)proteomic data analysis the expression of SMPD1 in paracancerous tissue and liver cancer tissue at the protein level;2.Referring to the seven-point sampling method,four points of "inner","middle","outer" and "side" were selected for the isolated liver cancer,where "inner" represents the tissue at the center of the tumor,and "middle" represents the tumor The tissue between the center and the tumor edge,"outer" represents the tumor edge tissue,"side" represents the adjacent tumor tissue 2 cm away from the tumor edge.Subsequently,Western blot experiments were performed to further verify the expression of SMPD1 in different parts of liver tissue;3.The SMPD1-knockout Hep G2 cell line was constructed by CRISPR/Cas9 method,and its effects on proliferation,migration,cycle and ROS were detected by scratch assay,plate cloning and flow cytometry,and Western blot experiments were used to study migration genes expression;4.The lipid changes in Hep G2 cells and Hep G2-SMPD1 knockout cells were measured by mass spectrometry,and the ceramide content was verified by immunofluorescence.Results:1.Analysis in the TCGA database found that there was no significant difference in the expression of SMPD1 in the cancer tissue and adjacent tissue of HCC patients,and there was no significant improvement in the survival prognosis of HCC patients;Clinical proteomic tumor analysis consortium(CPTAC)proteomic data analysis found that the expression of SMPD1 in paracancerous tissues was significantly higher than that in cancer tissues and had statistical significance;2.The expression of SMPD1 in different parts of liver tissue gradually increased from the inside to the outside,and the highest expression was in the adjacent part of the tumor;3.The results of plate cloning showed that the proliferation ability of Hep G2 cells was enhanced and the level of ROS was reduced after knockout of SMPD1,but their migration ability was inhibited to a certain extent,and the expression of EMT-related genes and proteins was decreased;4.When SMPD1 was knocked out,immunofluorescence showed that the total amount of ceramide was decreased,but lipidomic analysis found that there were differences in the increase and decrease of different carbon chain ceramide contents.In addition,metabolites such as dihydroceramide,free fatty acid,sphingomyelin,phosphatidylcholine and cholesterol ester were also found to have different degrees of change.Conclusions:1.There were differences in the expression of SMPD1 in different parts of liver cancer.The expression of SMPD1 was lower in the center of the tumor,while the expression in the periphery and adjacent to the tumor was higher;2.SMPD1 knockout resulted in accelerated proliferation of Hep G2 cells,decreased ROS,and inhibited cell migration to a certain extent,which may be related to changes in intracellular sphingolipid content. |
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