Study On The Role And Mechanism Of ApoM In The Occurrence And Development Of Liver Cancer

Objective: Use the TCGA database to analyze the correlation between ApoM and hepatocellular carcinoma and observe the effect of ApoM gene expression on the proliferation,apoptosis,migration and invasion of hepatocellular carcinoma from the in vivo and in vitro levels,and then to explore the occurrence of ApoM in hepatocellular carcinoma and its role in the development process.Methods: Use the TCGA database to analyze the difference in the expression level of ApoM gene in cancer and adjacent tissues,and verify it at the in vivo and cellular level;in order to explore the impact of ApoM expression level on liver cancer cells,use Crispr-Cas9 technology in mice ApoM gene deletion and overexpression cell models were established on the liver cancer cell line Hepa1-6 and the human liver cancer cell line Huh-7,and the Edu cell proliferation experiment,nude mouse tumor transplantation experiment and cell cycle experiment were used to explore the proliferation activity of ApoM gene on hepatocellular carcinoma cells In order to explore the specific links of the ApoM gene deletion affecting the cycle of liver cancer cells,firstly use the TCGA database to analyze the correlation between ApoM genes and related cell cycle factors in clinical samples of hepatocellular carcinoma and perform the Western-blot method.Validation;Analyze the effect of ApoM gene expression level on liver cancer cell apoptosis by cytometry and verify it by Western-blot method;use Transwell and Western-blot experiments to analyze the 12influence of ApoM gene on the invasion and migration ability of liver cancer cells;to explore The role of ApoM in the occurrence of primary liver cancer,using C57BL/6J mice and injecting N-nitrosodiethylamine to induce primary liver cancer,to observe the effect of ApoM gene knockout on the rate of primary liver cancer;use Blood biochemical analysis and oil red O staining were used to explore the effect of ApoM gene knockout on lipid metabolism of liver cancer cells;bioinformatics and Western-blot experiments were used to analyze the effect of ApoM gene knockout on lipid metabolism and related key proteins FASN and SREBP1.Results:1.TCGA database analyzes the expression level of ApoM in 50 matched samples of hepatocellular carcinoma and liver cancer patients’ cancer tissues and adjacent tissues(P=0.025),and immunohistochemical methods are used to detect the ApoM gene in cancer tissues and adjacent tissues of liver cancer patients.The expression level(P=0.00025),the results indicate that the expression level of ApoM in cancer tissues is lower than that in adjacent tissues;the expression level of ApoM in the normal mouse liver cell line AML12 detected by WB method is higher than that in the liver cancer cell line Hepa1-6(P=0.012).2.ApoM gene inhibits the proliferation,migration and invasion of liver cancer cells and up-regulates the level of apoptosis.2.1 Edu and nude mouse experiments showed that the proliferation of Hepa1-6 and Huh-7 liver cancer cells in the ApoM gene deletion group was significantly increased(P=0.00050,P=0.017),while the ApoM gene overexpression group decreased(P=0.070,P=0.032);cell cycle experiments suggest that ApoM gene knockout accelerates the transition of liver cancer cells from G1 phase to S phase,while overexpression of ApoM gene causes liver cancer cells to block in G1 phase and inhibits cells from turning to S phase.WB experiments indicate that ApoM gene knockout affects the expression of key regulatory proteins related to the G1/S transformation of liver cancer cells and hinders the formation of Cyclin E1 and CDK2 complexes.2.2 Flow cytometry and WB experiments showed that the key proteins 13Cleaved-caspase3,Cleaved-caspase9,and Bax/Bcl-2 related to the apoptosis of liver cancer cells in the ApoM gene deletion group were significantly increased;the overexpression group was the opposite(P<0.05).2.3 The invasion and migration ability of liver cancer cells in the ApoM gene deletion group was significantly increased,and the expression level of MMP-2 protein was significantly increased,while the overexpression group was the opposite(P<0.05).3.In the experiment of N-nitrosodiethylamine inducing primary liver cancer in mice,ApoM knockout mice have a faster tumor formation rate and lower survival rate than wild-type mice(P<0.05);At the same time,the apoptosis level of liver cancer tissue in the ApoM gene knockout group was significantly lower than that in the WT group(P<0.05).4.In the case of diethylnitrosamine induction,compared with normal wild-type mice,the ApoM gene knockout group mice had impaired liver function and significantly increased triglyceride levels(P<0.05);and protein network and The enrichment analysis results showed that ApoM has an interaction relationship with liver lipid metabolism-related proteins LPA,APOM,GPANK1,APOA2,APOC2,PON1,etc.,and may participate in cholesterol efflux,formation of extracellular structure,high-density lipoprotein particle assembly,and low-density lipoprotein particles.Density lipoprotein particle remodeling and other processes;WB detection indicated that compared with the control group,the expression levels of FSAN and SREBP1 in liver cancer cells of the ApoM gene knockout group were significantly increased(P<0.05).In conclusion:1.The expression level of ApoM in cancerous tissues in patients with liver cancer is lower than that in adjacent tissues.2.ApoM gene inhibits the proliferation,migration and invasion of liver cancer cells and up-regulates its apoptosis level.3.The deletion of ApoM gene promotes the rate of diethylnitrosamine-induced liver cancer in mice and promotes liver dysfunction.4.The deletion of ApoM gene up-regulates the expression of SREBP1 and FASN,causing lipid metabolism disorders,which in turn affects the occurrence and development of liver cancer.