The Correlation Between ApoM And Live Cancer And Its Possible Molecular Mechanisms

Objective:Firstly,the content of ApoM in serum of patients with liver cancer and its expression in liver cancer and adjacent tissues were detected.Then,the effects of ApoM gene deletion on hepatic autophagy,lipid metabolism and apoptosis were investigated by establishing ApoM knockout mice and cell models to analyze the association between ApoM and liver cancer.Methods:1.210 cases of liver cancer group and 245 serum samples of healthy control group were collected.The concentration of ApoM in serum was detected by ELISA.The concentrations of HDL,LDL,apoAl,apoB,and LPa associated with lipid metabolism were measured using an automated biochemical analyzer.176 cases of hepatocellular carcinoma were collected from paraffin-embedded specimens,and 76 specimens of adjacent tissues were selected.The expression of ApoM in tissues was detected by immunohistochemistry.2.ApoM knockout mouse model constructed by CRISPR/Cas9 technology,and wild-type C57BL/6J strain mice were mated,and the tail DNA was extracted for genotype identification.The progeny ApoM gene knockout heterozygotes were selected.The mice were then mated with the cage,and the progeny were selected as ApoM knockout homozygous mice.In this study,ApoM knockout male rats were used as experimental group,and wild-type male rats born at the same age or in the same litter were used as controls.After DNA identification,the liver was observed by transmission electron microscopy and optical microscopy.3.Western blot was used to detect the expression of related proteins involved in lipid synthesis in the liver tissues of wild-type mice and ApoM knockout mice:SREBP1,FASN and ACACA.4.The expression of autophagy-related proteins:ATG7,ATG5-12,Beclinl,P62 and LC3BII/LC3BI in the liver tissues of wild-type mice and ApoM knockout mice were detected by Western blot.5.Using the CRISPR/Cas9 technology to knock out the ApoM gene,construct a mouse ApoM gene-deficient AML 12 cell model,and detect the key proteins involved in lipid synthesis-related proteins and autophagy pathways by Western blot:SREBP1,FASN,ACACA,ATG7,ATG5-12,Beclinl,P62 and LC3BII/LC3BI.The mRNA levels of autophagy-related proteins:ATG7,ATG5-12,Beclin1,P62,LC3B,LC3A were detected by RT-PCR.6.Immunofluorescence technique was used to detect the expression of autophagy substrate P62 protein at the cellular level of ApoM knockout mice.7.After autophagy was induced by starvation induction,the expression of triglyceride in AML 12 cells was detected by oil red O staining and the expression of P62 protein in autophagy substrate was detected again by immunofluorescence technique.8.Western blot was used to detect the expression of related proteins involved in lipid synthesis and the key proteins involved in autophagy pathway after autophagy induced by starvation.9.Western blot was used to detect the key proteins of mitochondrial apoptosis pathway in the liver cells of ApoM knockout mice and ApoM knockout mice.Bad,p-Bad,Bax,Bcl2,Bclx1,clever-caspase3,clever-caspase9 And the expression of p-p53.Results:1.ApoM was expressed in hepatocellular carcinoma tissues and paracancerous tissues.The expression site was mainly located in the cytoplasm.The expression in hepatocellular carcinoma tissues was significantly higher than that in adjacent tissues.The difference was statistically significant(P<0.01).There was no significant difference in the expression of ApoM in high,medium and poorly differentiated hepatocellular carcinoma tissues(P>0.05).2.The level of ApoM in serum of patients with liver cancer was significantly higher than that of healthy control group(31.28±12.25ng/ml vs 28.62±8.78ng/ml,P<0.01).3.The results of DNA level and RNA level showed that the ApoM knockout mouse model and mouse normal liver cells were successfully modeled.4.ApoM knockout mice are fatter in terms of growth and development,while the general aspects of skin gloss and stress response are not significantly different from wild type.Observation by light microscopy and transmission microscopy revealed that the liver structure of the ApoM knockout mice group changed significantly,lipid deposition occurred,and the accumulation of lipid storage cells was accompanied.At the same time,the AML of the ApoM gene was knocked out.Lipid accumulation also occurs in normal liver cells.5.Western blot analysis of proteins involved in lipid synthesis showed that the expression of SREBP1,FASN and ACACA was higher in liver tissues of ApoM knockout mice than in wild type mice(P<0.05).6.Western blot analysis showed that the expression of key proteins involved in the autophagy pathway:ATG7,ATG5-12,Beclinl,LC3BII/LC3BI,and P62 was increased in the liver tissue of ApoM knockout mice compared with wild type mice(P<0.05).7.In the ApoM knockout cell model constructed by AML 12 normal hepatocytes,the expression levels of related proteins involved in lipid synthesis were significantly higher than those in the control group(P<0.05).The protein levels of the autophagy pathway were significantly higher in the protein level and mRNA level than in the control group(P<0.05).8.Immunofluorescence detection The autophagy substrate P62 protein showed that autophagy substrate accumulation occurred after knockdown of ApoM gene.9.At the level of mouse ApoM knockout cell model,autophagy was induced by starvation.The results showed that compared with the control group,the expression of SREBP1 protein in FASN involved in lipid synthesis decreased(P<0.05).The expression of P62,Atg5-12 and Beclinl protein in autophagy pathway decreased(P<0.05),while LC3BII/LC3BI protein was significantly increased(P<0.05).The results showed that starvation induced a reversal effect on the lipid synthesis pathway induced by ApoM gene knockdown and the expression of some proteins in the autophagy pathway.10.The deletion of ApoM gene increased the expression of apoptosis-related proteins Bad,p-Bad,Bax/Bcl2,clever-caspase3 and clever-caspase9 in mouse liver tissue and mouse liver cells(P<0.05);P53 expression(P<0.05).Conclusion:1.The level of ApoM in serum of patients with liver cancer is significantly increased,and the expression level of liver cancer tissue is higher than that of adjacent tissues,suggesting that the expression of ApoM in liver cancer patients is disordered.2.ApoM gene knockout can increase the synthesis of lipid metabolism related proteins in liver and liver cells of mice,leading to disorder of lipid metabolism;and hunger has a reversal effect.3.ApoM gene knockdown can up-regulate the expression of autophagy-related proteins in mouse liver and liver cells,which in turn causes autophagy.4.The deletion of ApoM gene leads to increased expression of apoptosis-related protein in mitochondrial pathway and up-regulation of p-p53 level in mouse liver and mouse hepatocytes.5.ApoM gene may participate in the occurrence and development of liver cancer by affecting lipid metabolism,autophagy and apoptosis.