Fig 2: ApoM gene promotes the expression of key apoptotic proteins. (A, B) Western blot analysis of the expression levels of key apoptotic proteins, the results showed that the deletion of ApoM gene reduced the expression levels of Cleaved-caspase-3, Cleaved-caspase-9 and Bax/Bcl-2 cell apoptosis-related proteins. The overexpression of ApoM gene increased the expression of Cleaved-caspase-3, Cleaved-caspase-9 and Bax/Bcl-2 apoptosis-related proteins, and statistical analysis was performed. *p<0.05, **p<0.03, ***p<0.01.

Fig 3: The expression level of ApoM in cancer tissues is lower than that in adjacent tissues. (A) 50 matched samples of primary liver cancer were selected from the TCGA database. The analysis showed that the expression level of ApoM gene in cancer tissues was lower than that in paracarcinoma tissues, and the difference was statistically significant (P=0.025). (B) Using immunohistochemical method to detect the expression level of ApoM in cancer tissues and adjacent tissues of liver cancer patients, the expression level of ApoM in cancer tissues decreased significantly (P=0.0063). (C) The expression level of ApoM in the normal mouse liver cell line AML12 was higher than that in the mouse liver cancer cell line Hepa1-6 (P=0.012) *p<0.05, **p<0.03, ***p<0.01.

Fig 4: The deletion of ApoM gene aggravate the migration and invasion of Hepa1-6 liver cancer cells. (A) The Transwell migration experiment indicated that the ApoM gene deletion group had stronger migration ability than the control group (P=0.000044), while the overexpression group decreased (P=0.00018). (B) The Transwell invasion experiment indicated that the ApoM gene deletion group had stronger invasion ability (P=0.000025), while the overexpression group decreased (P=0.00042). (C) Western blot analysis was used to analyze that the ApoM gene deletion group had a higher MMP-2 protein expression level (P=0.027), and the overexpression group was the opposite (P=0.027). All data were ± s of 3 independent measurements, n≥3, *p<0.05, **p<0.03, ***p<0.01.

Fig 5: The deletion of ApoM gene accelerates the tumorigenesis rate of C57BL/6J mouse liver cancer induced by N-nitrosodiethylamine. (A) Livers were taken at 0, 4, 8, 12, 16 and 20 weeks. Compared with WT mice, ApoM gene-deficient mice developed tumors earlier under N-nitrosodiethylamine induction. (B) Western blot assay showed that the ApoM level in the cancer tissue of WT mice in the liver cancer induction model group was lower than that in the paracarcinoma tissues (P=0.010). (C) Compared with the WT group, the ApoM gene deletion group had higher wet liver weight per month. At 12 weeks, the wet liver weight of the ApoM gene deletion group was higher than that of the WT group and was statistically significant (P=0.0048). (D) The body weight of mice in the WT group increased from 0 weeks to 12 weeks, and their body weight decreased after 12 weeks. The body weight of mice in the ApoM gene deletion group increased from 0 to 8 weeks, and their body weight decreased after 8 weeks. (E, F) Liver function ALT (P=0.00013) and AST (P=0.00061) levels of liver cancer induction model ApoM-/- group are higher than those of WT group, and have statistical significance. (G) Western blot analysis of liver cancer induction model, the key apoptosis proteins Cleaved-caspase9 (P=0.20), Cleaved-caspase3 (P=0.26), Bax/Bcl-2 (P=0.014) in wild mice were higher than ApoM gene deletion group. (P=0.0011). (H) Compared with the WT group, the ApoM gene deletion group has a faster time for liver tumors, which was statistically significant (P=0.028). (I) The survival of ApoM-/- mice was shorter than that of WT mice (P=0.011).