Akkermansia Muciniphila Derived Exosomal Protein Akk_M8 Suppress Hepatocellular Carcinoma

Primary liver cancer is the sixth most common cancer in the world and the third leading cause of cancer death.Hepatocellular carcinoma has an insidious onset,rapid progress,and is prone to intrahepatic and extrahepatic metastasis.It is of great significance for the treatment and prognosis of hepatocellular carcinoma to find more safe and effective therapeutic interventions.As one of the most complex components of the human body,the changes in the composition and function of intestinal flora are closely related to the occurrence and development of many human diseases.Research shows that the abundance change of Akkermansia muciniphila(Akk)is significantly related to the progression of liver cancer patients.In recent years,it has been found that bacterial extracellular vesicles(BEVs)released by intestinal bacteria may be one of the important communication channels between bacteria and the host.This paper aimed to explore whether Akk affected the occurrence and development of liver cancer by secreting BEVs into the systemic circulation and clarify the specific molecular mechanism of BEVs.1.Objectives(1)To elucidate the role of Akk in the occurrence and development of liver cancer;(2)Reveal the pathway how Akk exerts its effects,and clarify the correlation between Akk-EVs and both the initiation and progression of liver cancer.(3)Dig out the specific effector molecule of Akk-EVs and clarify its mechanism of action.2.Methods2.1 Research on the correlation between Akk and the occurrence and progression of liver cancer.(1)A naturally occurring liver cancer mouse model(NASH-HCC)was constructed,and mouse feces were collected to compare the intestinal Akk abundance of mice with liver cancer to those in normal mice;(2)Liver tumorigenesis in NASH-HCC liver cancer model mice was examined after intragastric supplementation with Akk.(3)A liver cancer metastasis model was constructed,and the mice were also gavaged with Akk.Then the relevant indicators such as tumor metastasis and survival rate of mice were detected.2.2 Determine whether Akk affects the progression of liver cancer by secreting EVs into the systematic circulation:(1)Liver cancer cells stimulated with Akk-EVs were transplanted subcutaneously into mice to detect the indicators of tumorigenesis such as tumor size.(2)Liver cancer cells were treated with Akk-EVs,and changes in the proliferation and migration,and invasion abilities of cells were examined in vitro.2.3 Reveal the effector molecules of Akk-EVs and clarify the relevant action mechanism:(1)Using genome-wide analysis and protein profiling to screen out potential functional molecules,then further verify the specific acting molecules of Akk-EVs by relevant in vitro and in vivo experiments;(2)Liver cancer cells were stimulated with the selected effector molecules of AkkEVs,followed by RNA sequencing to analyze gene expression changes,and then initial screening and validating of relevant pathways of action.3.Results3.1 The abundance of Akk was significantly decreased in the intestine of mice with liver cancer,whereas the incidence of tumors and metastasis decreased after Akk supplementation.3.2 Akk-EVs intervened liver cancer cells formed smaller tumors in vivo and exhibited high survival rates in mice.When hepatoma cells were co-cultured with Akk-EVs,the proliferation,metastatic and invasion abilities were significantly decreased,and EMT transformation was reduced.3.3 Through genome-wide and proteomic analyses,we initially hypothesized that Akk_M8 may be the effector molecule by which Akk-EVs exert its anti-hepatoma activity.As indicated by transcriptome and Western blot analyses,Akk_M8 may inhibit liver cancer progression in a manner that promotes ferroptosis in liver cancer cells.4.ConclusionIn vitro and in vivo experiments demonstrated that Akk and its secreted EVs reduced HCC proliferation,migration and invasion ability.Genome-wide,proteomic,and transcriptomic analyses revealed that the membrane protein Akk_M8 of Akk-EVs might be the functional molecule inhibiting liver cancer progression by promoting ferroptosis and thus inhibiting proliferation and metastasis of liver cancer cells.The above findings would open new sights in the study of communication between bacteria and the host,as well as provide new strategies for the development of Akkbased effective cancer vaccines or Akk adjuvant drug therapies.