The Role And Mechanism Study Of Ermap In Inhibiting Liver Metastasis Identified By Genome-wide CRISPR/Cas9 Screening

Tumor metastasis is the main cause of death in cancer,and liver metastasis occurs in many cancer types and is strongly correlated with poor prognosis.At present,there are various methods for studying the key molecules and mechanisms in the process of tumor liver metastasis.From the functional point of view,we use the CRISPR/Cas9 knockout library to screen the genes regulating tumor colonization and metastasis in mices,and further verify the function of genes and their mechanisms to regulate tumor metastasis.We use the spleen injection,a liver metastasis model,in which tumor cells suspension is injected into the spleen and transferred into the liver through the spleen sinus and portal vein system,to allow the tumor cells to colonize and form metastatic lesions.This model simulates the process of HCC intrahepatic metastasis through the portal vein and its branches to other places in the liver to form metastases.It is important to study the invasion ability and survival of HCC cells in the distal hepatic sinusoids during HCC intrahepatic metastasis.In this regard,we used the CRISPR/Cas9 gene screening technology to transfer the lentivirus,carrying the CRISPR/Cas9 knockout library information,into Hepa1-6,a mouse hepatoma cell line,and then operate spleen injection using mentioned Hepa 1-6.The experiment shows that the metastasis ability of Hepa1-6 KO by CRISPR/Cas9 is significantly improved.Therefore,we can say that the invasion and metastasize ability of Hepa1-6 could be enhanced by the deletion of certain genes.DNA from metastatic lesions are sequenced to get the sgRNA information contained in the metastases,and we further sequenced the sgRNA-targeted genomes to confirm the gene-shifting mutations.We presume that these genes lost their primary functions due to the mutations made by the frameshift cleavage by the CRISPR/Cas9 system.The lab and animal experiments show that the transformed Hepa1-6 with mutations have improved intrahepatic colonization and metastasis ability,therefore the liver become vulnerable to lesions and metastasis.A preliminary analysis of these gene information is conducted and after looking for information from the GEO database,we found that ERMAP has a difference in expression between liver cancer and adjacent tissue,and the expression of ERMAP is correlated with poor prognosis.We then performed qPCR analysis of clinical samples,like portal vein tumor thrombi,paracancerous tissue and cancer,and found that the ERMAP expression level :portal vein tumor thrombus <paracancerous tissue < cancer.This initially confirmed that the expression level of ERMAP is related to liver cancer,and ERMAP is a tumor suppressor gene.We then searched ERMAP gene for further information.It encodes a type I transmembrane protein that belongs to the butyrophilin family.The intracellular segment contains the B30.2 domain,and the extracellular segment contains the lgV domain,and thus belongs to the immunoglobulin superfamily.The ermap protein in human and mouse is slightly different,and the extracellular segment of the mouse has an lgC domain.Although the protein encoded by humans and mice differs,as the members of the immunoglobulin superfamily,the interaction is mainly determined by the distal domain lgV,so the human and mouse ermap proteins should function similarly.According to the literature analysis,the Butyrophilin family is similar to the B7 family,and participates in immune regulation through the interaction between membrane proteins,but its structure and function are more diverse.So far,its interacting proteins are difficult to find.ERMAP is highly expressed in red hematopoietic tissues such as fetal liver,suggesting that it participates in erythropoiesis.It is presumed that it may be involved in the formation of erythrocyte islands and interact with macrophage surface molecules.We synthesized and expressed the recombinant protein sErmap containing the extracellular domain of mouse ermap,which carries the human Fc segment tag.By Flow Cytmetry(FCM),it was found that sErmap has and only binds to Kupffer cells,the liver intrinsic macrophage.So it is initially believed that ermap exerts a tumor suppressing effect by binding to surface molecules on Kupffer cells,which act as macrophages in the hepatic sinusoids.Their main function is to clean up abnormal cells and microorganisms enter the hepatic sinus,like scavengers in hepatic sinus.Therefore,as cell regulator,we speculate that ermap can mediate Kupffer cells phagocytosis to tumor cells in the hepatic sinusoids and inhibit the colonization and growth of cancer cells in the liver.We knocked out the Ermap gene of Hepa1-6 cells by CRISPR/Cas9 technology and verified its knockout effect.Co-culture of hepa1-6 cells and Kupffer cells in vitro indicated that kupffer cells could phagocytose hepa1-6 cells,and the phagocytosis effect was reduced after the Ermap-KO.Therefore,we conclude that Ermap mediates the phagocytosis of Kupffer cells to tumor cells.In summary,Kupffer,the liver intrinsic macrophage,is an important component of the liver’s natural immunity,which can recognize and phagocytose tumor cells in the hepatic sinusoidal metastasis.Furthermore,ERMAP can mediate Kupffer cells to recognize and clear tumor cells.The current research on Kupffer cells could inhibit tumor cell metastasis also provides ideas for new possible clinical therapeutic methods.