Study On The Roles And Mechanism Of Differentially Expressed Genes SLC38A4 And GJA1 In Highly Colonized Hepatocellular Carcinoma Cell Lines In Intrahepatic Metastasis

Hepatocellular carcinoma(HCC),as a kind of malignant tumor of liver cancer,is one of the most malignant tumors in our country.Accumulating evidence indicates that intrahepatic metastasis is not only the primary site of HCC metastasis,but also the leading cause of death from HCC.Although the process of intrahepatic metastasis of HCC cells is well comprehending,the research is not yet in-depth and there is no effective drug to block the intrahepatic metastasis.Therefore,we need to further clarify the key molecules and mechanism of the process of intrahepatic metastasis of HCC.Despite the inhibitory and pro-oncogenes alterations,the interactions between cells and the microenvironment are crucial to normal tissue homeostasis and tumor tissue growth.Especially the interaction between tumor cells and stroma forms a strong network that influence tumorigenesis,tumor progression,prognosis and recurrence.Tumor metastasis is a complex,multi-step and multifactorial process that involves the formation of primary tumors,epithelial-mesenchymal transformation of tumor cells,infiltration into blood vessels and lymphatic vessels,survival in blood vessels and lymphatic vessels,extravasation of blood vessels and lymphatic vessels,in the distal foci survival.Tumor microenvironment including tumor hypoxic environment,tumor-associated macrophages(TAM),NK cells,dendritic cells,T cells,tumor-associated fibroblasts and endothelial cells plays an important role in tumor metastasis.The outcome of tumor metastasis is the formation of new tumors on the remotely implanted lesions,the survival and regeneration of the final implanted lesions has been a problem that scientists want to overcome.In order to study the final stage of the tumor metastasis,it is particularly important to screen for highly colonized cells.In order to study the final stage of the liver cancer metastasis process,we constructed a spleen injection-liver colonization(metastasis)model.Using this model,we screened a series of highly colonized(metastasis)HCC cells.Using profiling microarray analysis,we found 64 differentially expressed mRNAs in highly colonized cells.Further verification was performed in more highly colonized cells to ensure that at least two other highly colonized cells were consistent with the expression results of differential genes.As a result,we screened 31 genes,of which 17 were upregulated,14 were down-regulated.According to the relationship with the prognosis of HCC patients,we finally screened SLC38A4,GJA1 differentially expressed in highly colonized cell lines,and were differential expressed in multiple cohorts of HCC tissue samples,and were related to the high recurrence and poor prognosis of HCC patients.We found that SLC38A4 is not only down-regulated in HCC tumors,but also gradually decreased with the progression of hepatocellular carcinoma.It also shows a tendency of gradually up-regulated expression during liver development,and is positively correlated with the prognosis of HCC patients.We subsequently found that the expression of SLC38A4 was related to tumor size,TNM stage,BCLC stage,tumor metastasis,and serum AFP levels in patients with HCC.Further,we studied the biological function of SLC38A4,the formation of stem spheres confirmed that SLC38A4 depletion increases the stemness of HCC cells;CCK8,EDU and cell cycle assays confirmed that SLC38A4 depletion promotes proliferation of HCC cells;wound healing assay and transwell experiments confirmed that SLC38A4 depletion promotes the metastasis of HCC cells;the tumor formed in nude mice subcutaneously confirmed that SLC38A4 depletion promotes tumor proliferation.The above results proved that SLC38A4 is an anti-oncogene,and SLC38A4 depletion can promote the progression of HCC tumors both in vitro and in vivo.We found that both ERK and AKT phosphorylation were significantly higher in SLC38A4 depleted cells than control cells,demonstrating that SLC38A4 depletion activates MAPK and PI3K-AKT signaling pathways to promote HCC progression.Then we studied about GJA1 and found that GJA1 was highly expressed in highly colonized cells and multiple cohort of HCC tissues.Its expression was positively correlated with the high recurrence rate and poor prognosis of HCC patients.The in vivo experiment-spleen injection-liver transplantation(metastasis)in nude mice confirmed that GJA1 can promote the ability of intrahepatic colonization of HCC cells.First,we examined the function of GJA1 overexpression HCC cells.We used CCK8,clone formation,EDU immunofluorescence staining assays to detect HCC cells growth,flow cytometry to detect cell cycle,transwell and wound healing assay to detect cell invasion and metastasis,we found that GJA1 overexpression on cell proliferation,invasion and metastasis was not statistically significant.In order to explore the reason for the high colonization of the selected cell lines,we hypodermic the high colonization HCC cell lines in nude mice and found that the high colonization cell lines had stronger vascularization ability.GJA1 overexpression cells also promoted subcutaneous tumorigenesis.CD31 and CD34 staining showed that the GJA1 overexpressed cells resulted in a higher vascular density.To investigate the effect of GJA1 on angiogenesis,we found no difference in the stimulation of HUVECs with conditioned medium of GJA1 overexpression and the control group.Since GJA1 can make a gap junction between two kinds of cells to transmit substance to each other,we hypothesized that cells overexpressing GJA1 may promote tubule formation by direct contact with HUVEC.GJA1 overexpressed HCC cells were cocultured with HUVEC and GJA1 overexpression HCC cells could enhance the ability of HUVECs to form tube.In conclusion,we elucidated the final stage of the HCC metastasis process and found that the gene SLC38A4 and GJA1 in tumor cells play an important role in the final denouement of tumor cells survival in the distal colonization.This study provides a fresh idea and perspective for further understanding the role of tumor microenvironment in the process of HCC metastasis and provides a potential target of prevention and treatment for the metastasis of HCC.