A20 Gene Inhibits HCC-cell Motility Induced By TNFa

Hepatocelluar carcinoma (HCC) is the third leading cause of cancer death worldwide. The high metastasis and recurrence are the main causes of HCC death. Therefore, to identify molecules that can supress invasion and metastasis of tumor will provide novel targets for HCC therapies.A20 (Tumor necrosis factor a-induced protein 3, TNFAIP3) was initially identified as a primary gene product following tumor necrosis factor (TNF) a treatment in human umbilical vein endothelial cells. Homology comparisons have revealed the presence of an ovarian tumor (OTU) domain in the N-terminal part of A20, suggesting that A20 functions as a deubiquitinase, which has been confirmed biochemically A20 is a immunity negative molecule. The C-terminal domain of A20 contains seven zinc finger (ZnF) structure, it has been proposed that ZnF4 confers intrinsic ubiquitin ligase activity to A20. A20 maintain negatively both innate and adaptive immunity. The importance of A20 in limiting inflammation is underscored by the association of polymorphisms in the A20 genomic region with multiple human autoimmune and inflammatory diseases.Recent studies demonstrate that A20 is a central regulator of inflammation and functions as a ubiquitin-editing enzyme to potently downregulate NF-kB signalling induced by TNF, IL-6. A20 is inactivated in various haematological malignancies and results in constitutive NF-kB activation in tumors, therefore, A20 is a crucial tumor suppressor. However, the role and underlying mechanisms of A20 in HCC-cell motility remain to be investigated.In this study, we explore the role of A20 gene in HCC, especially its function on the motility of HCC induced by TNFa. The aim of this study is to explore new targets for the treatment of HCC and provide theoretical basis for clinical treatment.MethodsIn vitro studies:We selected 75 pathological sections derived from liver cancer patients to identify A20 expression. Observation of immunostaining was focused the carcinoma and adjacent tissues, where the expression of A20 was heterogeneous. Then, to observe the relationship between the expression of A20 and tumor cells infiltrating in vessel in HCC, double-immunostaining method was performed to identify A20 and CD34.Next, we picked human hepatocellular carcinoma cell lines (SMMC-7721 and Huh-7) in which A20 was down-regulated expression. Cells were transfected with empty plamid and A20 plamid, the experiment group was treated with TNFa. Then, we collected the cells to detect the migration and invasion capacity. To explore the molecular mechanism how A20 inhibits HCC-cell motility enhanced by TNFa, some molecules that are associated with cell motility, such as EMT-related molecules(E-cadherin, N-cadherin), FAK and RAC1, were detected on protein level and mRNA level. For scientific and academic rigour, we selected Hep3B, A20 over-expression cell line, to repeat experimental results.In vivo studies:Experimental Metastasis Mouse ModelExperimental metastasis mouse model was a simple and yet physiologically relevant metastasis model. In this model, tumor cells were injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Lung metastases were analyzed by inflation with ink, thus allowing easier visual observation and quantification.Results1-1、A20 expression was down-regulated in HCC tissuesBy observing the A20 expression of pathological sections, we found that the expression of A20 was heterogeneous among the carcinoma and adjacent tissues. The level of A20 expression in the carcinoma tissues was lower than the lever in the adjacent tissues. This result indicted that A20 was a tumor supressor.1-2、A20 expression of tumor cells infiltration in vessel was down-regulated in HCC.To clarify clinical relevance of our study, we analyzed the relationship between the expression of A20 and tumor cells infiltration in vessel in HCC. The level of A20 expression of tumor cells infiltration in vessel was lower than the lever in carcinima tissues. These results demonstrated that A20 could inhibit HCC-cell motility.2、A20 suppressed markedly migration and invasion of HCC cells induced by TNFa in vitro.To verify the validity of our hypothesis, we further detected the effect of A20 on migration and invasion of HCC cells in vitro via transwell experiment. We found that overexpression of A20 reduced HCC-cell motility enhanced by TNFa. Consistently, it could reverse the athletic ability when A20 was knockdown. The results indicated that A20 could effectively suppress the migration and invasion capacity enhanced by TNFa.3、A20 suppressed HCC-cell motility enhanced by TNFa via inhibiting NF-kB/EMT pathway.The results above suggested that A20 could inhibit HCC-cell motility enhanced by TNFa. So, we wanted to know the underlying mechanisms of A20 in HCC-cell motility. Since EMT is a vital process of tumor cell metastasis, E-cadherin and N-cadherin are crucial EMT-related molecules. Our findings revealed that A20 could cause a significant decrease in expression of N-cadherin and an increase in expression of E-cadherin treated by TNFa. However, it was of no effect for A20 gene without TNFa.4、A20 suppressed HCC-cell motility enhanced by TNFa via inhibiting RIPK1/FAK/RAC1 pathway.FAK can mediate cell-cell and cell-matrix adhesions. Also, RAC1 is known to inhibit cell migration and invasion by carcinoma cells, which is downstream molecule of FAK. So we hypothesized that A20 suppressed HCC-cell motility enhanced by TNFa via inhibiting FAK/RAC1 pathway. In this study, we tested the hypothesis that inhibition of A20 may reverse TNFa-induced FAK and RAC1. Many research revealed that RIPK1 could combine with FAK and activate p-FAK. We have proved the outcome that A20 could suppress the activity of FAK/RAC1 via effect on RIPK1.Conclusions1、A20 suppressed markedly migration and invasion of HCC cells induced by TNFa.2、A20 suppressed EMT progress of HCC cells dependent of NF-κB signaling pathway.3、A20 could suppress the activity of FAK/RAC1 via effect on RIPK1.