Study On The Role Of RIPK4 In Hepatocellular Carcinoma And Its Related Mechanism

Objective:Primary liver cancer is a common liver malignant tumor.Hepatocellular carcinoma(HCC)accounts for 90% of all primary liver malignant tumors,and its morbidity and mortality are high.Functional genes are one of the important reasons that affect the development of HCC.RIPK4 as a functional gene plays an important role in the development of various tumors,but the specific mechanism of action in liver cancer is not completely clear.We explored and verified the role of RIPK4 in the invasion and metastasis of HCC through the four levels of tissue,cell,molecule and animal,and explored the relationship between RIPK4 expression and the clinical patient prognosis and clinical factors.We hope to provide new therapeutic targets for the clinical treatment of HCC patients.Methods:First,we analyzed the expression of RIPK4 in HCC through public databases.We collected clinical specimens of liver cancer patients for immunohistochemical staining and real-time quantitative PCR experiments to verify the expression of RIPK4 in HCC and normal tissues.Second,we used a public database to analyze the relationship between RIPK4 and the prognosis of liver cancer patients,combined with clinical follow-up data and using Kaplan-Meier,single factor and multifactor risk proportional regression models to explore the relationship between of RIPK4 and clinical pathological factors,and we constructed a Nomogram model to predict the prognosis of patients.Then we screened out Huh7 and Hep3 B cell lines and constructed Huh7-LV-RIPK4 and Hep3B-LV-RIPK4 and their control cell lines by lentivirus infection.Then we used the wound healing test and Transwell test to explore the effect of RIPK4 on the migration and invasion ability of liver cancer cells.In addition,we also used Western blotting to study the effect of RIPK4 on the STAT3 pathway and the effect of RIPK4 on the process of hepatocellular carcinoma epithelial-mesenchymal transition(EMT).Finally,we conducted in vivo experiments by injecting Huh7-LV-RIPK4 cells into the tail vein of experimental mice to construct an in vivo metastatic tumor model to study the effect of RIPK4 on HCC invasion and metastasis in mice.Results:(1)Through public databases analysis of the expression level of RIPK4 in HCC,it was found that the expression level of RIPK4 in HCC tissue was significantly lower than that in normal liver tissue.At the same time,real-time quantitative PCR and immunohistochemical detection were used to find that the expression of RIPK4 in HCC tissues was down-regulated.(2)The prognosis of clinical HCC patients showed that the OS and DFS of HCC patients with high RIPK4 expression lived longer.In addition,combined with clinicopathological factors,we found that the expression level of RIPK4 was an independent risk factor for overall survival and disease-free survival of HCC patients,and the expression of RIPK4 in HCC patients was negatively correlated with Microvascular tumor thrombus(χ2= 4.795,P <0.05).(3)Through the wound healing test and Transwell test,we found that RIPK4 can affect the invasion and metastasis of HCC cells in vitro.Huh7-LV-RIPK4 and Hep3B-LV-RIPK4 cells showed a weaker invasion and metastasis ability than Hep3B-LV-RIPK4 cells.(4)Western blot experiments confirmed that RIPK4 can affect affect the expression of MMP-2 and MMP-9 proteins through the STAT3 pathway to change the invasion and metastasis ability,by reducing the epithelial protein N-cadherin and Zeb-1 changes the EMT process.Rescue experiments combined with STAT3 pathway activators confirmed that RIPK4 indeed inhibited the invasion and metastasis of HCC through STAT3 pathway.(5)Through in vivo experiments in animal mice,we found that the lung metastasis nodules in mice injected with Huh7-LV-RIPK4 cells through tail vein were significantly lower than those in control mice.Conclusions:Our study revealed that RIPK4,as a differentially expressed gene,is downregulated in HCC tissues and leads to poor prognosis in HCC patients.The specific mechanism was that RIPK4 inhibited epithelial mesenchymal transformation and inhibited the invasion and metastasis of hepatocellular carcinoma by reducing the phosphorylation of STAT3,which may provide a new target and a new therapeutic strategy for the clinical treatment of patients with hepatocellular carcinoma.