The Mechanism Of CD97 Promoting The Proliferation,Invasion And Metastasis Of Hepatocellular Carcinoma By Regulating PI3K/AKT/mTOR Pathway

Objective(s):Primary liver cancer is one of the most common malignant tumors in the world,and its high morbidity and mortality seriously threaten human health.Hepatocellular carcinoma is the most common pathological type in primary liver cancer.So far,the prognosis of advanced hepatocellular carcinoma patients is still poor.The selection of novel tumor biomarkers and effective therapeutic targets is conducive to the individualized and precise treatment of hepatocellular carcinoma.In this study,clinical specimen detection,a series of in vitro and in vivo experiments were used to explore the role of CD97 in hepatocellular carcinoma proliferation,invasion and metastasis,aiming to provide a new direction for the diagnosis and treatment of hepatocellular carcinoma.Methods:1.The hepatocellular carcinoma data in the public database was explored,q RTPCR,Western blot and IHC techniques were used to identify the expression difference of CD97 in hepatocellular carcinoma tissue specimens between cancerous tissue and pericancerous tissue.Secondly,the clinical data of patients were sorted out to analyze the correlation between CD97 and the clinicopathological features of hepatocellular carcinoma patients,and to compare the influence of high or low expression of CD97 on the survival of patients.Cox proportional regression models were used to identify potential factors associated with overall survival and disease-free survival.2.The hepatocellular carcinoma cell lines with CD97 overexpression and knockdown were constructed.The effects of CD97 on proliferation,invasion and metastasis of hepatocellular carcinoma were explored by CCK8,colony-forming unit assays,wound healing test,transwell,q RT-PCR,Western blot,immunofluorescence assay,xenograft model of nude mice and IHC assay.In addition,PI3 K pathway inhibitor LY294002 and activator 740Y-P were used to verify the regulation of CD97 on PI3K/AKT/m TOR signaling pathway.3.The targeting relationship between miR-21-5p and CD97 was clarified by double luciferase reporting assay.The effects of miR-21-5p-CD97-PI3K/AKT/m TOR axis on the biological behavior of hepatocellular carcinoma were explored by CCK8,colony-forming unit assays,wound healing test,transwell,q RT-PCR and Western blot assay.Results:1.The results of HCC cohorts in TNMplot,TCGA and GEO databases show that the transcriptome level of CD97 in hepatocellular carcinoma tissues was significantly higher than paracarcinoma tissues.The m RNA and protein levels of CD97 in hepatocellular carcinoma tissues in our central were also significantly higher than paracarcinoma tissues.The m RNA and protein levels of CD97 in hepatocellular carcinoma cells were also higher than normal hepatocyte.Secondly,hepatocellular carcinoma patients with high CD97 expression had higher pathological grade and TNM stage,as well as higher tumor recurrence rate.Cox risk regression model showed that CD97 expression was an independent factor affecting overall survival and disease-free survival of hepatocellular carcinoma.Kaplan-Meier survival curve analysis showed that hepatocellular carcinoma patients with high expression of CD97 had shorter overall survival and disease-free survival.2.Overexpression of CD97 in hepatocellular carcinoma cell lines up-regulates the expression of cyclin PCNA and Cyclin D1,inhibited the protein levels of E-cadherin,and increased the expression levels of N-cadherin and Vimentin.Thus,the cell cycle and epithelial mesenchymal transformation were promoted,and the proliferation,migration and invasion of cells were enhanced.Knockdown of CD97 down-regulated the expressions of PCNA and Cyclin D1,increased the expressions of E-cadherin,and decreased the protein levels of N-cadherin and Vimentin,which inhibited the cell cycle and epithelial mesenchymal transformation.Ultimately,the cells’ ability to proliferate,migrate,and invade was weakened.Overexpression of CD97 activates the PI3K/AKT/m TOR signaling pathway by enhancing the expression of phosphorylated molecules p-m TOR(2448),p-PI3K(p85)and p-AKT(473).Knockdown of CD97 can reduce the activity of PI3K/AKT/m TOR signaling pathway by down-regulating the protein levels of p-m TOR(2448),p-PI3K(p85)and p-AKT(473).Our research was further demonstrated that CD97 is the upstream regulator of PI3K/AKT/m TOR signaling pathway through the PI3 K pathway inhibitor LY294002 and activator 740 YP,and CD97 mediates the proliferation,invasion and metastasis of hepatocellular carcinoma by regulating PI3K/AKT/m TOR signaling.The results of the constructed xenograft model of hepatocellular carcinoma in nude mice showed that the tumor volume and mass of the CD97 overexpression group increased significantly,and the protein levels of CD97,PCNA,Ki67,Cyclin D1,p-m TOR(2448),p-PI3K(p85)and pAKT(473)in the tumor tissues were up-regulated.However,the tumor volume and mass of the CD97 knockdown group were significantly reduced,and the expressions of CD97,PCNA,Ki67,Cyclin D1,p-m TOR(2448),p-PI3K(p85)and p-AKT(473)in tumor tissues were down-regulated.Combined with the results of in vitro experiments,it was clarified that CD97 promoted the proliferation of hepatocellular carcinoma by regulating cell cycle,and was involved in regulating the activity of PI3K/AKT/m TOR signaling pathway.3.The mRNA levels of miR-21-5p and CD97 were negatively correlated in hepatocellular carcinoma.the negative relationship between miR-21-5p and CD97 in hepatocellular carcinoma cells was verified by using miR-21-5p mimics and inhibitor.The targeted regulatory relationship between miR-21-5p and CD97 was clarified by double luciferase reporting assay.At the same time,it was observed that the proliferation,migration and invasion of hepatocellular carcinoma cell lines treated with miR-21-5p mimics were inhibited,but this inhibition could be compensated by the overexpression of CD97.The proliferation,migration and invasion of hepatocellular carcinoma cell lines treated with miR-21-5p inhibitors were significantly enhanced,but this enhancement could be reversed by the knockdown of CD97.These results indicated that miR-21-5p could target and regulate the expression of CD97,thereby affecting the proliferation,invasion and metastasis of hepatocellular carcinoma cells.In addition,miR-21-5p mimics inhibited the expression of p-m TOR(2448),p-PI3K(p85)and pAKT(473)in hepatocellular carcinoma cells,but this inhibition was compensated by the overexpression of CD97.miR-21-5p inhibitor up-regulated the expression of these molecules,which was reversed by the knockdown of CD97.These results indicate that miR-21-5p affects the activity of PI3K/AKT/m TOR signaling pathway by targeting CD97.Conclusion(s):1.The expression of CD97 in human hepatocellular carcinoma tissues was significantly higher than paracancer tissues.The expression level of CD97 is negatively correlated with the prognosis of hepatocellular carcinoma patients,and acts as an independent influencing factor of the prognosis.2.CD97 affects the proliferation,migration and invasion of hepatocellular carcinoma cells by regulating the cell cycle and the occurrence of epithelial-interstitial transition.CD97 mediates the proliferation,invasion and metastasis of hepatocellular carcinoma by activating the downstream PI3K/AKT/m TOR signaling pathway.3.miR-21-5p regulates the cell cycle and the occurrence of epithelial-interstitial transition by down-regulating the expression of CD97,and inhibits the proliferation,migration and invasion of hepatocellular carcinoma.miR-21-5p is involved in the activation of PI3K/AKT/m TOR signaling pathway by targeting the expression of CD97.