The Biological Characteristic Of AGTR1 Expression In Hepatocellular Carcinoma And Its Molecular Targeted Therapy

In recent years,with the rapid progress of genome sequencing technology,has accelerated the researchers’understanding of tumor heterogeneity.The database,containing a large number of tumor clinical samples,represented by Oncomine and TCGA,has attracted wide attention from different countries scholars,and is increasingly applied to the study of tumor.Evidence-based medicine has replaced experiential medicine as the starting point for scientific diagnosis and treatment gradually.The individualized medical-based"precision medicine"model emerges as the times require,which classifies the disease by traditional symptoms and signs and the molecular classification of the disease to find the most appropriate drug or treatment at the molecular level,and ultimately to achieve the purpose of personalized and precise treatment of the disease and the specific patients.Hepatocellular carcinoma（HCC）is one of the common malignant tumors,which has become the second leading cause of cancer death in China and the third in the world,China is a country with high incidence of liver cancer.Surgical resection is considered as a possible treatment for newly diagnosed HCC patients.However,only about 10%to 20%of patients’tumor can be resected,and surgical patients are facing the possibility of tumor recurrence.The 5-year survival rate in our country is only 14.1%according to the statistics from 2000 to 2014.In recent years,the development of targeted therapeutic drugs for hepatocellular carcinoma has gradually become a hot topic.However,due to a large number of different oncogenes and tumor suppressor genes involved in the occurrence and development of hepatocellular carcinoma,the exact molecular mechanism is not fully understood.Hepatocellular carcinoma has strong heterogeneity,large individual difference and low drug targeting.At present,sorafenib is the first FDA approved oral multi-kinase inhibitor for advanced HCC,but its efficiency is only 25%.In 1995,Lever published a large seminal retrospective study in The Lancet,found that patients who took angiotensin converting enzyme inhibitors（ACEIs）and angiotensin receptor blockers（ARBs）had lower risk of developing breast and lung cancer than those who did not take the drugs.But other scholars’study did not give the similar results.The different study results aroused the attention of many scholars.In these different studies,except for different factors such as race,population and medication cycle,patients’gene expression profiles were not considered.In further study,it was found that AGTR1,encoded by the key receptor type-1 angiotensin II（AT1R）in the renin-angiotensin system,was expressed heterogeneously in multiple tumors and that overexpression of this gene would cause a variety of tumors,including the poor prognosis of breast cancer,ovarian cancer,prostate cancer,gastric cancer,but the expression of AGTR1 in hepatocellular carcinoma and its impact on HCC has rarely been reported.Based on this,this study carried out relevant research.Part I Effect of Differential Expression of AGTR1 on the Development of Hepatocellular CarcinomaBased on 347 clinical RNA chip datas from all three hepatocellular carcinomas of the Oncomine database,we found that the AGTR1 gene was located in the top 5%of overexpressed genes in all three of these data sets and was highly expressed in some patients with hepatocellular carcinoma.At the same time we analyzed the differences of all the 374 hepatocellular carcinoma clinical samples in the TCGA database,and found that the relative expression level of AGTR1 in the first high 80 samples and the last low 80samples of AGTR1 were significantly different.Further combined with the prognosis data of hepatocellular carcinoma samples in the database,using the Kaplan-Meier survival analysis,we found that the lifetime of low expression group was significantly longer than that of the high expression group.The above results indicate that AGTR1 is highly expressed in some hepatocellular carcinoma patients,and this high expression is related to patients’survival.Therefore,it is preliminarily deduced that AGTR1 may play an important role in the occurrence and development of hepatocellular carcinoma.In order to prove the role of AGTR1 in the different stages of HCC,we screened the low-expression AGTR1 HCC cells SMMC-7721 and BEL-7404 and established the stable overexpression AGTR1 HCC cells,meanwhile we selected high-expression AGTR1 HCC cell MHCC-97H and established the stable interference expression AGTR1 HCC cells.Then we investigated the effect of AGTR1 on hepatocellular carcinoma in vivo and vitro level.We found that AGTR1 can promote cell proliferation,colony formation,cell invasion and migration in vitro and can also accelerate the formation and growth of the subcutaneous tumor formation in nude mice in vivo.In addition,we used the vector containing the luciferine fragments,built sh-AGTR1,and screened stable low-expression AGTR1MHCC-97H cells and control cells.We used small animal live imaging system to observe dynamically the metastasis of MHCC-97H cells with different AGTR1 expression in the spleen liver metastasis model,and found that inhibiting AGTR1 can reduce the metastasis ability of MHCC-97H HCC cells from spleen to liver.Part II Study on the Mechanism of the Differential Expression of AGTR1 on the Development of Hepatocellular CarcinomaIn order to study the mechanism of AGTR1 on the development and metastasis of HCC cells,we continued to overexpress or interfere with the expression of HCC cells.Further studied by Westen Blot,we found that AGTR1 in overexpression/high expression HCC cells can promote the phosphorylation of JAK2,STAT3 and ERK,while inhibiting AGTR1 can reduce the phosphorylation of corresponding molecules.AG490,a specific inhibitor of JAK2,can inhibit the growth,clonogenicity,migration and invasion ability of HCC cells due to the change of AGTR1 expression.Moreover AG490 can also inhibite the expression of VEGFA due to the over/high expression of AGTR1 in vitro.Therefore,we concluded that the over/high expression of AGTR1 probably activated the phosphorylation of JKA2,and then promote the phosphorylation of STAT3,ERK and other molecules in the cytoplasm.These phosphorylated molecules entered the nucleus and initiated the transcription,translation and synthesis of VEGF and other molecules,and promoted the secretion of VEGF and other cytokines.A large number of newly secreted VEGF maybe can bound with receptors on the cell surface to activate the intracellular signaling pathways such as STAT3,MAPK,and to promote the generation of neovascularization and the proliferation and migration of tumor cells.Part III:Study on the Anti-tumor Effect of AGTR1 Inhibitor on HCC CellsIn this study,we chose losartan,which is the longest clinical application time and the most frequent application drug,as the inhibitor of targeting AGTR1 to investigate its effect.In vitro experiments,we found that when the concentration of losartan was 1¹00μM,it can inhibit proliferation,colony formation,migration and invasion process of AGTR1over/high expession SMMC-7721 or MHCC-97H cells in vitro.However it did not have the above effects on AGTR1 low/interference expression SMMC-7721 or MHCC-97H cells.In the subcutaneous tumor-bearing model of nude mice experiment,after 4 weeks of administration of lostartan,we found that losartan with administration of 20mg·Kg^-180mg·Kg^-11 can inhibit the growth of subcutaneous tumors of MHCC-97H cells in nude mice compared with the blank control group.Immunohistochemical analysis showed that with the increase of the dose,the expression of AT1R,VEGFA and CD31 in the tumor decreased significantly respectively.Thus we found that losartan can inhibit the growth of AGTR1 over/high expresses HCC cells in vitro and vivo level.This study revealed that the AGTR1 can promote the proliferation,colony formation,migration and invasion of AGTR1 over/high expession HCC cells through JAK2/STAT3pathway,investigated the effect of losartan as a targeted inhibitor of AGTR1 over/high expression HCC cells,and provided experimental datas for the molecular mechanism of AGTR1 on the occurrence and development of hepatocarcinogenesis,finding effective targeted therapeutic drugs,and the treatment level of hepatocellular carcinoma especially the AGTR1 overexpression/high expression hepatocellular carcinoma.