YAP1 Inhibition In HUVECs Is Associated With Released Exosomes And Increased Hepatocarcinoma Invasion And Metastasis

Objective: Hepatocellular carcinoma(HCC)is one of the most common malignant tumors of the digestive system in the world,and the incidence and mortality of liver cancer remain high all year round.In China,the annual mortality rate of liver cancer is second only to gastric cancer and lung cancer.The onset of liver cancer is hidden,and early symptoms are not obvious.70% of patients with liver cancer have lost the opportunity of surgery when they were diagnosed and can only receive conservative treatment.Therefore,exploring the mechanism of liver cancer development and effective treatment methods is the key to improve the current status of liver cancer treatment.Tumor neovascularization is one of the important characteristics of tumors.It plays an important role in the rapid proliferation of tumor cells and metastasis to distant places.The important role of tumor blood vessels in tumor progression makes anti-tumor neovascularization therapy Current effective methods for treating tumors.In recent years,drugs that target tumor neovascularization as therapeutic targets have made great progress in tumor treatment.Along with the clinical application of anti-tumor neovascularization,more and more studies have found that antiangiogenesis has achieved good results in the treatment of liver cancer.However,there are still some patients who cannot benefit from anti-neoplastic neovascularization.Some patients have achieved significant results in early anti-vascular therapy,but with the extension of the treatment time,the phenomenon of tumor metastasis to distant places has appeared.Therefore,more and more effective therapeutic targets are needed to improve the anti-tumor neovascular therapy in Therapeutic effect in liver cancer.Hippo signaling pathway is a highly conserved signaling pathway discovered in recent years.It can participate in the regulation of organ size in vivo and cell proliferation and apoptosis through its core transcriptional regulatory factor Yes-associated protein 1(YAP1).Studies have confirmed that YAP1 is highly expressed in a variety of tumors and is closely related to tumor progression and prognosis.Especially in recent years,studies have confirmed that the YAP1 inhibitor Verteporfin has achieved good results in tumor treatment,suggesting that YAP1 is expected to become a new target for tumor treatment.So,whether YAP1 can be used as a target for anti-neovascular neovascularization and its mechanism is worthy of further study.Previous studies have confirmed that YAP1 exerts its important mechanism by regulating the expression of long non-coding RNA in cells.So,whether YAP1's role in the regulation of liver cancer angiogenesis is related to lnc RNA is worthy of our in-depth study.Especially in the course of anti-neovascular therapy,vascular endothelial cells remodel the tumor microenvironment,and whether this remodeling has an effect on tumor cell proliferation,invasion,and other biological behaviors are the focus of our attention.MALAT1(metastasis-associated lung adenocarcinoma transcript 1,MALAT1)belongs to the lnc RNA family and was first discovered in non-small cell lung cancer research.Increasing evidence shows that MALAT1 plays an important regulatory role in a variety of tumors.Recent studies suggest that MALAT1 can not only play an important regulatory role in cells,but also MALAT1 can be released into the tumor microenvironment and taken up by other cells through the exosomes,and then play its important biological role.MALAT1 not only plays an important regulatory role in tumor progression,but also has important regulatory functions in the process of angiogenesis.So,in vascular endothelial cells,whether the effect of YAP1 deletion on endothelial cells is related to MALAT1,and what role the deletion of vascular endothelial cells YAP1 plays in the remodeling of tumor microenvironment deserves our attention.In summary,what function YAP1 plays in vascular endothelial cells and whether it can be an effective target for anti-tumor vascular therapy deserves further study.In this part of the study,we intend to detect the expression of YAP1 in liver cancer tissues and the role of YAP1 in angiogenesis of liver cancer by Western blot,real-time PCR,immunohistochemistry and other experimental methods to provide data support for the next molecular mechanism experiments.Methods: 1)Western blot was used to detect the expression level of YAP1 in Hepatocellular carcinoma tissues,and the relationship between YAP1 expression and angiogenesis factor was analyzed by real-time PCR and TCGA database;the relationship between YAP1 and angiogenesis was detected by bioinformatics;Tipofin and transfection of si RNA inhibited YAP1 expression.CCK-8,transwell,and angiogenesis experiments were used to detect the effects of YAP1 deletion on endothelial cells.Transfection of YAP5 SA was used to inhibit YAP1 expression.CCK-8,transwell,and blood vessels Generation experiment to detect the effects of YAP1 activation on endothelial cells;GSEA to analyze changes in lnc RNA expression in endothelial cells after YAP1 deletion in endothelial cells;PCR verification to detect changes in lnc RNA expression in endothelial cells after YAP1 deletion;knock down MALAT1 by transfection si RNA The function of MALAT1 in endothelial cells was detected by CCK-8,transwell,and angiogenesis experiments;the mechanism of YAP1 regulating endothelial cell function was clarified by co-transfection of YAP5 SA and Si MALAT1 2)Collect conditioned medium after endothelial cell YAP1 deletion,and test the effect of conditioned medium on the biological behavior of liver cancer cells;use ultracentrifugation to extract exosomes in conditioned medium after YAP1 deletion,and detect the exosomes by NTA.The diameter and distribution of exosomes were detected by western blot and transmission electron microscopy,respectively.Exosome surface markers and morphology were analyzed.The biological process of YAP1-deleted endothelial cells was analyzed by bioinformatics.The expression of MALAT1 in exosomal contents was detected by PCR.;Click-i T was used to detect the expression of newly generated MALAT1 in hepatocellular carcinoma cells after exosomal induction;Western blot and immunofluorescence experiments were used to detect the mechanism of exosome release after endothelial cell YAP1 deletion;analysis of exosomes by bioinformatics methods The mechanism to promote the invasion and metastasis of liver cancer.The activation of ERK1 / 2 in hepatocellular carcinoma after exosome induction was detected by western blot,and the mechanism of exosomes to promote the invasion and metastasis of liver cancer cells was verified by giving PD98059,an ERK1 / 2 inhibitor.Results:(1)The expression of YAP1 gene is closely related to multiple angiogenic factors(CD31,SPHK1,SPHK2,VEGF);(2)After YAP1 deletion,vascular endothelial cells increase capacity,migration and angiogenesis ability decrease significantly;(3)Lnc RNA MALAT1 is modulated by YAP1 deficiency in angiogenesis;(4)YAP1 deficiency increases the number of exosomes released from HUVEC cells and promotes HCC invasion and migration;(5)YAP1 deficiency leads to exosomal MALAT1 accumulation and promotes the expression of MALAT1 in HCC;(6)The expression of MALAT1 closely related to TNM stage,degree of differentiation, portal vein infiltration and lymph node metastasis;(7)Bioinformatics analysis confirmed MALAT1 is closely related to the invasion of liver cancer;(8)Patients with MALAT1 expression had nothing to do with patient OS,but were significantly related to patient DFS;(9)The ability of hepatocellular carcinoma to invade and migrate was significantly inhibited after knocking down MALAT1 expression;(10)VAMP3 mediates the release of exosomes caused by the absence of YAP1 in endothelial cells;(11)Exosomes released from endothelial cell YAP1 deletion promote the invasion and metastasis of liver cancer cells through the ERK1/2 pathwayConclusion: Altogether,our data demonstrate that vascular endothelial cell YAP1 deficiency promotes release of MALAT1-containing exosomes and effectively inhibits angiogenesis.Released exosomes promote the distant migration of hepatoma tumor cells by activating the ERK1/2 pathway;accordingly,ERK1/2 inhibitors can reverse the metastasis-promoting effect of YAP1 deficiency.Our results suggest that the combination of Verteporfin and ERK1/2 inhibitor may be an effective new approach for antiangiogenesis therapy.