Mechanism Of YAP Promoting The HCC Development By Regulating The PGE2 Signaling Pathway

Background:Hepatocellular carcinoma(HCC)is one of malignant tumors with the highest mortality rate in the world.The incidence of HCC in China is is higher than most of countries and regions.HCC is prone to early metastasis and recurrence.Furthermore,there is currently a lack of effective treatments.As such,it is very important to better understanding of special genes and pathways of HCC development in order to improve the treatment of HCC and survival of HCC patients.Hippo signaling pathway is a cell growth inhibitory pathway,which is highly conserved during evolution.Yes-Associated Protein(YAP),an important oncogene,is the key downstream effector of Hippo pathway.YAP is involved in the regulation of proliferation and apoptosis-related target genes,and is closely related to the occurrence and development of human tumors,including HCC.Accordingly,the Hippo-YAP pathway is a significant target for cancer therapy.Inflammation is a major factor promoting in tumor growth.PGE2,a typical inflammatory factor,could enhance tumor cell survival,growth,metastasis,invasion,angiogenesis,and immune suppression,thereby promoting the development of cancer.15-PGDH is the key catabolic enzyme of PGE2.COX is a key regulatory enzyme in PGE2 synthesis.And its inhibitors,including aspirin and celecoxib,have attracted much attention for the anticancer effect.Accumulating evidences suggest that the Hippo signaling pathway is associated with the PGE2 signaling pathway.For instance,both of them have been shown to could promote the proliferation of tumor cells,inhibit the apoptosis of tumor cells,and participate in the regulation of tissue growth.However,the relationship between Hippo signaling pathway and PGE2 signaling pathway in the occurrence and development of HCC remains an open question.The aims of this study were to investigate the role of PGE2 signaling pathway in the process of YAP promoting the development of HCC and the potential value of PGE2 inhibitors in the treatment of HCC.Method:1 The expression of YAP was examined by immunohistochemistry in HCC tissues and paired adjacent normal tissues.The relationship between the expression of YAP and the prognosis of HCC patients was analyzed by Kaplan-Meier test.The relationship between the expression levels of YAP and pathological parameters was analyzed by Chi square test.2 MST1-/-MST2fl/fl knockout mice were injected with Cre-adenovirus to achieve the MST2 liver specific knockout.Through transient transfection,lentiviral infection to achieve the differential expression of YAP in HCC cell lines.The expression level of YAP and the key molecules in PGE2 signaling pathway was detected by Western Blot and RT-PCR in mouse liver and HCC cell lines.The levels of cell line PGE2 were observed by ELISA kit.Furthermore,the regulatory effect of YAP on PGE2 signaling pathway was analyzed.3 After treating HCC cell lines with activators of PGE2 signaling pathway,the levels of YAP and the ability of cell proliferation were observed.After overexpression of YAP in HCC cell lines,the inhibitor of PGE2 signaling pathway were added,the expression of YAP and PGE2 signaling pathway was detected and the abilities of cellular proliferation,EMT and stem cell-like behaviors were observed.Through subcutaneous tumor to observe the effect of PGE2 signaling pathway inhibitors on HCC cell lines in vitro.4 The correlation between the expression level of YAP and PGE2 signaling pathway was analyzed in human HCC samples through IHC,Western Blot,and RT-PCR.Result:1 YAP was highly expressed in the most of HCC tissues,whereas its expressions in the most of paired adjacent normal tissues are low.The high expression level of YAP was positively associated with the poor prognosis of HCC patients,the degree of tumor differentiation,metastasis,and TNM grade of HCC.2 After the specific knockout of MST1/2 gene in liver of mice,the liver volume increased and the tumor formed.The level of YAP protein was negatively correlated with the expression of 15-PGDH protein,but positively correlated with the level of COX-2 RNA in liver tissue of the mice and HCC cell lines.3 After treating HCC cell lines with activators of PGE2 signaling pathway,the expressions of YAP protein and its target genes were increased.Moreover,the proliferation ability of HCC cell lines.PGE2 signaling pathway inhibitors reversed the increased expression of YAP and the abilities of cellular proliferation,EMT and stem cell-like behaviors after overexpression of YAP.4 The level of YAP was positively correlated with COX-2,but negatively correlated with 15-PGDH in human HCC samples.Conclusion:YAP promoted the occurrence and development of HCC.There was a closely relationship between Hippo signaling pathway and PGE2 signaling pathway in HCC.In liver-specific MST1-/-MST2fl/fl knockout mice and HCC cell lines,the increase of YAP level was accompanied by the change of PGE2 signaling pathway.In particular,15-PGDH protein level was down-regulated,whereas the level of COX-2 RNA and the PGE2 were up-regulated.The stimulation of PGE2 signaling pathway activators increased the level of YAP and enhanced the transcriptional activity of YAP.In contrast,PGE2 signaling pathway inhibitors down-regulated the level of YAP,and inhibited the cell proliferation,cell colony formation rate,and the levels of EMT and stem cell associated gene.Moreover,the expression level of YAP was correlated with 15-PGDH and COX-2 in clinical HCC samples.In conclusion,Inhibition of COX-2 may be an effective treatment for HCC patients with high expression of YAP.