RACO-1 Modulates Hippo Signaling In Esophageal Squamous Cell Carcinoma

Background:Esophageal cancer ranks among the top 10 malignant tumors in the world in terms of morbidity and mortality,showing significant regional distribution differences.It is one of the malignant tumors with the highest mortality in Henan province and other regions.More than 95% of esophageal cancer in China are esophageal squamous cell carcinoma(ESCC),which is quite different from the majority of esophageal adenocarcinomas in the west.Although the specific pathogenesis of carcinogenic mechanisms is not completely clear,recent genomic sequencing data showed dysregulation of Hippo signaling could be a critical factor in the development of ESCC.In our previous study,the silencing of RACO-1(Ring Domain AP-1 Co-Activator 1)in the esophageal squamous cell carcinoma cell lines can regulate the stability of YAP protein,and it is believed that RACO-1 is a new regulator of the Hippo signaling pathway.Therefore,further study on the regulation of RACO-1 on the Hippo signaling pathway and the YAP protein transport mechanism is of great significance for improving the early diagnosis and targeted treatment of esophageal squamous cell carcinoma.Objective:To elucidate the mechanism of RACO-1 in regulating Hippo signaling pathway in ESCC,and provide new ideas for early screening and specific therapeutic targets of ESCC.Methods:1.Silencing RACO-1 in ESCC cells,using Real-time quantitative polymerase chain reactionquantitative(q RT-PCR)and Western blot to detect m RNA and protein levels in the silent group cells and the control group cells to determine the silencing efficiency.2.Useing Wound healing experiments and Transwell experiments to evaluate the effects of RACO-1 on the repair ability and migration and invasion ability of ESCC cells.3.Using WST assay to detect the effect of RACO-1 on proliferation ability of ESCC cells.4.Co-immunoprecipitation and immunofluorescence co-localization experiments are worked to determine the localization of RACO-1 and YAP in ESCC cells and whether the two are related.5.Silencing RACO-1 in ESCC cells,using Western blot and q RT-PCR to detect the expression of YAP protein and YAP target gene m RNA.6.Through the Rescue experiment,determining whether the change of invasion and migration of ESCC by RACO-1 knocking-down is due to the change of Hippo signaling pathway.7.Using MG132,CHX and ubiquitination experiments,determining whether RACO-1affected the stability of YAP protein through ubiquitination.Results:1.Silencing RACO-1 in the ESCC cells,the results showed that the expression levels of its protein and m RNA were decreased compared with the control group(P<0.05),indicating successful silencing.2.The Wound healing experiment observed that after the silencing of RACO-1 in esophageal squamous cell lines,the healing rate and the migration ability of the experimental group were increased compared with that of the control group(P<0.05),and the Transwell experiment presented that the migration ability of the silencing group was significantly increased compared with that of the control group(P<0.05).3.WST experiment proved that the loss of RACO-1 inhibited the proliferation ability of ESCC cells.4.Co-immunoprecipitation experiments displayed that RACO-1 and YAP were endogenous in ESCC cells.The co-localization of RACO-1 and YAP in ESCC cells was demonstrated by immunofluorescence.5.RACO-1 deletion in ESCC cells increased the expression of YAP protein level,and the levels of CTGF and CYR61 m RNA,which are the target genes of YAP,were also increased(P<0.05).6.The increased cell invasion and migration by RACO-1 depletion in ESCC cells could be rescued by further knocking-down of YAP.7.MG132,CHX and ubiquitination experiments showed that RACO-1 could regulate the stability of YAP protein through ubiquitination.Conclusions RACO-1 as an endogenous inhibitor for YAP/TEAD axis of Hippo signaling in ESCC.RACO-1 inhibits migration and invasion of esophageal cancer cells by regulating Hippo/YAP signaling pathway through ubiquitination.Regulation of RACO-1 activity or expression may be a feasible strategy for the treatments of esophageal cancer patients.