VEPH1 Suppresses The Progression Of Gastric Cancer By Regulating Hippo-YAP Signaling Pathway

Gastric cancer(GC)is among the most common malignancies,which has the fifth highest incidence among all cancer types and leads to the third leading cause of cancer-related deaths worldwide.Currently,most GC patients are already in the advanced stage when they are diagnosed due to the atypical symptoms of early GC,resulting in the loss of the best chance of surgery and a poor 5-year survival rate.Ventricular zone-expressed PH domain-containing protein homolog 1(VEPH1)is an intracellular adaptor protein that is widely involved in the pathophysiological process of the body.VEPH1 mainly contains two functional domains,the N-terminal supercoiled domain and the C-terminal PH domain,which regulate cellular signaling and function by interacting with different molecules.Studies have shown that VEPH1 is differentially expressed in various cancers,including ovarian cancer,lung squamous cell carcinoma,and renal clear cell carcinoma,which affects the prognosis of patients.Recent studies have found that the expression of VEPH1 is reduced in hepatocellular carcinoma tissues,which is closely associated with the poor prognosis of patients.The low expression of VEPH1 unleashes the m TORC1 signal and then affects the progression of hepatocellular carcinoma.However,the expression and function of VEPH1 in GC are still unclear.This study mainly studies the role and molecular mechanism of VEPH1 in GC from the human body,cell and animal levels,which may provide certain theoretical basis for the clinical diagnosis and treatment of GC.Part1 Low expression of VEPH1 in GC is closely associated with the clinical characteristics of patientsAims: The expression level of VEPH1 in GC tissues was investigated,and the correlation between its expression and the clinical characteristics of patients was analyzed.Methods:(1)We collected clinical specimens from 135 patients with GC who were surgically resected and confirmed by pathology.The expression of VEPH1 protein in tumor tissues and adjacent normal tissues was analyzed by immunohistochemistry(IHC).In addition,we randomly selected 60 patients diagnosed with GC after endoscopic biopsy and collected GC tissue and adjacent normal tissue samples by endoscopy.The expression of VEPH1 in GC tissues and adjacent normal tissues was analyzed by real-time quantitative polymerase chain reaction(q RT-PCR)and western blot(WB).(2)The correlation of VEPH1 protein expression and the clinical characteristics of GC patients was statistically analyzed.(3)The relationship between the expression level of VEPH1 and the survival prognosis of GC patients was analyzed by Kaplan-Meier survival analysis.Results:(1)The expression levels of both VEPH1 m RNA and protein were significantly reduced in GC tissues compared to adjacent normal tissues(p<0.05).(2)The low expression of VEPH1 was negatively correlated with the tumor node metastasis(TNM)stage of GC patients,patients with lower VEPH1 expression had a higher TNM stage(p<0.05).There was no obvious correlation between VEPH1 expression level and the sex,age,tumor size and pathological grading of the patients(p>0.05).(3)The results of survival analysis showed that the 5-year survival rate of GC patients with low VEPH1 expression was significantly lower than those with high VEPH1 expression(p<0.05).Conclusions: VEPH1 is downregulated in GC tissues,and the expression of VEPH1 was closely related to the TNM stage and survival prognosis of GC patients.Part2 VEPH1 inhibit GC cell proliferation,migration,invasion,and EMTAims: To explore the effects of VEPH1 on GC cell proliferation,migration,invasion and EMT,as well as on subcutaneous neoplasia and abdominal metastasis in nude mice.Methods:(1)The expression of VEPH1 protein was determined in the normal gastric cell line GES-1 and the GC cell lines(MKN45,MKN 28,MKN 7,AGS,MGC803,SGC7901 and BGC823)by WB.(2)Immunofluorescence assay examined the localization of VEPH1 expression in GC cells SGC-7901 and MKN45.(3)SGC-7901 and MKN45 cells with VEPH1 knockdown or overexpression were constructed by lentivirus transfection,and the effects of VEPH1 on cell proliferation,migration and invasion were assessed by Cell counting kit-8(CCK-8)assay,Wound healing assay and Transwell assays.(4)After knockdown and overexpression of VEPH1,WB measured the expression levels of epithelial-mesenchymal transition(EMT)related proteins E-cadherin,N-cadherin,Twist and Snail in SGC-7901 and MKN45 cells.(5)Subcutaneous tumorigenesis model of BALB/c mice was used to further verify the effect of VEPH1 on tumor growth.(6)Peritoneal graft tumor model of BALB/c mice was used to further verify the effect of VEPH1 on tumor invasion and metastasis.Results:(1)The protein expression of VEPH1 was significantly lower in GC cell lines(MKN45,MKN28,MGC803,SGC-7901,and BGC823)than those in GES-1 cells(p<0.05).(2)Immunofluorescence experiments showed that VEPH1 was mainly present in the cytoplasm in the GC cells SGC-7901 and MKN45(p<0.05).(3)Interference of VEPH1 expression promoted the proliferation,migration and invasion of SGC-7901 and MKN45 cells(p<0.05).Overexpression of VEPH1 inhibited the proliferation,migration and invasion of SGC-7901 and MKN45 cells(p<0.05).(4)E-cadherin protein expression was decreased in MKN45 cells with VEPH1 knockdown,while N-cadherin,Twist and Snail were significantly increased.E-cadherin protein expression was increased in SGC-7901 cells with VEPH1 overexpression,while N-cadherin,Twist and Snail were significantly decreased(p<0.05).(5)The enforced expression of VEPH1 significantly inhibited the sizes and weights of the xenografts,whereas VEPH1 knockdown caused opposite results(p<0.05).(6)VEPH1 upregulation significantly decreased the number of xenografts in the abdomen of nude mice,whereas VEPH1 downregulation caused opposite results(p<0.05).Conclusions: VEPH1 plays its functional role as a tumor suppressor gene in GC.VEPH1 is able to inhibit the proliferation,migration,invasion and EMT of GC cells in vitro,as well as to reduce the tumorigenic and invasion capacity of GC cells in vivo.Part3 VEPH1 suppresses proliferation,migration,invasion and EMT of GC cells by regulating Hippo-YAP signaling pathwayAims: To investigate the molecular mechanism of VEPH1 suppresses proliferation,migration,invasion and EMT in GC cells by regulating Hippo-YAP signaling pathway.Method:(1)Signaling pathways associated to VEPH1 inhibition of GC progression by RNA sequencing and KEGG analysis.(2)According to the results of RNA sequencing and KEGG analysis,we evaluated the effect of VEPH1 knockdown on the expression of landmark proteins of AKT,Hippo and RAP1 signaling pathways such as AKT,Last1,YAP and RAP1 in SGC-7901 and MKN45 cells.(3)After interference and overexpression of VEPH1,the expression of the downstream target genes CYR61,FOXM1,EGFR,and AREG of the Hippo-YAP signaling pathway was examined by q RT-PCR.(4)After treatment with Hippo-YAP signaling pathway inhibitor(YAP/TAZ inhibitor-1),the changes in cell proliferation,migration and invasion ability of each group were evaluated by CCK-8 assay,Wound healing assay and Transwell assays.We also detected changes in the expression of EMT-related proteins,such as E-cadherin,N-cadherin,Twist,and Snail by WB.(5)The expression of YAP and EMT related proteins,such as E-cadherin,N-cadherin,Twist and Snail,were detected in nude mice with VEPH1 knockdown and VEPH1 overexpression groups,respectively.(6)The expression levels of VEPH1,YAP and EMT-related proteins E-cadherin,N-cadherin,Twist and Snail in human GC tissues and adjacent normal tissues.(7)Co-immunoprecipitation(Co-IP)assay examined the interaction between VEPH1 and YAP.Results:(1)By RNA sequencing and KEGG analysis,it was found that Hippo,RAP1 and PI3K/AKT signaling pathways may be important signaling pathways for VEPH1 to inhibit the biological behavior of GC cells.WB experiments further confirmed that the most obvious changes in the expression of the marker proteins Last1 and YAP in the Hippo signaling pathway(p<0.05).(2)The expression of Last1 was decreased,and the expression of YAP and TAZ were increased in SGC-7901 and MKN45 cells with VEPH1knockdown(p<0.05).In contrast,the expression of Last1 was increased,and the expression of YAP and TAZ were decreased in SGC-7901 and MKN45 cells with VEPH1 overexpression(p<0.05).The Co-IP results showed no binding of VEPH1 and YAP protein.(3)The expression of downstream target genes of the Hippo-YAP signaling pathway such as CYR61,FOXM1,EGFR,and AREG were increased in SGC-7901 and MKN45 cells with VEPH1 knockdown,while the expression of downstream target genes were decreased in SGC-7901 and MKN45 cells with VEPH1 overexpression(p<0.05).(4)Inhibition of YAP/TAZ,a key downstream molecules of the Hippo-YAP signaling pathway,was able to reverse the promotion effect of VEPH1-knockdown on cell proliferation,migration,invasion,and EMT in SGC-7901 and MKN45 cells(p<0.05).(5)The results of the subcutaneous tumorigenesis experiment in nude mice confirmed that the protein expression level of E-cadherin in the tumor tissue of the VEPH1-knockdown group was significantly lower than that of the control group,while the protein expression level of YAP,N-cadherin,Snail and Twist were significantly higher than that of the control group(p<0.05).However,the VEPH1-overexpression group showed the opposite result to the knockdown group(p<0.05).(6)The expression of VEPH1 and E-cadherin was decreased,and YAP,N-cadherin,Twist,and Snail were significantly increased in human GC tissues compared with adjacent normal tissues(p<0.05).Conclusions: VPEH1 can inhibit the proliferation,migration,invasion and EMT of GC cells by blocking the Hippp-YAP signaling pathway,and VPEH1 may become a potential target for the diagnosis and treatment of GC.