Effect And Mechanism Of FNDC1 In Promoting Gastric Cancer Invasion And Peritoneal Metastasis

Background and objective Gastric cancer(GC)has a high degree of malignancy and poor prognosis,and peritoneal metastasis(PM)is the most common and aggressive type of metastasis of GC and the main cause of death in patients with advanced GC.The mechanism of PM of GC has not been fully clarified,and due to the lack of effective predictitors,its diagnosis is difficult,while the treatment is even more limited,and the efficacy is unsatisfactory.Intraperitoneal chemotherapy(IPC)is a common method for clinical prevention and treatment of PM,but IPC also has therapeutic risks.Therefore,it is worth paying attention to differentiating the risk factors of PM,searching for specific molecular markers,and performing IPC prophylactically in high-risk patients to delay or even avoid peritoneal recurrence and metastasis.In our previous study,we screened multiple genes potentially associated with PM of GC by whole exon-capture sequencing(WES),in which FNDC1 was mutationally enriched in patients with PM,suggesting that it may be associated with PM of GC.On this basis,the aim of this study was to analyze the correlation between FNDC1 and PM and elucidate its specific mechanism of action by bioinformatics analysis,gene transfection,RNA interference,xenograft tumor and proteomics.Methods1.Bioinformatics analysis was performed using the gene expression database(GEO)to screen genes related to PM of GC,and the analysis results were compared with the previous WES detection results to verify that FNDC1 can be used as a relevant gene for PM for further study.2.Immunohistochemical staining(IHC)was used to detect the localization distribution and expression level of FNDC1 protein in 74 cases of GC and paired adjacent normal tissues,and its correlation with the clinical data and prognosis of patients with GC was analyzed.3.Real-time PCR(RT-PCR)and Western Blot(WB)were used to detected the levels of expression of FNDC1 in human GC cell lines and GES-1,and the cell lines with FNDC1 gene knockdown and overexpression were screened according to the results.4.Lentivirus-mediated FNDC1 gene knockout vectors were constructed subjected to viral packaging,followed by transfected into the GC cells AGS and MGC803 with relatively high FNDC1 expression.The cell lines stably knocked down FNDC1 gene were screened and identified,and then cell function experiments were performed to verify the role of FNDC1 in the invasion and metastasis process of GC.5.The impact of FNDC1 knockdown on the subcutaneous tumor formation ability was observed by xenograft assay.6.Lentiviral vectors were constructed to knockdown FNDC1 in AGS and MGC803 cells and to overexpress FNDC1 in BGC823 cells with relatively low FNDC1 expression.Then GC cells and xenograft tumor tissues were used to detect the changes of key proteins of epithelial cell transformation(EMT)and Wnt/?-catenin signaling pathway-related proteins by WB and co-immunoprecipitation(Co-IP),respectively.Thus,the role of FNDC1 in regulating the Wnt/?-catenin signaling pathway,thereby promoting EMT in GC cells was clarified.7.By lentiviral transfection,?-catenin was overexpressed in FNDC1-knockdown MGC803 cells,followed by cell invasion and scratch assay to observe the effects of FNDC1 knockdown and ?-catenin overexpression on the invasion and migration of GC cells.Results1.The FNDC1 gene was obtained using public database screening,and bioinformatics analysis suggested that FNDC1 was over-expression in GC and PM groups,which was correlated with reduced prognosis.Gene enrichment analysis(GSEA)showed a positive correlation between high expression of FNDC1 and EMT.2.The up-regulated of FNDC1 was correlated with tumor volume,tumor infiltration,lymph node metastasis,tumor stage,and PM.The expression of FNDC1 and was associated with reduced progression-free survival(PFS)and overall survival(OS).The expression level and depth of invasion of FNDC1 are independent risk factors leading to poor prognosis in patients with GC.3.The m RNA and protein levels of FNDC1 were significantly higher than those of GES-1 in all groups of GC cells,with MGC803 and AGS having the highest expression levels and BGC823 having relatively low expression levels.4.FNDC1 knockdown inhibited the proliferation,colony formation,invasion and migration ability of GC cells AGS and MGC803,and cell cycle analysis showed G2/M arrest.5.Xenograft assay revealed that the subcutaneous tumor formation ability of MGC803 was attenuated after FNDC1 knockdown.6.After FNDC1 knockdown,E-cadherin and Krt12 protein levels were significantly up-regulated,and vimentin,survivin,Snail and Slug protein levels were significantly decreased in MGC803 cell and xenograft tumor tissues;However,after FNDC1 overexpression,E-cadherin and Krt12 protein levels were significantly decreased,and vimentin,survivin,Snail and Slug protein levels were significantly increased.7.After FNDC1 knockdown,the total ?-catenin and nuclear ?-catenin protein levels in AGS and MGC803 were significantly decreased,and the ?-catenin ubiquitination level was significantly increased.8.The invasion and migration of GC cells were attenuated after FNDC1 knockdown,while overexpression of ?-catenin in GC cells with FNDC1 knockdown was able to reverse the trend.ConclusionFNDC1 is associated with GC invasion and PM.FNDC1 is overexpressed in GC tissues and cells and is associated with reduced prognosis.The expression of FNDC1 was an independent risk factor for prognosis.FNDC1 knockdown could significantly inhibit the proliferation,invasion and migration activity,and G2/M arrest of GC cells.Pathway analysis showed that FNDC1 may induce EMT in GC cells by regulating the Wnt/?-catenin signaling pathway and promote the invasion and metastasis of GC.FNDC1 has the potential to be used as a predictor of PM and may also be studied in depth as a therapeutic target for PM of GC,which has the translational application value and is worthy of further validation.