| Background and objective:Ovarian cancer(OC)is the fifth leading cause of cancer death in women worldwide.Its high mortality rate is mostly due to the occult clinical symptoms of early OC,which often progresses to the middle and late stages of the disease when diagnosed.The treatment of OC mainly includes surgical resection,chemotherapy and radiotherapy,but the surgical treatment effect of patients with advanced OC is not ideal,and they are at higher risk of recurrence,metastasis,drug resistance and worse prognosis.If effective and accurate tumor markers or new gene targets can be found,it is expected to improve the survival rate and prognosis of patients.Leucine rich repeat and fibronectin type Ⅲ domain containing 1(LRFN1)is a synapse-associated protein located on chromosome 19q13.2.Some studies have established gene marker panels including LRFN1 or tumor mutation load risk models,suggesting that they can help identify OC patients with poor prognosis,but the specific mechanism is unknown.However,there are few studies on LRFN1 in OC.Through bioinformatics data analysis and subsequent basic experimental verification,this study aims to explore:(1)the role of LRFN1 in the occurrence and development of OC and the possible related pathways;(2)The potential value of LRFN1 as a diagnostic marker and prognostic indicator of OC..Methods:1.Bioinformatics analysis(1)Using TCGA and GTEx databases,all datas were standardized by R software to screen differential genes.Combined with the clinical data of OC patients in TCGA database,the relationship between LRFN1 expression in OC and 5-year overall survival(OS)was analyzed.The ROC curve of the clinical diagnostic value of LRFN1 in OC was drawn using the ’ pROC ’ package in R language.(2)The GEPIA database was used to verify the expression of LRFN1 in different tumors and OC.The 5-year OS analysis of patients with different expression of LRFN1 in ovarian cancer was verified in the GEPIA database.(3)The median of LRFN1 expression was set as the standard,and the "Limma"package in R software was used to analyze the difference of OC tissue sample gene matrix(divided into LRFN1 high/low expression group)in TCGA database.GO analysis and KEGG pathway enrichment analysis of differential genes were performed using the DAVID database.(4)The relationship between LRFN1 and tumor immune infiltration in OC was further analyzed by TIMER and TISIDE databases..(5)The cBioportal database was used to explore the relationship between LRFN1 gene mutation and OC and related pathway analysis.2.Basic experimental verification(1)The clinicopathological information of 60 patients with epithelial ovarian cancer and 15 patients with benign tumors who underwent total resection in Bethune First Hospital of Jilin University from June 2012 to September 2022 was collected.According to the process and requirements,the pathological wax blocks of ovarian cancer and normal ovarian tissue were obtained and sliced.The expression level of LRFN1 in clinical patients’ pathological tissue sections was detected by immunohistochemical staining,and SPSS software was used for data statistical analysis..(2)The IOSE-80,TOV-112D,OVCAR3,SKOV3 and A2780 cell lines were cultured,and the expression of LRFN1 in normal ovarian cell line OC cell lines was detected by Western Blotting(WB).3.Statistics and data analysisIBM SPSS Statistics 25 software was used to analyze the data.Graphpad Prism 8 was used for chart drawing,and student-t test was used for data analysis,p<0.05 was statistically significant.Comparisons between groups for categorical data were made using the χ2 test or Fisher test(when a two-sidedp<0.05 was statistically significant).Results:(1)The combined analysis of TCGA and GTEx database showed that LRFN1 was highly expressed in ovarian cancer tissues(adj.p=9.28E-57 and |log2FC|=1.25).In addition,the overall survival rate of ovarian cancer patients with high LRFN1 expression was lower than that of patients with low LRFN1 expression(pHR=0.0085).AUC(area under the ROC curve)was 92.8%,suggesting that high expression of LRFN1 may have a certain value in the diagnosis of ovarian cancer(p<0.05).(2)The analysis results in the GEPIA database showed that LRFN1 was highly expressed in various cancer tissues(including OC),and high expression of LRFN1 was not conducive to the overall survival rate of OC patients(pHR=0.016).(3)Immunohistochemical results of clinical pathological sections showed that the expression of LRFN1 in OC tissues was higher than that in normal ovarian tissues(p<0.001).(4)Western Blotting results showed that the expression of LRFN1 in OC cell lines TOV-112D,OVCAR3,SKOV3 and A2780 was higher than that in normal ovarian cell line IOSE-80(p<0.001).(5)The results of pathway enrichment analysis suggested that LRFN1 might be enriched in immune-related pathways such as peptide antigen assembly of MHC classⅡ protein complex,TH1 and TH2 cell differentiation(p<0.05).(6)The results of TIMER database analysis showed that LRFN1 was positively correlated with the infiltration of B cells,immature B cells,neutrophils,NK cells,immature CD4+T cells and other immune cells in OC(p<0.005).The results of TISIDE database analysis showed that LRFN1 expression was positively correlated with immunosuppressive factors PVRL2(r=0.281,p=6.37e-07)and CD276(r=0.186,p=0.00106)in OC.(7)The results of cBioportal database analysis showed that in OC,LRFN1 mutations were mainly concentrated in amplification mutations;when LRFN1 was mutated,the 5-year OS of patients decreased significantly(pHR<0.005).When LRFN1 and TP53 genes were co-expressed,the probability of gene mutation was high(56.4%).This mutation might affect biological processes such as cell proliferation,survival and apoptosis.Conclusion:(1)The expression of LRFN1 in OC is higher than that in normal ovary,which may be related to the occurrence and development of OC.(2)The prognosis of OC patients with high expression of LRFN1 is worse.When LRFN1 is mutated,the prognosis of patients is worse,suggesting that LRFN1 has certain clinical value in the application of potential diagnosis and prognosis of OC.(3)LRFN1 is enriched in immune-related pathways such as TH1 and TH2 cell differentiation.The expression of LRFN1 in OC is related to the level of immune cell infiltration,which may be used as a potential diagnostic marker and prognostic indicator of OC.LRFN1 expression may be related to omentum metastasis and disease location in patients with epithelial ovarian cancer(4)The mutation of LRFN1 in OC is mainly amplified variation.When co-expressed with TP53 gene,the probability of mutation is increased significantly. |
