Expression And Clinical Significance Of ARMCX1 In Endometrial Carcinoma

【 Objective 】To investigate the expression level and role of Armadillo repeat containing X-linked 1(ARMCX1)in Endometrial Cancer(EC),and to determine the relationship between ARMCX1 expression and EC by comprehensive bioinformatics analysis.It provides new ideas and basis for clinical diagnosis and treatment.【 Method】 The study was divided into two parts :1.Bioinformatics Analysis The study was performed using the GEOquery package of R software(version 3.6.3)from Gene Expression Omnibus(Gene Expression Omnibus).Four endometrial cancer datasets(GSE115810,GSE17025,GSE63678 and GSE23518)were downloaded from GEO database,including 142 endometrial cancer tissues and 20 corresponding normal tissues.The limma package(version 3.42.2)was used to analyze the difference between the two groups and draw the volcano diagram.The Venn Diagram package of R software(version 4.2.1)was used to analyze the unique and co-expressed genes between each dataset,and the target gene ARMCX1 was determined.cluster Profiler(4.4.4)package was used to perform enrichment analysis of the co-expressed genes to explore more roles and functions of ARMCX1 in tumors.GEPIA 2 database was used to explore the expression level of ARMCX1 in pan-cancer,and UALCAN database was used to explore the m RNA and protein expression levels of ARMCX1 in endometrial cancer.The Survival Analysis online analysis module of GIPA2 database was used to analyze the relationship between ARMCX1 expression level and the prognosis of EC patients.c Bio Portal database was used to further explore the genetic changes of ARMCX1 in different tumors and the relationship between ARMCX1 mutation and the prognosis of patients with different tumors.The RNAseq data of TCGA-UCEC(endometrial cancer)project STAR process were downloaded and sorted from TCGA database using R software(version 4.2.1),and the data in FPKM format and clinical data were extracted.The correlation between the main variables in the data and the immune invasion matrix data was analyzed.Immune infiltration algorithm: Based on the ss GSEA algorithm provided in the R package-GSVA(1.46.0),24 immune cell markers provided in immune-related articles were used to calculate the immune infiltration of the corresponding cloud data.Data processing method: log2(value+1);Statistical method:spearman.R software(version 4.2.1)was used to download and collate RNAseq data of TCGA-UCEC(endometrial cancer)project STAR process from TCGA database and extract data in FPKM format and clinical data.ROC analysis was performed on the data using p ROC package(version 1.18.0).log2(value+1);All the above results were visualized using the ggplot2 package(version 3.3.6).2.Immunohistochemistry(IHC)From August 2020 to December 2022,17 cases of endometrial cancer(EC),17 cases of atypical endometrial hyperplasia(AEH)and 17 cases of normal proliferative endometrium(NP)were collected by IHC.The clinical information of the patients was obtained,and the expression level of ARMCX1 in the samples was measured by IHC.The expression of ARMCX1 in EC patients was verified.3.Statistical methods The basic data of experimental samples were analyzed by using R software(4.2.1)version stats(4.2.1)package.Because age,height,weight and BMI did not meet the normal distribution,Kruskal-Wallis method was used.Chisq test was used for comparison between menopausal groups.Yates’ correction test was used for comparison between hypertension groups.Fisher’s exact test was used for comparison between diabetic groups.The expression level of ARMCX1 in each clinical sample group was tested by overall test(Welch one-way ANOVA test).ROC analysis was performed with p ROC(1.18.0)package,and the results were visualized with ggplot2(3.3.6).The expression level of ARMCX1 in different clinical characteristics of EC group was analyzed by SPSS version 25.0.The metastasis and infiltration degree were analyzed by t-test,and the pathological type group was analyzed by Welch one-way ANOVA.【 Result 】1.using limma(3.42.2 version)package,with | log2 FC | > 1,the rectified P values< 0.05 for the standard in four data sets were screened 3305 different genes,including1202 high expression of 2103 lower expression.Among them,25 were highly expressed and 280 were low-expressed in GSE115810 dataset.GSE17025 dataset had 904 high expression numbers and 1630 low expression numbers.GSE23518 dataset had 15 high expression numbers and 16 low expression numbers.In GSE63678 dataset,258 were highly expressed and 177 were low-expressed.ARMCX1 belonged to the low expression group.2.The online functional analysis of UALCAN database showed that the maximum m RNA expression level of ARMCX1 in normal tissues was 75.924 TPM,the minimum was 13.12 TPM,and the median was 41.626 TPM.In EC,the maximum m RNA expression level was 22.214 TPM,the minimum expression level was 0.131 TPM,and the median was 4.057 TPM.The maximum protein expression level of ARMCX1 in normal tissues was 2.179,the minimum was 0.498,and the median was 1.319.The largest protein expression level in EC was 2.924,the smallest was-1.817,and the median was 0.3.The results of genetic variation analysis showed that ARMCX1 was mainly changed into "mutation"(3.97%)and "amplification"(0.38%)in EC,and there was no correlation between ARMCX1 mutation and the prognosis of EC patients(P > 0.05).4.Survival analysis showed that ARMCX1 expression level was not related to the prognosis of EC(P > 0.05).5.The results of immune infiltration showed that the expression of ARMCX1 was positively correlated with Tcm,macrophages and NK cells,and negatively correlated with Th17 cells,CD56 bright cells,NK CD56 dim cells,TReg and DC cells(P < 0.05).6.Immunohistochemistry showed that the mean expression level of ARMCX1 in the normal group was 6.1176 ± 2.713,the mean expression level in the atypical hyperplasia group was 6.2353±3.7338,and the mean expression level in the endometrial cancer group was 3.6471 ± 1.6179.The average expression level in the normal and atypical hyperplasia groups was higher than that in the tumor sample group.7.The diagnostic ROC showed that the area under the ROC curve of the data from the TCGA database was 0.965[95% confidence interval(CI): 0.946%-98.4%],the cut-off value was 3.447,the sensitivity was 0.82671,and the specificity was 1.The area under the ROC curve of clinical samples was 0.734[95% confidence interval(CI):54%-87.8%],the cut-off value was 7,the sensitivity was 1,and the specificity was0.38889.The area under the curve of ARMCX1 and ARMCX1 were both above 0.7,indicating that ARMCX1 had a certain diagnostic value in EC.【 Conclusion 】1.The protein and m RNA expression levels of ARMCX1 in EC were lower than those in normal tissues.2.ARMCX1 is mainly changed into "mutation" and "amplification" in EC,and there is no correlation between ARMCX1 mutation and prognosis of EC patients.3.ARMCX1 expression was positively correlated with Tcm,macrophages and NK cells,and negatively correlated with Th17 cells,CD56 bright cells,NK CD56 dim cells,TReg cells and DC cells.4.The expression level of ARMCX1 m RNA in EC was significantly lower than that in the normal group and the atypical hyperplasia group,while its expression level in the normal group and the atypical hyperplasia group was not statistically significant.5.ARMCX1 can be used as a novel marker for EC diagnosis and a potential therapeutic target.