| BackgroundEndometrial cancer(EC)is one of the most common gynecologic malignancies in the world,accounting for 7%of the total number of malignant tumors in women,and about 20%to 30%of the female reproductive system tumors.It is estimated there will be 66570 new cases of uterine tumors and 12940 deaths in the United States in 2021[1].In addition,the incidence rate and mortality rate of EC has steadily increased over the past ten years.According to the International Federation of Obstetrics and gynecology(FIGO)staging system,at present,EC is mainly classified according to FIGO stage,tumor histology classification and histological type.It is important to note that EC patients with similar clinicopathological characteristics often have different disease outcomes,the molecular heterogeneity is one of the important reasons for this phenomenon may affect of tumor invasion and metastasis[2].Nucleolin(NCL)is the most abundant non ribosome phosphorylation family in nucleolus,mainly distributed in the nucleus,and participates in regulating various cell processes.In recent years,many studies have found that NCL may be related to tumor development[3-7].Some studies have pointed out that the NCL is usually located in the nucleus in normal tissues,and in some malignant tumors,NCL is often found to show abnormal high expression in cytoplasm and cell membrane,and this abnormal expression may be closely related to the poor prognosis of tumor patients.In this study,the expression of NCL in EC was detected by bioinformatics to the relationship between the expression of NCL mRNA and the Clinicopathologic Characteristics and prognosis of EC patients;The expression level of NCL protein was detected,and the correlation between the expression level and distribution of NCL protein and prognosis of type I EC was analyzed.Objective1.To explore the expression of NCL mRNA in EC and its correlation with clinicopathologic characteristics,and to explore its impact on the prognosis of patients.2.To detect the expression and localization of NCL protein in type I EC and relationship with prognosisMethods1.To detect the NCL mRNA expression in EC and its clinical significance.Downloaded the data of NCL mRNA expression in EC tissues and adjacent normal tissues from The Cancer Genome Atlas(TCGA).Analyzed its correlation with Clinicopathologic Characteristics of patients.Survival analysis was used to test its relationship with prognosis2.To detect the protein expression and localization of NCL in EEC and relationship with prognosis.Immunohistochemistry was used to detect the protein expression of NCL in 82 EEC tissues and 15 normal tissues,and to analyze its correlation with the prognosis of patients.Results1.The expression and significance of NCL mRNA in EC.The RNA sequencing data and clinicopathological characteristics of 494 EC patients were downloaded from the official website of TCGA Uterine Corpus Endometrial Carcinoma(UCEC)project.The R software package "Deseq2" was used to standardize the RNA sequencing data and integrate the NCL mRNA data,clinicopathological characteristics and survival data.According to the mRNA expression level of NCL,494 patients with endometrial carcinoma were divided into three groups:low expression group(n=165),medium expression group(n=165)and high expression group(n=164).23 patients had RNA sequencing expression data of adjacent tissues;those were recorded as adjacent control group(n=23).The results showed that there was no significant difference between the adjacent control group and the medium NCL expression group(P>0.05),but the NCL expression of adjacent control group was significantly lower than the high NCL expression group(P<0.001),and significantly higher than the low NCL expression group(P<0.001).The results showed that there might be over-expression of NCL in the high expression group and down-regulation of NCL in the low expression group.Downloaded the data of NCL mRNA expression of EC tissues and adjacent normal tissues from TCGA.Analyzed the relationship between NCL mRNA expression and clinicopathologic characteristics.The increased expression of NCL mRNA was significantly correlated with serous endometrial carcinoma(P<0.001),advanced EC(FIGO Ⅲ-Ⅳ)(P=0.029)and low-grade EC(P<0.001).2.The relationship between the expression level of NCL mRNA in EC and clinicopathological features.2.1 Clinicopathological features of patients in TCGA.The average age of 494 patients with EC is(63.61±11.84)years old.There were 377(76.3%)patients with type I EC,95(19.4%)patients with type Ⅱ EC and 24(4.3%)patients with mixed EC.56.3%(278/494)patients were diagnosed with G3 tumors,23.3%(115/494)with G2,18.4%(91/494)with G1.62.6%(309/494)patients with FIGO stage I tumors,stage Ⅱaccounted for 10.1%(50/494),stage Ⅲ accounted for 22.5%(1 11/494)and stage Ⅳfor 4.8%(24/494).2.2 Relationship between the expression of NCL mRNA and clinicopathological features.High expression of NCL mRNA was significantly correlated with pathological type(P<0.001),FIGO stage(P=0.029)and histological grade(P<0.001),but not with surgical method and age(P>0.05).The difference of NCL mRNA expression between type I EC group and type Ⅱ EC group was compared.The results showed that the expression of NCL mRNA in type Ⅱ EC was significantly higher than that in type Ⅰ EC(P<0.001).Compared with FIGO Ⅰ+Ⅱ group,the expression of NCL mRNA in FIGO Ⅲ+ Ⅳ group was significantly higher than that in FIGO Ⅰ+Ⅱ group(P<0.01).The patients in TCGA were divided into three groups:G1,G2 and G3.The expression of NCL mRNA in G3 group was significantly higher than that in G1 and G2 groups(P<0.001).It is suggested that the up regulation of NCL expression may related to tumor progression.3.The relationship of expression level of NCL mRNA in EC with prognosis.3.1 The relationship between the expression of NCL mRNA and the survival time of EC patients.The overall survival curve and disease-free survival curve of high,medium and low expression groups of NCL mRNA were drawn by Kaplan-Meier survival analysis,and the prognostic value of NCL mRNA expression in EC patients was evaluated by Log-rank-Mantel-Cox test.The results showed that the expression of NCL mRNA was significantly correlated with the prognosis of patients.The overall survival(OS)of high expression group of NCL mRNA was significantly lower than that of medium expression group and low expression group(P=0.001).The disease-free survival(DFS)of high expression group was significantly lower than that of medium expression group and low expression group(P=0.006).3.2 The relationship between the expression of NCL mRNA and the survival time of patients with type Ⅰ EC.The overall survival curve and disease-free survival curve were drawn by Kaplan-Meier survival analysis,and the prognostic value of NCL in patients with type Ⅰ EC was evaluated by Log-rank-Mantel-Cox test.Compared with the high expression group,the middle and low expression groups showed better OS(P=0.019)and DFS(P=0.010).3.3 The relationship between NCL mRNA expression level and survival time of patients with type Ⅱ EC.Kaplan-Meier survival analysis was used to draw the overall survival curve and disease-free survival curve of NCL mRNA high expression group,medium and low expression group in patients with type Ⅱ EC,and Log-rank-Mantel-Cox test was used to evaluate the prognostic value of NCL in patients with typeⅡ EC.The results showed that there was no significant difference in OS and DFS among high expression group,medium expression group and low expression group.3.4 Subgroup analysis based on pathological grade and FIGO stage.Subgroup analysis were used to explore the expression and significance of NCL in subgroup.Subgroup analysis based on pathological grade and FIGO staging showed that the OS(P=0.037)and DFS(P=0.013)of high NCL expression group were poor for pathological grade 1 or 2 tumors.In FIGO stage Ⅰ or Ⅱ tumors,there was a significant negative correlation between OS and NCL expression.Compared with the medium and low expression groups of NCL,the high expression group of NCL showed a poor OS(P=0.012).3.5 Prognostic value of NCL mRNA expression level.3.5.1 Cox proportional hazards model was used to analyze the relationship between NCL mRNA expression and prognosis of EC patients.Hazard ratio(HR)and 95%confidence interval(CI)of different clinicopathological factors were calculated by univariate Cox proportional hazards model.Results showed that the expression of NCL was significantly correlated with OS(HR=1.717,95%CI=1.250-2.359,P=0.001)and DFS(HR=1.404,95%CI=1.134-1.739,P=0.002)in EC patients.Other clinicopathological features associated with OS in EC patients included pathological type(HR=2.215,CI=1.104-4.444,P=0.025),FIGO stage(HR=4.416,95%CI=2.328-8.377,P<0.001)and histological grade(HR=2.839,95%CI=1.293-6.234,P=0.009).Other clinicopathological features associated with DFS in EC patients included FIGO stage(HR=3.090,95%CI=2.021-4.723,P<0.001).Multivariate Cox proportional hazards model showed that NCL was identified as an independent prognostic marker of DFS in EC patients(HR=1.282,CI=1.027-1.601,P=0.028).3.5.2 Cox proportional hazards model was used to analyze the relationship between the expression of NCL mRNA and prognosis of patients with type I EC.Univariate Cox regression analysis showed that high expression of NCL mRNA(HR=1.490,CI=1.145-1.938,P=0.003)and high FIGO stage(HR=2.736,CI=1.619-4.623,P<0.001)were related to DFS in patients with type I EC.Multivariate Cox regression analysis showed that high expression of NCL mRNA(HR=1.411,CI=1.083-1.840,P=0.011)and FIGO stage(HR=2.516,CI=1.483-4.270,P=0.001)were independent prognostic factors for DFS.4.The protein expression of NCL in type I EC and the clinicopathological characteristics of the patients.4.1 Clinicopathological characteristics of patients.82 patients with type I EC were evaluated by immunohistochemical staining on paraffin sections.The average age is(59.22±10.62)years old.15.9%(13/82)patients were diagnosed with G3 tumors,34.1%(28/82)with G2 tumors,50%(41/82)with G1 tumors.86.6%(71/82)with FIGO stage Ⅰ+Ⅱ tumors,13.4%(11/82)with Ⅲ+Ⅳ tumors.28%(23/82)patients had deep myometrial invasion and 9.8%(8/82)patients had cervical invasion.8.5%(7/82)patients had pelvic lymph node and/or para-aortic lymph node metastasis.89.0%(71/82)underwent open surgery,11%(9/82)underwent mini-invasive surgery.15 patients with non-malignant diseases were included in our study.The average age is(55.16±9.73)years old.7%(7/15)patients were diagnosed with leiomyoma,33.3%(5/15)were diagnosed with adenomyosis,20%(3/15)with abnormal uterine bleeding.86.7%(13/15)patients underwent open surgery.4.2 Expression level and location of NCL protein in type I EC.Immunohistochemical results showed that NCL was stained in the nuclei of both non-malignant and cancer tissues,and high nuclear NCL expression was observed in 55 tumor tissues(67.1%).The expression of extra-nuclear NCL was negative in non-malignant tissues and positive in 34 tumor tissues(41.5%).The results showed that NCL might migrate to the outside of nucleus in some tumor cells.5.The relationship between the expression level and location of NCL protein and the prognosis of patients with type I EC.5.1 The relationship between the expression level and location of NCL protein and the survival time of patients with type I EC.The survival curve was drawn by Kaplan-Meier survival analysis,and the prognostic value of the expression level of NCL protein in the nucleus was evaluated by Log-rank-Mantel-Cox test.Compared with the patients with high nuclear NCL expression,the DFS of patients with low nuclear NCL expression was significantly poor(P=0.001).Kaplan-Meier survival analysis and Log-rank-Mantel-Cox test were used to evaluate the prognostic value of extra-nuclear NCL protein expression.The results showed that there was a significant correlation between the expression of NCL and DFS in patients with type I EC(P=0.031).High expression of NCL in nucleus and negative extra-nuclear NCL expression were associated with better prognosis.5.2 Cox proportional hazards model was used to analyze the relationship between NCL protein expression and prognosis in patients with type I EC.In the univariate Cox proportional hazards model,patients with positive expression of extra-nuclear NCL had a higher risk of adverse outcomes(DFS:HR=3.398,95%CI=1.046-1 1.038,P=0.042),and patients with high expression of intra-nuclear NCL had a better prognosis(DFS:HR=0.178,95%CI=0.055-0.580,P=0.004).The other clinicopathological feature associated with DFS of type I EC patients was FIGO stage(HR=3.647,95%CI=1.121-11.864,P=0.032).The clinicopathological features with P<0.05 in univariate Cox regression were included in the multivariate Cox proportional hazards model.The results showed that positive extra-nuclear NCL expression(HR=3.377,95%CI=1.029-11.186,P=0.046)and low nuclear NCL expression(HR=0.233,95%CI=0.068-0.796,P=0.020)and FIGO stage(HR=3.435,95%CI=1.009-1 1.690,P=0.048)were independent prognostic factors of DFS in type 1 EC patientsConclusion1.The overexpression of NCL mRNA was significantly correlated with advanced(FIGO III-IV)EC and low-grade EC,suggesting that the overexpression of NCL mRNA may be associated with tumor progression.2.The high expression of NCL mRNA in tumor tissues of EC patients was associated with poor clinical prognosis.3.In normal tissues,NCL protein was mainly expressed in the nucleus,but not in the extra-nuclear.NCL protein can be expressed both in and out of the nucleus in some tumor cells of patients with type I EC.4.In tumor tissues of patients with type I EC,the positive expression of extra-nuclear NCL protein is associated with poor clinical prognosis,while the low expression of intra nuclear NCL protein is associated with poor prognosis. |
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