2.1 Gene expression analysis in different tumors.
We applied the TIMER2 approach to analyze the expression status of ANLN across various cancer types of TCGA. As shown in Figure 1, the expression level of ANLN in the tumor tissues of BLCA、BRCA、CHOL、COAD、ESCA、HNSC、 KIRC、KIRP、LIHC、LUAD、LUSC、PRAD、READ、SKCM、STAD、etc is higher than the corresponding control tissues (P < .01).
2.2. The relationship between ANLN and clinicopathological characteristics in patients with lung adenocarcinoma
The clinical characteristics of 594 patients were obtained from the TCGA-LUAD cohort, including age, gender, tumor stage and TNM classification. According to the median of ANLN mRNA levels, patients were divided into 268 cases of high ANLN expression group and 267 cases of low ANLN expression group. As shown in Table 1, the expression of ANLN was significantly correlated with the grade (P<0.001), gender (P=0.017), age (P=0.001), T stage (P<0.001) and N stage (P<0.001) of lung adenocarcinoma Related. The expression of ANLN has nothing to do with clinicopathological factors such as M staging (P=0.122).
2.3 Expression analysis
We analyzed the expression level of ANLN in 535 cases of lung adenocarcinoma tissues and 59 cases of adjacent lung tissues, and found that ANLN was highly expressed in lung adenocarcinoma tissues (P<0.001, Figure 2A). At the same time, we also analyzed the expression of ANLN in 57 cases of lung adenocarcinoma tissues and their paired adjacent tissues. The results showed that ANLN was highly expressed in lung adenocarcinoma tissues (P<0.001, Figure 2B). We detected the expression of ANLN protein in the UALCAN database and found that it was significantly highly expressed in tumor tissues (Figure 2C). Overall, these results indicate that the expression of ANLN in lung adenocarcinoma tissues is higher than that in normal tissues.
In addition, we also used the ROC curve to analyze the effectiveness of ANLN expression levels in distinguishing lung adenocarcinoma tissues from non-tumor tissues. The area under the curve (AUC value) of ANLN is 0.978, suggesting that ANLN can be used as an ideal biomarker to distinguish lung adenocarcinoma from non-tumor tissues (Figure 2D).
2.4 Prognostic analysis
In order to understand the correlation between ANLN expression and the prognosis of LUAD patients, we used Kaplan-Meier survival curve to analyze the prognostic value of ANLN expression in lung adenocarcinoma patients. According to the median of ANLN mRNA expression, the cohort was divided into low expression subgroup and high expression subgroup. The overall survival of patients with low ANLN expression (HR=1.90, 95%CI: 1.42−2.55, P<0.001) was significantly higher than that of the high expression group (Figure 3A). Similarly, the disease- specific survival of the low expression group (HR=1.98, 95%CI: 1.37−2.88, P<0.001) was also significantly higher than that of the high expression group (Figure 3B). These results indicate that the expression of ANLN is significantly associated with a poor prognosis.
A Cox univariate and multivariate proportional hazard model for the overall survival of LUAD patients was constructed. As shown in (Figure 4A), in the COX univariate regression model, T stage (P=0.002), N stage (P<0.001), and M stage (P=0.006) (P<0.001), pathological grade (P<0.001), ANLN (P<0.001) affect the prognosis of LUAD patients. Multivariate analysis further showed that T stage (P=0.046), N stage (P=0.003), pathological grade (P=0.022), ANLN (P=0.032) expression levels have a significant impact on the prognosis of patients with lung adenocarcinoma(Figure 4B). As mentioned, ANLN is a potential independent risk factor for LUAD.
2.5 Genetic alteration analysis
We observed the genetic alteration status of ANLN in different tumor samples of the TCGA cohorts. As shown in (Figure 5A). Only "mutation" is the type were Uterine Carcinosarcom、Diffuse Large B-Cell Lymphoma、Kidney Renal Clear Cell Carcinoma、Kidney Renal Papillary Cell Carcinoma . The main types of mutations are Uterine Corpus Endometrial Carcinoma 、Bladder Urothelial Carcinoma 、Skin Cutaneous Melanoma 、Lung Adenocarcinoma、Adrenocortical Carcinoma 、Stomach Adenocarcinoma 、Head and Neck Squamous Cell Carcinoma、Lung Squamous Cell Carcinoma、Cervical Squamous Cell Carcinoma、Colorectal Adenocarcinoma. Only the "amplified" type is Pheochromocytoma and Paraganglioma, The main type of amplification are sophageal Adenocarcinoma、Glioblastoma Multiforme 、Ovarian Serous Cystadenocarcinoma、Prostate Adenocarcinoma、Sarcoma 、Brain Lower Grade Glioma、Pheochromocytoma and Paraganglioma. In LUAD, "Mutation" accounted for 2.12%,“Amplification”accounted for 0.71%;"Deep Deletion" 0.35%;“Multiple Alterations”accounted for 0.18%.The types and sites of the ANLN genetic alteration are further presented in( Figure 5B), and case number of the ANLN genetic alteration are further presented in table3. We found that the missense mutation (R153Q/L) of ANLN was the major type of genetic change, and mutation sites could be observed in the 3D structure of ANLN protein (Figure 5C). In addition, we investigated the potential association between ANLN gene changes and clinical survival outcomes in patients with LUAD. Compared with LUAD patients without ANLN changes, LUAD patients with ANLN changes had poorer overall outcomes (p-value: 0.0238) (Figure 5D) and disease-specific survival (P-value: 0.309)(Figure 5E).
2.6 Correlation analysis between the expression of ANLN and six main infiltrating immune cells
The TIMER database was used to analyze the correlation between the expression of ANLN in LUAD and the six main infiltrating immune cells (B cells, CD4 T cells, CD8+T cells, neutrophils, macrophages and dendritic cells). The analysis showed that the expression level of ANLN was significantly correlated with the levels of B cells (r=−0.227, p value=4.33e-07) and neutrophils (r=0.16, p value=4.00e−04) (Figure 6A) .
In addition, we evaluated the prognostic value of six immune cells through Kaplan-Meier analysis and found that B cells (P<0.001) and ANLN expression (P<0.001) were significantly correlated with the prognosis of LUAD (Figure 6B). It is suggested that ANLN has a strong regulatory effect on the immune cell infiltration of lung adenocarcinoma, especially the regulation of B cell infiltration.
2.7 Co-expression analysis
In order to better understand the expression mechanism of ANLN in LUAD, use the "LinkFinder" module in LinkedOmics to check the co-expression mode of ANLN. The related genes co-expressed by ANLN were obtained by Pearson test, and the results are shown in a volcano graph (Figure 7A). (Figures 7B and 7C) show the heat maps of the top 50 genes that are positively and negatively correlated with ANLN, respectively.
GSEA is used to identify signal pathways related to ANLN. The GO annotation of GSEA shows (Figure 8A) that ANLN co-expressed genes mainly involve chromosome segregation, mitotic cell cycle phase transition, DNA replication, DNA recombination, and RNA localization. On the contrary, the drug catabolism process is inhibited, and the protein activates the cascade process. KEGG enrichment analysis shows (Figure 8B) that genes are mainly enriched in the cell cycle, RNA transport, pyrimidine metabolism, spliceosome, ubiquitin-mediated proteolysis, and Epstein-Barr virus infection.
In addition, the top 5 significant genes related to ANLN, including KIF4A, KIF23, CKAP2L, TPX2, KIF2C, were selected for further analysis, and their correlation with ANLN was verified in the TIMER database. The results show that ANLN and KIF4A (COR=0.888, p=3.4e−175), KIF23 (COR=0.889, p=1.09e−175), CKAP2L (COR=0.883, p=2.74e−170), TPX2(COR =0.876, p=9.24e−165), KIF2C (COR=0.87, p=8.31e−160) were significantly correlated (Figure 9A-E).