IL-11RA is a Prognostic Biomarker and Correlated with Immune Inltration in Lung Adenocarcinoma

Background: Interleukin-11 receptor subunit alpha (IL-11RA) contributes to multiple biological processes in various tumors. However, the role of IL-11RA in Lung adenocarcinoma (LUAD) is still undetermined. The study aims to explore the role of IL-11RA in LUAD via an integrated bioinformatics analysis. Methods: TIMER, GEPIA, TCGA and HPA databases analysis were used to detect IL-11RA expression. UALCAN database was used to analysis the correlation between IL-11RA expression and clinicopathological parameters of LUAD. Kaplan-Meier Plotter, TCGA and GEO databases were used to analysis overall survival (OS) and progression-free survival (PFS) of the LUAD patients. Univariate Cox regression analysis was used to assess the prognostic value of IL-11RA in different clinical characteristics. GSEA, and TIMER were used to investigate the relationship between IL-11RA and immune inltration. Results: The expression of IL-11RA was down-regulated in LUAD tissues. Furthermore, IL-11RA expression was closely associated with clinical stage, lymph node stage and smoking habits. The patients with lower IL-11RA expression had poorer overall survival (OS) and progression-free survival (PFS). Lower IL11RA expression was signicantly associated with its hypermethylation, and the hypermethylation of CpG site at cg14609668 and cg21504624 was obviously correlated with poorer OS. Then, we found that IL-11RA may play an important role in LUAD progression and immune regulations. Notably, High expression of IL-11RA may suppress the progression of LUAD through inhibiting cell proliferation and immune cell inltration, especially in B cells, CD4 + T cells, and Dendritic Cell. Conclusions: Decreased IL-11RA expression correlates with poor prognosis and immune inltration in LUAD. Our work highlights IL-11RA might be a potential biomarker for prognosis and provide a new therapeutic target for LUAD patients.


Introduction
Lung cancer is one of the most prevalent types of malignancies worldwide, with a high morbidity and mortality [1]. Histologically, lung cancer is divided into several different types. Lung adenocarcinoma (LUAD) is the most frequent type, accounting for more than 50% of all lung cancers [2]. Currently, surgical resection combined with chemotherapy and radiotherapy is a primary method for treating LUAD [3]. In recent years, lung cancer immunotherapy has become a new research hot spot, which can activate the patients' immune system, exert an antitumor effect [4], and improve the clinical symptoms and overall survival [5]. Despite the advance of immunotherapy, the prognosis of patients with LUAD remains poor [6]. Therefore, in the era of precision medicine, it is necessary to explore a reliable immune-related biomarker that can predict the prognosis of LUAD and become a novel target for LUAD immunotherapy.
IL-11 is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for the treatment of various cancers. Permyakov and his colleagues found that aside from interleukin-11 receptor subunit alpha (IL-11RA) and gp130 receptors, IL-11 interacts with calcium sensor protein S100P [7]. IL-11RA is a member of the cytokine receptors family, which can be found in cytoplasm and membrane, indicating that IL-11RA not only has functions that are ligand-dependent but also ligandindependent [8][9]. Currently, IL-11RA is known to be associated with tumor progressivity, cell growth, and differentiation from several malignant tumors [10][11], suggesting its potential involvement in human tumorigenesis. However, the relationship between IL-11RA and LUAD is still poorly understood.
Here, we used publicly available databases, such as the TCGA, GEO, GEPIA, TIMER, Kaplan-Meier plotter, HPA and UALCAN, to evaluate the expression of IL-11RA in LUAD and its prognostic role as well as its possible molecular mechanism. IL-11RA expression in LUAD was validated by TIMER, GEPIA, TCGA and HPA databases. Prognosis and clinical characteristics were analyzed using Kaplan-Meier plotter, TCGA and GEO databases. Subsequently, TCGA databases were used to analyze the relationship between methylation and IL-11RA expression in LUAD. Moreover, we also investigated the association of IL-11RA expression with tumor-in ltrating immune cells in LUAD. The present study revealed the important function of IL-11RA in LUAD and explain the potential relationship and mechanism of the interaction between IL-11RA and tumor progressivity.

Data collection
The TCGA database containing transcriptomic data and clinical data of 59 normal and 526 lung adenocarcinoma tissues were downloaded from the UCSC Xena website (http://xena.ucsc.edu/). After excluding duplicate patients, 59 normal and 513 lung adenocarcinoma tissues and 57 pairs of lung adenocarcinoma and normal lung tissues were included for IL-11RA expression level analysis, and 504 LUAD samples had complete OS survival information. Moreover, the DNA methylation data of 32 normal and 471 lung adenocarcinoma tissues was also downloaded from the UCSC Xena website, and 460 LUAD patients were included for OS survival analysis. The GSE72094 data set containing the expression and survival data of 398 LUAD patients was downloaded from The Gene Expression Omnibus dataset (GEO, https://www.ncbi.nlm.nih.gov/geo/).

GEPIA database analysis
Gene Expression Pro ling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/detail.php) is a newly developed interactive web server for analyzing the RNA sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and the GTEx projects, using a standard processing pipeline [12].
This study performed a differential IL-11RA mRNA expression analysis in 483 LUAD and 347 normal tissues.

HPA database analysis
The Human Protein Atlas (HPA, http://www.proteinatlas.org) database provides abundant transcriptome and proteome data via immunohistochemistry and RNA-sequencing analyses. In this study, the protein expression levels of IL-11RA were determined by immunohistochemistry.
UALCAN database analysis the UALCAN database (http://ualcan.path.uab.edu) uses TCGA level 3 RNA-seq and clinical data from 31 cancer types [13]. In this study, we used the UALCAN database to study the correlation between IL-11RA mRNA expression level and clinicopathological parameters of LUAD.

Kaplan-Meier plotter database analysis
The correlation between IL-11RA expression and survival in LUAD was analyzed by Kaplan-Meier plotter (http://kmplot.com/analysis/). 719 LUAD patients were divided into high expression groups and low expression groups based on the median expression levels of the IL-11RA. The prognostic value of IL-11RA was assessed by overall survival (OS) and progression-free survival (PFS) using the hazard ratio (HR), 95% con dence intervals (CI), and log-rank p-value. Meanwhile, univariate cox regression analysis was used to assess the prognostic value of IL-11RA in different clinical characteristics. Besides, the prognostic signi cance of IL-11RA in LUAD was also analyzed in TCGA-LUAD and GSE72094 data set through "survminer" and "survival" packages in R.

Analysis of IL-11RA methylation
The methylation level of IL-11RA between 32 normal and 471 LUAD patients and the prognostic value of IL-11RA DNA CpG sites in 460 LUAD patients were analyzed by R software. The correlation between IL-11RA gene expression and its methylation levels were analyzed by the Pearson method.
Gene set enrichment analysis In order to clarify the possible mechanism of IL-11RA in lung adenocarcinoma, TCGA datasets of LUAD with a functional gene set le (c5.all.v7.2) gene set were analyzed by GSEA. The 504 LUAD patients were divided into a high IL-11RA expression group and a low IL-11RA expression group based on the median expression levels. Gene sets with adjust pvalue < 0.05 were considered statistically signi cant. The normalized enrichment score (NES) was acquired by analyzing with 1000 times permutations [14].

Calculation of Immune Scores
Immune scores were calculated by the ESTIMATE algorithm of the downloaded data for each LUAD sample [15]. Subsequently, comparison of Immune scores between the high and low IL-11RA expression groups was analyzed.

TIMER database analysis
Tumor Immune Estimation Resource (TIMER, https://cistrome.shinyapps.io/timer/) is a comprehensive resource for systematical analysis of immune in ltrates across diverse cancer types. We used TIMER to analyze the expression of IL-11RA in multiple tumors and its association with several immune in ltrated cells (B cells, CD4 + T cells, CD8 + T cells, neutrophils, Myeloid Dendritic cell and macrophages) in LUAD. In addition, the correlation analysis between IL-11RA and relate genes and markers of immune cells were analyzed in TIMER.
Statistical analysis R version 4.0.3 software were used for statistical analyses. Paired t-test and unpaired t-test were used to compare the expression of IL-11RA between normal and tumor groups. Survival analyses in the TCGA-LUAD and GSE72094 cohort were conducted between high and low IL-11RA expression groups through Kaplan-Meier analysis with the log-rank test. Univariate Cox analysis was used to evaluate the in uence of IL-11RA expression and other clinicopathological factors (gender, stage, smoke) on survival. The correlation between IL11-RA expression and methylation was assessed by pearson's correlation. The correlation between IL11-RA expression and immune cells and its markers was assessed by spearman's correlation. P-value < 0.05 was set up as the cut-off criterion.

The expression of IL-11RA is down-regulated in lung adenocarcinoma
To evaluate the IL-11RA expression in various tumours, we searched Transcriptome-seq data in the TIMER database (Fig. 1A). The result revealed that the IL-11RA mRNA expression in liver hepatocellular carcinoma (LIHC), pheochromocytoma, and paraganglioma (PCPG) were higher compared to normal tissues, while it was considerably lower in bladder urothelial carcinoma (BLCA), invasive breast carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA) and uterine corpus endometrial carcinoma (UCEC) than in the normal tissue. These data show that IL-11RA has a signi cant differential expression between tumour tissues and normal tissues, and it may be regarded as oncogenic gene, particularly in LUAD. To further investigate the relevance of IL-11RA expression level with human LUAD, we performed a detailed analysis of RNA-Seq data from the GEPIA and TCGA databases. The quantitative evaluation of the expression level of IL-11RA mRNA in LUAD tissues was signi cantly lower than that in normal tissues ( Fig. 1B and Fig. 1C). Data from tumor and matched normal tissues of LUAD patients in the TCGA further veri ed these results (Fig. 1D). Besides, we further analyzed the protein level of IL-11RA in LUAD tissues from the Human Protein Atlas (HPA) database. Immunohistochemical results indicated that the protein expression level of IL-11RA exhibited signi cant upregulation in LUAD tissues. Overall, these results suggest that the expression of IL-11RA is decreased in various types of cancer, particularly in LUAD, demonstrating that IL-11RA may suppress LUAD tumorigenesis.
The correlation between IL-11RA mRNA expression level and clinicopathological parameters of LUAD In order to clarify the correlation between IL-11RA mRNA expression level and clinicopathological parameters of LUAD, we investigated IL-11RA expression on the basis of patients' different clinical pathological parameters, such as age, gender, stage, lymph -node status, smoking habits using UALCAN database. Results showed that IL-11RA expression was much lower in LUAD patients than normal tissues in subgroup analysis based on age and gender ( Fig. 2A and Fig. 2B). For cancer stages and lymph node status, IL-11RA expression was lower expressed in different subclasses than that in normal lung tissues ( Fig. 2C and Fig. 2D). It is highly intriguing that the expression of IL-11RA decreases as the stage increases between stage 1 and stage 3 (Fig. 2C), and IL11RA also decreases with the increase of the lymph node stage between N0 and N2 (Fig. 2D). Besides, IL-11RA expression was closely associated with smoking habits (Fig. 2E). An analysis of subclass showed that IL-11RA expression was much lower in lung adenocarcinoma patients who smoke or those who have smoked for more than 15 years than lung adenocarcinoma patients who do not smoke or those who have smoked for less than 15 years. All these data together indicated that low IL-11RA expression was widely correlated with lung adenocarcinoma.
To better understand the effect of decreased expression of IL-11RA on survival, the Kaplan-Meier plotter database was accessed to analyze the correlation between IL-11RA expression and clinical characteristics of patients by univariate cox regression analysis ( Table 1). The low expression of IL-11RA corresponded with worse OS and PFS in women, male, smoking or nonsmoking patients (P < 0.05). For clinical staging, downregulation of IL-11RA was linked to worse OS in stage 1, stage N0, and stage M0 in LUAD (P < 0.05) and worse PFS in stage 1 (P < 0.05).  Analysis of IL-11RA methylation pro le in LUAD patients To further identify the potential mechanism leading to the downregulation of IL-11RA in LUAD, we rstly analyzed the DNA methylation levels of IL-11RA in the 471 LUAD samples and 32 normal samples from TCGA database. We found that the DNA methylation levels of IL-11RA were signi cantly higher in LUAD tissues than in normal lung tissues (Fig. 4A, P = 2.079e-12). Consecutively, the correlation between DNA methylation level and expression of IL-11RA in LUAD was assessed. The result indicates that the mRNA expression level of IL-11RA was negatively correlated with its DNA methylation level (Fig. 4B, Cor = -0.22, P = 2.2e-06). These results showed that the hypermethylation of IL-11RA might contribute to the downregulation of IL-11RA in LUAD. Furthermore, we discovered that LUAD patients with the higher methylation level of CpG sites cg14609668 (Fig. 4C) and cg21504624 (Fig. 4D) had a worse OS. These results suggest that the methylation and expression level of IL-11RA could in uence the prognosis of LUAD patients.

IL-11RA is involved in immune in ltration and proliferation in LUAD
To further explore the biological function of IL-11RA in LUAD, we performed Gene Set Enrichment Analysis (GSEA) analysis. Previously, research has shown that the occurrence and development of tumor are closely related to immune in ltration levels [16]. Therefore, we utilize GSEA to explore the correlation of IL-11RA and immune in ltration. Enrichment results showed that many gene sets related to immune activation, such as activation of immune response (Fig. 5A), immune receptor activity (Fig. 5B), leukocyte mediated cytotoxicity (Fig. 5C), T cell activation (Fig. 5D), B cell activation (Fig. 5E), Macrophage activation (Fig. 5F), were enriched in the IL-11RA high expression group, which suggests that IL-11RA low expression may promote the progression of LUAD through immune inhibition. Furthermore, we found that several cell cycle-related gene sets were enriched in the IL-11RA low expression group (Fig. 5G-5I), which indicates that IL-11RA low expression may also be involved in facilitating cell proliferation in LUAD.

Correlation analysis between IL-11RA expression and six main in ltrating immune cells and its immune marker genes
To further clarify the correlation between IL-11RA expression and immune in ltration of LUAD, we rstly calculated the immune scores of the LUAD samples by the ESTIMATE algorithm using the data from TCGA database to predict the presence of in ltrating immune cells. The results found that the immune scores were lower in the IL-11RA low group (Fig. 6A), which indicated that IL-11RA low expression might be involved in the immune inhibition of LUAD.
Then, considering that IL-11RA is a crucial mediator of immune cell activation, a correlation analysis between IL-11RA expression and six types of immune-in ltrating cells, including B cells, CD4 + T cells, macrophages, CD8 + T cells, neutrophil and myeloid dendritic cell was performed via the TIMER database. The results showed that IL-11RA expression levels were signi cantly positively correlated with levels of in ltrating B cells, CD4 + T cells, and myeloid dendritic cell in LUAD (Fig. 6B-6H). Taken all together, the above evidence indicates that IL-11RA may be involved in the immune response of patients with LUAD by affecting the immune cells.
Lastly, to expand the understanding of the crosstalk between IL-11RA and multiple marker genes of immune cells, we did correlation analysis between them via the TIMER database. The results found that 44/52 (84.62%) immune cell markers were signi cantly associated with IL-11RA expression (P < 0.05), of which the number of positive correlations was 40/52 (76.92%), and the negative was 4/52 (7.69%). The results showed that the expression level of IL-11RA was signi cantly correlated with most immune markers of immune cells in LUAD.

Discussion
With the aggravation of environmental pollution and abuse of tobacco products, the incidence of LUAD has increased year by year, which is a subject of concern [17]. Although remarkable advances were achieved in the diagnosis and therapeutic strategies over the past decades, the overall prognosis of LUAD patients is still poor [18]. Multiple studies have reported that abnormal gene expression is involved in cancer-related processes and correlated with these patients' prognosis [19][20]. However, the potential role of IL-11RA in the development and prognosis of LUAD remains ambiguous. In the present study, we demonstrated that IL-11RA is signi cantly downregulated in patients with LUAD via bioinformatics analysis based on multiple databases and signi cantly predicts a poor prognosis; also, the higher tumor stage got a lower expression. Univariate Cox analyses indicated that the lower expression of IL-11RA might be de ned as a risk factor that affects the OS and PFS of LUAD patients. Moreover, the hypermethylation of IL-11RA might contribute to the downregulation of IL-11RA and poorer OS in LUAD. Lastly, we analyzed a correlation analysis between IL-11RA and immune in ltration or immune markers, nding that IL-11RA was related to most of the immune marker genes, which may be the mechanism by which IL-11RA affects prognosis. These results suggest that IL-11RA may play a role in the development of LUAD and serve as a possible prognostic biomarker and a novel immune-related therapeutic target in LUAD.
IL-11RA was previously reported as a molecule that is highly associated with tumorigenesis [9]. The abnormal expression of IL-11RA was observed in multiple tumor tissues, such as gastric carcinoma, colon cancer, breast cancer, prostate cancer, osteosarcoma, and melanoma [21][22]. Our results found that the mRNA and protein expression level of IL-11RA is signi cantly downregulated in patients with LUAD. The expression of IL-11RA decreases with the increase of stages between stage 1 and stage 3 or between stage N0 and N2, but no further decreases in the stage 4 or N3, which may be due to the extremely limited advanced cases, so expanding the advanced sample size for further study is needed.
Furthermore, the relationships between lung cancer and duration of smoking and type of smoking have quanti ed by epidemiologist [23]. We found that compared with non-smokers, the expression of IL-11RA in smokers was signi cantly lower, and there was a consistent trend in the comparison between patients who had smoked for less than 15 years and those who had smoked for more than 15 years. We guess that the down-regulation of IL-11RA may be one of the potential mechanisms of lung cancer caused by smoking.
Regarding prognosis, in the Kaplan-Meier plotter, TCGA and GEO databases, the survival analysis found matching prognostic value that low IL-11RA expression was associated with poor outcomes in LUAD.
Moreover, downregulation of IL-11RA was linked to worse OS of LUAD in stage 1, stage N0, and stage M0. These results robustly re ected that IL-11RA serve as a possible prognostic biomarker in lung adenocarcinoma.
Abnormal DNA methylation occupies an essential role in the induction and progression of LUAD [24].
DNA methylation is correlated with carcinogenesis by repressing the tumor suppressor gene's expression and promoting oncogenes' expression [25][26]. Therefore, we guess that hypermethylation of antioncogenes such as IL-11RA led to their low expression, and may be an important cause of tumorigenesis.
In our analysis, a negative correlation between IL-11RA methylation and IL-11RA mRNA expression existed in LUAD tissues. In addition, we found that hypermethylation of CpG sites cg14609668 and cg21504624 had a worse OS. These results well demonstrated our guess.
Another signi cant nding from this study is that IL-11RA expression correlates with immune in ltration levels in LUAD. In the tumor microenvironment of lung cancer, the landscape of immune cell types is T cells, followed by B cells, macrophages, dendritic cells and natural killer cells [27]. A great variety of immune cells provide an anti-tumor effect by migrating close to tumors when tumour arise [28]. Our results demonstrate that the immune in ltrating was lower in the IL-11RA low group and IL-11RA expression has signi cantly positive relationships with the in ltration level of CD4 + T cells, B cell and myeloid dendritic cell. IL-11RA expression also strongly correlated with majority of the molecular markers of immune in ltrating cells in LUAD. In addition, the results that IL-11RA may be involved in immune response and cell proliferation was validated by GSEA. Most studies indicate that dendritic cells contribute to the activation of antitumor T lymphocytes [29][30], and higher levels of T cells and B cells were closely related with better prognosis in cancer patients [31][32][33]. Therefore, these ndings indicate that IL-11RA expression is closely related with recruitment and regulation of immune in ltrating cells and may be involved in the immune response to LUAD development, resulting in a worse prognosis in LUAD patients.
It is worth noting that some inevitable limitations exist in our analysis. First, although we preliminarily investigated the biological process of IL-11RA in LUAD through GSEA enrichment analysis, the detailed mechanism that correlation between IL-11RA low expression and LUAD progression requires further biomedical experiments. Then, more prospective data were needed for proving the clinical utility of it.

Conclusions
In conclusion, we suggest that decreased IL-11RA expression correlates with poor prognosis and immune in ltration in LUAD by applying integrated bioinformatics approaches. Our work highlights IL-11RA might be a potential biomarker for prognosis and provide a new immunotherapeutic target for LUAD patients.