CXCL2 as a Prognostic Marker in Breast Cancer is Associated with Immune Inltration and Regulated by miR-215

Background: CXCL2 is a part of chemokine superfamily, which encodes secretory proteins involved in immune regulation and inammation. The correlation between CXCL2 and prognosis of different cancers, tumor inltrating lymphocytes are not clear. Methods: We analyzed the expression of CXCL2 and its effect on clinical prognosis through Oncomine database, Tumor Immune Estimation Resource (TIMER) website, Kaplan-Meier plotter, PrognoScan database and Gene Expression Proling Interactive Analysis (GEPIA). TIMER and GEPIA were used to analyze the correlation between CXCL2 and the gene marker of immune inltration. StarBase was used to predict the miRNA that may regulate CXCL2. The relationship between miR-532-5p and CXCL2 was detected by qRT-PCR. Kaplan-Meier plotter was used to evaluate the impact of miR-532-5p on clinical prognosis. Results: PrognoScan, Kaplan-Meier plotter and GEPIA database analysis showed that low expression of CXCL2 was associated with poor disease-specic survival time (DSS), relapse-free survival time (RFS) and overall disease survival (OS) in breast cancer patients. In addition, low expression of CXCL2 was associated with poor OS and RFS in patients with lymph node positive breast cancer. CXCL2 expression was positively correlated with the inltration of B cells, CD4+T and CD8+T cells, neutrophils and dendritic cells (DCs) in BRCA, mainly in Luminal breast cancer. MiR-532-5p can directly regulate CXCL2 expression. High miR-532-5p expression is signicantly correlated with HER2 negative, grade 2 and 3 and poor OS in patients with HER+ER- breast cancer. Conclusion: CXCL2 is closely related to the prognosis and immune inltration level of breast cancer patients, it can be regulated by miR-532-5p. 19 In ovarian cancer and endometrial cancer, calcitriol and progesterone can cause the low expression of CXCL2 protein, which can reduce the invasiveness of cancer cells and inhibit the expression of metastasis related proteins. 20 These ndings indicate that CXCL2 plays an important role in the invasion and metastasis of cancer. Some studies have shown that CXCL2 has the effect of chemokine, which can attract myeloid-derived suppressor cells (MDSC) into the tumor cells in mouse ovarian cancer cells, while MDSC can inhibit the proliferation of CD8 + T cells. 21 Another study found that CXCL2, produced by M2 macrophages, 22 was suppressed in PD-L1 KO ID8-inoculated mice. This mechanism may support tumor progression being suppressed as a result of an altered macrophage subset from M2 macrophages producing CXCL1 and CXCL2 to M1 macrophages. 23 Spleen T lymphocytes of tumor-bearing mice were not only produced by DA-3 breast tumor cells, but also secreted CXCL2. 24 These ndings indicate that CXCL2 has multiple functions in tumor inltrating lymphocytes. However, the potential function and mechanism of CXCL2 in tumor progression and tumor immunology are still unclear. In this study, we analyzed the expression of CXCL2 and its correlation with the prognosis of cancer patients through Oncomine, PrognoScan, GEPIA and Kaplan-Meier plotter. At the same time, we also discussed the correlation between CXCL2 and tumor inltrating immune cells in different tumor microenvironment through Tumor Immune Estimation Resource (TIMER). The results of this study elucidate the important role of CXCL2 in BC, and provide the potential relationship and mechanism between CXCL2 and tumor immune interaction. mediated T-cell exhaustion. These results further conrm that CXCL2 is related to the immune inltrating cells in BRCA, suggesting that CXCL2 plays an important role in immune escape in BRCA microenvironment. of different types of cancer. Low expression of CXCL2 is associated with poor prognosis in BC patients. CXCL2 expression level can affect the prognosis of patients with lymph node metastasis of gastric cancer and BC, indicating that CXCL2 expression can be used as a predictor of tumor metastasis. In addition, the analysis showed that the expression of CXCL2 was related to the level of immune inltration and different immune marker sets in BC. Our research provides new insights into the potential role of CXCL2 in tumor immunity and its application as a biomarker of tumor. In this study, we used different databases to detect the expression level and prognosis of CXCL2 in various types of cancer. The expression of CXCL2 was different in different cancer tissues and normal tissues. In the Oncomine database, the expression of CXCL2 in colorectal cancer, esophageal cancer, head and neck cancer, and lymphoma was higher than that in normal tissues. In other data sets, the expression of CXCL2 was lower in bladder cancer, BC, colorectal cancer, leukemia, lung cancer, liver cancer, lymphoma and sarcoma. But in TIMER database, CXCL2 expression was signicantly lower in BLCA, BRCA, CHOL, HNSC, KICH, LIHC, LUAD, LUSC, and PRAD, but it was signicantly higher in COAD, KIRC, and READ. The differences of CXCL2 expression levels in different types of cancer in different databases may reect the data collection methods and potential mechanisms related to different biological characteristics. Analysis of data from the PrognoScan and Kaplan-Meier plotter databases showed that low expression of CXCL2 was associated with poor prognosis in colorectal, breast and lung cancer. In datasets of PrognoScan, Low expression of CXCL2 may be an independent risk factor for poor prognosis in colorectal cancer, BC and lung cancer. Kaplan-Meier plotter showed that the abnormal expression of CXCL2 was signicantly related to the prognosis of gastric cancer, BC and lung cancer. These ndings strongly suggest that CXCL2 is a prognostic biomarker for colorectal cancer, gastric cancer, BC and lung cancer. Another important aspect of this study is that the expression of CXCL2 is associated with different levels of immune inltration in BC. We found that the CXCL2 expression was positively correlated with the inltration of CD8 + T cells, CD4 + T cells, neutrophils and DCs in BRCA. In STAD, there were negative correlations with inltration of B cells, CD4 + T cells, macrophages, and DCs. Moreover,CXCL2 plays an important role in tumor immunomodulation of BRCA, we found that most markers of monocytes, TAMs and M2 macrophage were strongly correlated with the expression of CXCL2 in BRCA, such as the CD115 of monocytes, CCL-2 and IL10 of TAMs, CD163, VSIG4 and MS4A4A of M2 phenotype. These results reveal the potential regulatory role of CXCL2 in the polarization of tumor-associated macrophages. At the same time, CXCL2 was signicantly correlated with Treg and T cell exhaustion marker genes, such as CCR8, STAT5b, PD-1, CTLA4, LAG3 and GZMB. In addition, there was a signicant correlation between the expression of CXCL2 and the regulation of several markers of T helper cells in BRCA. These correlations may indicate the potential mechanism of CXCL2 regulating T cell function in BRCA. These results suggest that CXCL2 plays an important role in the recruitment and regulation of BRCA immune inltrating cells. We further conrmed that CXCL2 was regulated by miR-532-5p in BC. The expression of miR-532-5p was signicantly correlated with HER2 negative, HER2 + ER - in molecular subtype and 2 and 3 Grades.

Quantitative real-time PCR of CXCL2 CXCL2 cDNA was reverse-transcribed according to the manufacturer's instructions (Takara, Dalian, China). GAPDH was used as the reference gene.
Quantitative real-time PCR was carried out with SYBR Green (Solarbio, China) in a total volume of 20 µL using an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea). The reactions were incubated at 94 °C for 10 min; followed by 40 cycles of 94 °C for 10 s, 60 °Cfor 20 s, and 72 °C for 30 s.

Oncomine database analysis
The expression level of CXCL2 gene in various types of cancer was determined in Oncomine database (https://www.oncomine.org/). 25 The threshold was determined according to the following values: P value was 0.001, fold change was 1.5, and gene ranking of all.

PrognoScan database analysis
The correlation between CXCL2 expression and the survival rate of various types of cancer was analyzed by PrognoScan (http://dna00.bio.kyutech.ac.jp/PrognoScan/). 26 Searching for the relationship between gene expression and patient prognosis in PrognoScan, such as overall survival (OS), relapse free survival (RFS) and disease free survival (DFS). Adjust the threshold value to Cox P < 0.05.

Kaplan-Meier plotter database analysis
Kaplan-Meier plotter can assess the effect of 54 k genes on survival in 21 cancer types. The datasets include 6,234 breast, 2,190 ovarian, 3,452 lung, and 1,440 gastric cancer. The system includes gene chip and RNA-seq data-sources for the databases include GEO, EGA, and TCGA. The correlation between CXCL2 expression and survival in breast, ovarian, lung and gastric cancers was analyzed by Kaplan-Meier plotter (http://kmplot.com/analysis/). 27 The hazard ratio (HR) with 95% con dence intervals and log-rank P-value were also computed. TIMER database analysis TIMER is a comprehensive resource for systematical analysis of immune in ltrates across diverse cancer types (https://cistrome.shinyapps.io/timer/). 28 The abundances of six immune in ltrates (B cells, CD4 + T cells, CD8 + T cells, Neutrophils, Macrophages, and Dendritic cells) are estimated by TIMER algorithm.
We analyzed the expression of CXCL2 in different types of cancer and the correlation between CXCL2 expression and immune in ltration. In addition, the correlation between CXCL2 expression and gene markers of tumor in ltrating immune cells was explored. The correlation module generates a scatter plot of expression between a pair of user-de ned genes in a given cancer type, together with the Spearman's correlation and the estimated statistical signi cance.
CXCL2 is used for X-axis with gene symbol, and the related marker gene is expressed as gene symbol on Y-axis. The gene expression level was displayed by log2 RSEM.

GEPIA analysis
Gene Expression Pro ling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html) 29 is used to further identify the signi cant related genes in TIMER GEPIA is a web-based tool for analyzing the RNA sequencing expression data of 9,736 tumors and 8,587 normal samples based on the TCGA and the GTEx data. GEPIA was used to generate survival curves including OS and DFS based on gene expression and log rank test and Mantel-Cox test for 33 different types of cancer. The correlation analysis of gene expression was carried out for a given TCGA expression data set. The correlation coe cient was determined by Spearman method. CXCL2 is used for the x-axis, and other genes of interest are expressed on the y-axis. Tumor and normal tissue data sets are used for analysis.

Statistical analysis
The survival curve was drawn by PrognoScan and Kaplan-Meier plotter. The results generated in Oncomine are shown as P values, fold changes, and ranks. The results of Kaplan-Meier plotter, PrognoScan and GEPIA are displayed together with the HR and P or Cox P-values of the log-rank test. The correlation of gene expression was evaluated by Spearman's correlation and statistical signi cance. P < 0.05 was considered statistically signi cant.

Expression level of CXCL2 in different malignant tumors
We analyzed CXCL2 expression level in cancer and normal tissues of a variety of malignant tumors through Oncomine database to clarify the expression difference. The results showed that the expression of CXCL2 in colorectal cancer, esophageal cancer, head and neck cancer, and lymphoma was higher than that in normal tissues. In other data sets, the expression of CXCL2 was lower in bladder cancer, BC, colorectal cancer, leukemia, lung cancer, liver cancer, lymphoma and sarcoma (Fig. 1A).
We further evaluated the expression of CXCL2 in cancer by TIMER database, and detected the expression of CXCL2 by RNA sequence data of various malignant tumors in TCGA. The differential expression of CXCL2 in all TCGA tumors is shown in Fig. 1B. Compared with adjacent normal tissues, CXCL2 expression was signi cantly lower in BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL (cholangiocarcinoma), HNSC (head and neck cancer), KICH (kidney chromophobe), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (Lung squamous cell carcinoma), and PRAD (prostate adenocarcinoma), but it was signi cantly higher in COAD (colon adenocarcinoma), KIRC (kidney renal clear cell carcinoma), and READ (rectum adenocarcinoma).
In order to further study the prognostic effect of CXCL2 on different cancers, Kaplan Meier plotter database was used to evaluate the prognostic value of CXCL2. The results showed that the poor prognosis of gastric cancer was related to the decreased expression of CXCL2 (OS HR = 0.62, 95% CI = 0.51 to 0.75, P = 1.1e-06; PFS HR = 0.67, 95% CI = 0.53 to 0.84, P = 0.00047) ( Abnormal expression of CXCL2 affects the prognosis of gastric cancer and breast cancer patients with lymph node metastasis To better understand the potential mechanism of CXCL2 expression in cancer, we used the Kaplan-Meier plotter database to study the relationship between the expression of CXCL2 and the clinical characteristics of gastric and breast cancer patients. In gastric cancer, the abnormal expression of CXCL2 was correlated with OS and PFS in male and female patients and moderate differentiation, and with OS in Lauren classi cation (P < 0.05). Especially, CXCL2 expression was correlated with OS and PFS in stage 3 (OS HR = 0.62, P = 0.0014; PFS HR = 0.58, P = 0.005), but not with OS and PFS of stage 1 to 2 and stage N1 patients (OS HR = 0.68, P = 0.0792; PFS HR = 0.77, P = 0.2025) ( Table 1). In addition, the abnormal expression of CXCL2 has the highest HR values of N2 in the N categories.
In BC, we see more that the abnormal expression of CXCL2 has more effect on RFS, but in the lymph node status, we found that the expression of CXCL2 has signi cant effect on OS and RFS of lymph node positive patients (OS HR = 0.65, P = 0.0335; PFS HR = 0.76, P = 0.0058) ( Table 2). These results suggest that CXCL2 expression level can impact the prognosis in gastric and breast cancer patient with lymph node metastasis.

CXCL2 expression is correlated with immune in ltration in gastric and breast cancers
Tumor-in ltrating lymphocytes are an independent predictor of sentinel lymph node status and survival in patients with cancers. 36, 37 Therefore, we further investigated whether the expression of CXCL2 was related to the levels of immune in ltration in different cancers. The purity of tumor is an important factor affecting the analysis of immune in ltration in clinical tumor samples, so we choose the cancer types whose expression level of CXCL2 is negatively correlated with the purity of tumor and is signi cantly correlated with the prognosis. We found that the expression of CXCL2 was correlated with better prognosis and high immune in ltration of BRCA. CXCL2 expression was positively correlated with the in ltration of CD8 + T cells (r = 0.098, P = 2.10e-03), CD4 + T cells (r = 0.173, P = 6.30e-08), neutrophils (r = 0.133, P = 3.81e-05) and DCs (r = 0.115, P = 3.73e-04) in BRCA (Fig. 3A). In STAD, there were negative correlations with in ltration of B cells (r=-0.175, P = 7.13e-04), CD4 + T cells (r=-0.316, P = 6.54e-10), macrophages (r=-0.238, P = 3.54e-06), and DCs (r=-0.185, P = 3.37e-04) (Fig. 3D). In COAD, CXCL2 expression has no signi cant correlations with tumor purity and in ltrating levels of B cells, CD8 + T cells, and dendritic cells (Fig. 3B). In addition, CXCL2 expression has no signi cant correlations with in ltrating levels of B cells, CD8 + T cells, CD4 + T cells, macrophages, and dendritic cells in LUAD (Fig. 3C).
Then, according to the different types of BC, we further analyzed the correlation between the expression of CXCL2 and the immune in ltration of different types of BC. The results showed that in luminal BC, the expression of CXCL2 was positively correlated with the in ltration of CD8 + T cells (r = 0.188, P = 1.21e-05), CD4 + T cells (r = 0.226, P = 1.16e-07), macrophages (r = 0.089, P = 3.87e-02), neutrophils (r = 0.146, P = 7.04e-04) and DCS (r = 0.136, P = 1.61e-03) (Fig. 4D). In the other two types of BC, the expression of CXCL2 was not signi cantly correlated with immune in ltration. These ndings suggest that CXCL2 plays a speci c role in the immune in ltration of breast and gastric cancers, especially in BC.

Relationship between CXCL2 expression and immune markers
To further explore the relationship between CXCL2 and various immune in ltrating cells, we studied the relationship between CXCL2 and immune markers of different immune cells in BRCA, STAD and LUAD through TIMER and GEPIA databases (Table 3 and Table 4). We also analyzed T cells with different functions, such as Th1 cells, Th2 cells, Tfh cells, Th17 cells and Tregs, as well as exhausted T cells. After purity related adjustment, CXCL2 expression level was signi cantly correlated with most of the immune markers of different immune cells and different T cells in BRCA. However, CXCL2 expression level was signi cantly correlated with only 28 gene markers in STAD and 20 in LUAD (Table 3).
In addition, we found that the expression levels of most markers of monocytes, TAMs and M2 macrophage were strongly correlated with the expression of CXCL2 in BRCA (Table 3). We also found that CD86 of monocytes, CCL-2 and IL10 of TAMs, PTGS2 of M1 phenotype, CD163, VSIG4 and MS4A4A of M2 phenotype were signi cantly correlated with the expression of CXCL2 in BRCA (P < 0.05; Fig. 5). We further analyzed the correlation between CXCL2 expression and the above markers in monocytes, TAMs and M2 macrophage by GEPIA database. The results between CXCL2 and markers of monocytes and TAMs in BRCA were similar to those in TIMER (Table 4). These suggested that CXCL2 may regulate the polarization of macrophages in BRCA.
The expression of CXCL2 was related to the in ltration level of Natural killer cell and DCS in BRCA, and the expression of Natural killer cell markers KIR2DL4, KIR3DL1 and KIR3DL2, as well as DC markers HLA-DPB1, HLA-DQB1, BDCA-1 and BDCA-4 were also signi cantly correlated with the expression of CXCL2 (P < 0.01) ( Table 3). These results further reveal the close relationship between CXCL2 and the in ltration of Natural killer cell and DCS. At the same time, CXCL2 was signi cantly correlated with Treg and T cell exhaustion marker genes, such as CCR8, STAT5b, PD-1, CTLA4, LAG3 and GZMB. CCR8 plays an important role in Treg cells. PD-1, as one of the markers of T-cell exhaustion, is signi cantly related to the expression of CXCL2, suggesting that the expression of CXCL2 plays an important role in PD-1 mediated T-cell exhaustion. These results further con rm that CXCL2 is related to the immune in ltrating cells in BRCA, suggesting that CXCL2 plays an important role in immune escape in BRCA microenvironment. CXCL2 expression is regulated by miR-532-5p The prediction results of StarBase database 38 show that CXCL2 may be the target gene of miR-532-5p. Jiang and song et al respectively con rmed that CXCL2 is the target gene of miR-532-5p in HCC, and con rmed the combination of the two binding sites by double luciferase reporter gene system. 39,40 Huang et al con rmed the abnormal expression of miR-532-5p in BC, and played a role as an oncogene. 41 Our experimental results con rmed that after overexpression of miR-532-5p, the level of CXCL2 mRNA decreased, and after inhibition of miR-532-5p expression, the level of CXCL2 mRNA increased, with signi cant statistical signi cance (Fig. 6B,C). Then we analyzed the in uence of miR-532-5p on the prognosis of patients with different clinicopathological characteristics by Kaplan-Meier plotter database ( Fig. 6A and Table 5). The results showed that the expression of miR-532-5p was signi cantly correlated with HER2 negative (P = 0.0013), HER2 + ER-(P = 0.034) in molecular subtype and 2 and 3 Grades (P = 0.0396 and P = 0.032). These results con rmed that CXCL2 was regulated by miR-532-5p in BC. MiR-532-5p has signi cant correlation with prognosis of patients in HER2 status, molecular subtype and grade.

Discussion
CXCL2 is a member of chemokine superfamily, which mainly comes from activated macrophages, 42 and also known as growth-related gene product (GROβ).
It has a strong chemotaxis effect on neutrophils 43 and abnormal expression in a variety of tumor cells. Our analysis showed that CXCL2 expression level was related to the prognosis of different types of cancer. Low expression of CXCL2 is associated with poor prognosis in BC patients. CXCL2 expression level can affect the prognosis of patients with lymph node metastasis of gastric cancer and BC, indicating that CXCL2 expression can be used as a predictor of tumor metastasis. In addition, the analysis showed that the expression of CXCL2 was related to the level of immune in ltration and different immune marker sets in BC. Our research provides new insights into the potential role of CXCL2 in tumor immunity and its application as a biomarker of tumor.
In this study, we used different databases to detect the expression level and prognosis of CXCL2 in various types of cancer. The expression of CXCL2 was different in different cancer tissues and normal tissues. In the Oncomine database, the expression of CXCL2 in colorectal cancer, esophageal cancer, head and neck cancer, and lymphoma was higher than that in normal tissues. In other data sets, the expression of CXCL2 was lower in bladder cancer, BC, colorectal cancer, leukemia, lung cancer, liver cancer, lymphoma and sarcoma. But in TIMER database, CXCL2 expression was signi cantly lower in BLCA, BRCA, CHOL, HNSC, KICH, LIHC, LUAD, LUSC, and PRAD, but it was signi cantly higher in COAD, KIRC, and READ. The differences of CXCL2 expression levels in different types of cancer in different databases may re ect the data collection methods and potential mechanisms related to different biological characteristics.
Analysis of data from the PrognoScan and Kaplan-Meier plotter databases showed that low expression of CXCL2 was associated with poor prognosis in colorectal, breast and lung cancer. In datasets of PrognoScan, Low expression of CXCL2 may be an independent risk factor for poor prognosis in colorectal cancer, BC and lung cancer. Kaplan-Meier plotter showed that the abnormal expression of CXCL2 was signi cantly related to the prognosis of gastric cancer, BC and lung cancer. These ndings strongly suggest that CXCL2 is a prognostic biomarker for colorectal cancer, gastric cancer, BC and lung cancer.
Another important aspect of this study is that the expression of CXCL2 is associated with different levels of immune in ltration in BC. We found that the CXCL2 expression was positively correlated with the in ltration of CD8 + T cells, CD4 + T cells, neutrophils and DCs in BRCA. In STAD, there were negative correlations with in ltration of B cells, CD4 + T cells, macrophages, and DCs. Moreover,CXCL2 plays an important role in tumor immunomodulation of BRCA, we found that most markers of monocytes, TAMs and M2 macrophage were strongly correlated with the expression of CXCL2 in BRCA, such as the CD115 of monocytes, CCL-2 and IL10 of TAMs, CD163, VSIG4 and MS4A4A of M2 phenotype. These results reveal the potential regulatory role of CXCL2 in the polarization of tumor-associated macrophages. At the same time, CXCL2 was signi cantly correlated with Treg and T cell exhaustion marker genes, such as CCR8, STAT5b, PD-1, CTLA4, LAG3 and GZMB. In addition, there was a signi cant correlation between the expression of CXCL2 and the regulation of several markers of T helper cells in BRCA. These correlations may indicate the potential mechanism of CXCL2 regulating T cell function in BRCA. These results suggest that CXCL2 plays an important role in the recruitment and regulation of BRCA immune in ltrating cells. exhaustion and Tregs, while CXCL2 as a target gene is regulated by miR-532-5p. Therefore, miR-532-5p may play an important role in immune cell in ltration and prognosis biomarkers of breast cancer patients by regulating CXCL2.      Scatterplots of correlations between CXCL2 expression and gene markers of Monocytes in the above four cancers.