ABSTRACT
Objective:To investigate the value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma.Methods:The mRNA sequencing data of 471 lung adenocarcinoma patients and corresponding methylation data of 413 cases were downloaded from The Cancer Genome Atlas (TCGA) database, the methylation levels of SHOX2 and RASSF1A gene promoter regions were calculated, and the difference in methy lation level between normal lung tissues and tumor tissues was analyzed. The clinical data of 54 patients with early-stage lung adenocarcinoma and 31 patients with benign lung tumors who underwent surgery at Drum Tower Hospital Affiliated to Nanjing University Medical School from January 2018 to January 2019 were retrospectively analyzed. The methylation status of SHOX2 and RASSF1A in tumor tissues and normal lung tissues (>5 cm from the edge of the tumor foci) (called clinical samples) was detect, and a positive methylation in the promoter region of either gene was considered as a combination of two genes methylation positivity. Using pathological diagnosis as the gold standard, the efficacy of gene methylation positivity in diagnosing early-stage lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve. Patients with >80% of tumor cells in paraffin samples were screened, and mRNA high-throughput sequencing was performed in their tumor tissues and normal lung tissues. The relationship between positive methylation of the two genes and clinicopathological features was analyzed, and the correlation between the promoter region methylation level of the two genes and mRNA expression levels in clinical samples and TCGA database samples was analyzed by Spearman method. Gene set variance analysis (GSVA) method was used to analyze the differences in Kyoto Encyclopedia of Genes and Genomes enrichment pathways between two-gene methylation-positive clinical lung adenocarcinoma samples and corresponding methylation-negative lung adenocarcinoma.Results:In TCGA database, the SHOX2 promoter region methylation island contained 6 sequenced methylation sites, of which sites cg04532033 and cg01557547 methylation levels were higher in lung adenocarcinoma tissues than in normal lung tissues (both P < 0.05); the RASSF1A gene promoter region methylation island contained 11 sequenced methylation sites, and the methylation levels of 6 of these sites in lung adenocarcinoma tissues were higher than those in normal lung tissues (all P < 0.05). Compared with normal lung tissues, the methylation level of SHOX2 promoter region was higher in stage Ⅰ and Ⅱ lung adenocarcinoma tissues (both P < 0.05); the methylation level of RASSF1A promoter region was higher in all stages of lung adenocarcinoma ( P < 0.001). Among 54 patients with early-stage lung adenocarcinoma, 28 were positive for SHOX2 promoter region methylation in tumor tissues, 21 were positive for RASSF1A promoter region methylation, and 40 were positive for combined methylation of both genes; 31 benign lung nodules were negative for SHOX2 and RASSF1A methylation. ROC curve analysis showed that the sensitivity of positive SHOX2 promoter region methylation for diagnosing early-stage lung adenocarcinoma was higher than that of RASSF1A promoter region methylation positivity (51.8% vs. 38.9%), and the area under the curve (AUC) for diagnosis by two-gene methylation positivity was larger than that for diagnosis by SHOX2 or RASSF1A gene methylation positivity alone (0.870 vs. 0.759 and 0.694). The circulating thresholds (Ct) of SHOX2 and RASSF1A methylation tested by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in stage Ⅰ and Ⅱ lung adenocarcinoma were lower than those in normal lung tissues (all P < 0.05); patients with two-gene methylation positivity were characterized by older age, longer tumor longest diameter and more advanced pathological stage compared with patients with two-gene methylation negativity (all P < 0.05). In clinical stage Ⅰ-Ⅱ lung adenocarcinoma samples, the Ct of SHOX2 and RASSF1A promoter region methylation tested by qRT-PCR was negatively correlated with their mRNA relative expression levels ( r=-0.43, P = 0.003; r = -0.48, P = 0.001); in TCGA database stage Ⅰ-Ⅱ lung adenocarcinoma samples, the level of SHOX2 promoter region methylation was negatively correlated with its mRNA relative expression level ( r = -0.23, P < 0.001), and the level of RASSF1A promoter region methylation was also negatively correlated with its mRNA relative expression level, but without statistical difference ( r = -0.05, P = 0.310). In two-gene promoter methylation-positive lung adenocarcinoma samples, the pathways related to folate metabolism and DNA stability were upregulated, and the pathways related to vasoconstriction and cell growth and differentiation were downregulated. Conclusions:The combined detection of SHOX2 and RASSF1A promoter region methylation can be used as an indicator for screening and diagnosis of early-stage lung adenocarcinoma. Abnormal promoter region methylation of the two genes may affect multiple tumor-related pathways and promote the occurrence and progression of early-stage lung adenocarcinoma.
ABSTRACT
Objective:To analyze the difference in immune microenvironment between primary tumor tissues and metastatic tumor tissues of metastatic colorectal cancer, and to screen specific immune-related differentially expressed genes (DEG) related to prognosis of metastatic colorectal cancer via bioinformatics methods.Methods:The GSE131418 microarray dataset of colorectal cancer and metastases was downloaded from gene expression omnibus (GEO) database, including 517 samples from the MCC cohort and 618 samples from the Consortium cohort in Moffitt Cancer Center. Immune-related gene sets were downloaded from immunology database and analysis portal IMMPORT, including 2 483 immune-related genes. A total of 695 cases of RNA sequencing data and 627 cases of clinical information of colorectal cancer tumors and adjacent tissues were downloaded from Cancer Genome Atlas (TCGA) data. The stroma cell score, immune cell score and stromal immune total score of metastatic tumor tissues and primary tumor tissues were calculated by using ESTIMATE algorithm, and 22 kinds of immune cell infiltration in primary tumor and metastatic tumor tissues of colorectal cancer were compared and analyzed by using CIBERSORT deconvolution algorithm. Immune-related DEG were screened to make Kyoto Encyclopedia of Genes and Gnomes (KEGG) signaling pathway enrichment analysis. The patients were divided into high and low expression groups according to the median expression levels of immune-related DEG. The Kaplan-Meier method and Cox regression risk model were used to analyze immune-related DEG, and the genes significantly related to prognosis in the results of the two methods were screened (all P < 0.01), and multivariate analysis was performed by using Cox regression method. The expression differences of each gene in tumor tissues, adjacent tissues, primary tumor tissues and metastatic tissues in GSE131418 data sets of TCGA database and GEO database were compared, and survival analysis was also performed. Results:The stroma cell score, immune cell score and stromal immune total score of colorectal cancer metastatic tissues were lower than those of primary tumor tissues (all P < 0.001). Compared with primary tumor tissues, the proportion of activated natural killer (NK) cells, monocytes, CD8 + T cells, T cells, activated dendritic cells in metastatic colorectal cancer tissues was increased, while the proportion of inactive mast cells, inactive dendritic cells, inactive NK cells, activated memory CD4 + T cells, M1 macrophages, and neutrophils was decreased. There were 289 immune-related DEG in metastatic tissues and primary tumor tissues of metastatic colorectal cancer, including 101 up-regulated genes and 188 down-regulated genes. KEGG signaling pathway enrichment analysis showed that in the immune microenvironment of metastatic tissues in metastatic colorectal cancer, vascular endothelial growth factor (VEGF) signaling pathway, programmed death ligand 1 (PD-L1) expression and programmed death 1 (PD-1) checkpoint pathway, T helper cell (Th) 1, Th2 and Th17 cell differentiation, NF-kappa B signaling pathway, interleukin 17 (IL-17) signaling pathway, chemokine signaling pathway, T cell receptor signaling pathway, MAPK signaling pathway, and NK cell-mediated cytotoxicity pathways enrichment were detected. Immune-related DEG related to prognosis including ANGPTL5, FPR1, HSPA8, NR2E3, PSMD2, PSMD8 and SBDS were screened out. Cox regression multivariate analysis showed that immune-related DEG ANGPTL5 ( HR = 2.69, 95% CI 1.22-5.92, P < 0.05), HSPA8 ( HR = 0.57, 95% CI 0.33-0.97, P < 0.05), and SBDS ( HR = 2.23, 95% CI 1.18-4.21, P < 0.05) were independent prognostic factors for metastatic colorectal cancer. The expression of ANGPTL5 in tumor tissues was lower than that in normal tissues, and the expression of ANGPTL5 in metastatic tissues was higher than that in primary tumor tissues. Patients with high expression of ANGPTL5 in tumor tissues had worse prognosis. The expression of HSPA8 in tumor tissues was higher than that in normal tissues, and the expression of HSPA8 in metastatic tissues was lower than that in primary tumor tissues. Patients with high expression of HSPA8 in tumor tissues had a better prognosis. The expression of SBDS in tumor tissues was lower than that in normal tissues, and the expression of SBDS in metastatic tissues was lower than that in primary tumor tissues. Patients with high expression of SBDS in tumor tissues had worse prognosis. Conclusions:Immune microenvironment of metastatic colorectal cancer is quite different from that of primary tumor. The degree of immune cell infiltration is reduced and the whole is immunosuppressed. The specific immune-related DEG related to prognosis of metastatic colorectal cancer may be new therapeutic targets of metastatic colorectal cancer.
ABSTRACT
Objectvie:To investigate the infiltration patterns of immune cells in colorectal cancer, and to explore the correlation of immune cells infiltration with clinical characteristics and overall survival (OS) of patients.Methods:The RNA sequencing data of 615 patients with colorectal cancer were downloaded from The Cancer Genome Atlas (TCGA) database. The data was updated on July 19, 2019, and included 571 colorectal cancer tissues and 44 paracancerous tissues. There were 552 cases with clinical data, such as survival time, survival status, age, gender, clinical stage, grade, tumor location and so on. Using CIBERSORT deconvolution algorithm, the relative amounts of 22 immune cell types were calculated based on standardized gene expression data. According to the results of CIBERSORT algorithm, the samples with high accuracy of deconvolution result were selected ( P < 0.05), and they were used for analysis and graphing. The correlations between the infiltration patterns of immune cells and the clinical characteristics and OS of patients were analyzed. Results:After the CIBERSORT method was used to filter and remove samples with P ≥ 0.05, a total of 282 tumor tissue samples and 16 paracancerous tissue samples were screened. In 293 cases with clinical information, there were 277 tumor tissue samples and 16 paracancerous tissue samples. In 293 samples, M0 macrophages, M1 macrophages, M2 macrophages, CD8 + T cells and unactivated CD4 memory T cells accounted for a higher proportion of total immune cells; in tumor tissue samples, the expressions of M0 macrophages, M1 macrophages, activated CD4 memory T cells, and unactivated natural killer (NK) cells were higher; in paracancerous tissues, the expressions of naive B cells, M2 macrophages, activated NK cells, unactivated dendritic cells, unactivated mast cells and plasma cells were higher; with the increase of clinical stage, the expressions of follicular helper T cells, activated CD4 memory T cells, activated NK cells, M1 macrophages decreased, and the expressions of plasma cells and regulatory T cells increased, and the differences were statistically significant (all P < 0.05). M1 macrophages, unactivated mast cells, activated CD4 memory T cells, CD8 + T cells, and follicular helper T cells were highly expressed in right colon cancer, while M0 macrophages and activated mast cells were highly expressed in left colon and rectal cancer, and the differences were statistically significant (all P < 0.05). The patients were divided into high infiltration group and low infiltration group based on the median expression level of infiltrated immune cells, and the survival analysis was performed. The result of survival analysis showed that patients with high initial B cell infiltration had good OS; however, patients with high infiltration of M2 macrophages, activated mast cells, and neutrophils had poor OS. Conclusions:There are different types of immune cell infiltration patterns in the colorectal cancer samples of different stages and locations, which are closely related to tumor progression and OS of patients. They are expected to be applied to the development of therapeutic targets and prognosis prediction.