| Tumor-associated macrophages(TAM)are macrophages that have evolved from the infiltration of peripheral blood mononuclear cells into solid tumor tissues and play an important role in Tumor microenvironment(TME)by secreting cytokines and growth factors to stimulate the growth and metastasis of tumor cells.Colony stimulating factor-1 receptor(CSF1R)is a macrophage surface receptor that can bind colony stimulating factor-1(CSF1).The CSF1/CSF1 R signaling pathway can regulate the differentiation and function of macrophages and plays a key regulatory role in the transformation of normal macrophages into tumor-associated macrophages.At present,a few studies have dealt with the molecular mechanism of CSF1 R in the development of Hepatocellular carcinoma(HCC),moreover,whether the level of DNA methylation in the CSF1 R promoter region is related to the occurrence of liver cancer is still unknown.In this study,160 cases of HCC patients were selected,tumor and Adjacent non-cancerous tissues(ANT)were used as our research objects.Infinium HumanMethylation450 Bead Chip sequencing platform was used,genome methylation sequencing of 160 samples were analyzed.Data analysis showed a large number of genes had significant differences in DNA methylation in tumor tissues and adjacent tissues of liver cancer,especially CSF1 R.So CSF1 R was used as the research object in this study.The methylation level of its promoter region at 160 pairs of liver cancer tumor tissues and adjacent tissues was further analyzed.At the same time,the changes of mRNA and protein levels of CSF1 R gene were studied in tumor tissues and adjacent tissues of liver cancer.Finally,the relationship between the DNA methylation level of the promoter region of CSF1 R gene and the clinicopathological characteristics of patients was analyzed.The potential effect of methylation of the promoter region of CSF1 R gene on the development of HCC were studied,which provides a theoretical basis for screening markers for clinical diagnosis of liver cancer.The research results are as follows:1.Analysis of clinical and pathological characteristics of patients.160 patients with liver cancer were selected for research,including 131 male patients and 29 female patients.There were 55 patients with drinking habits,20 patients with diabetes,37 patients with hyperlipidemia,136 patients were associated with hepatitis B virus,88.8% of the patients had cirrhosis.15 had multiple tumors,14 had tumors larger than 10 cm in diameter,108 had capsule breakthroughs,29 had tumor necrosis.2.Genome methylation sequencing.DNA was extracted from tumor tissues and adjacent tissues of 160 patients with liver cancer,treated with bisulfite,and subjected to whole genome methylation sequencing by Illumina's HumanMethylation450 Bead Chip sequencing chip.A large number of genes with significant differences in DNA methylation in tumor tissues and adjacent tissues were screened.One of the genes that had significant differences and played a key role in the transformation of macrophages to tumor-associated macrophages was chose as the research object for in-depth analysis.3.The methylation level of the CSF1 R promoter region in liver cancer patients was significantly higher than that in HCC tumor tissues.The methylation of CSF1 R promoter region in the adjacent tissues and tumor tissues of 160 patients with liver cancer was analyzed by differential analysis.There was a significant difference in the methylation level of the CSF1 R promoter region in adjacent tissues and tumor tissues,which was significantly higher than in adjacent tissues than that in HCC tumor tissues(65.32 vs.57.26%,P <0.0001).4.The methylation site of the CSF1 R promoter region in adjacent cancer tissues has potential significance as a biologic site for liver cancer diagnosis.By making ROC curves of the methylation sites of the CSF1 R promoter region in 160 samples of cancerous tissue,and calculating the area under the line,the potential value of methylation of the CSF1 R promoter region in the tissues adjacent to cancerous tissue as a biomarker site was determined.The results showed that the area under the ROC curve of methylation sites in all CSF1 R promoter regions was greater than 0.5,indicating that the methylation levels of different CpG sites in the CSF1 R promoter region in tissues adjacent to liver cancer have the potential as biomarkers for liver cancer diagnosis.5.The mRNA expression level of CSF1 R in adjacent tissues is significantly higher than that in tumor tissues.Mining the transcriptome sequencing data of liver cancer patients obtained earlier by our research group found that the expression level of CSF1 R mRNA in adjacent tissues was significantly higher than that in tumor tissues.At the same time,the analysis of other liver cancer-related transcriptome data in TCGA got the same conclusion.6.The protein expression level of CSF1 R in adjacent tissues is significantly higher than that in tumor tissues.Tissue in situ hybridization was performed on 16 pairs of 160 patient samples,and the results showed that CSF1 R protein was mainly present in the cytoplasm,and the expression level of CSF1 R protein was significantly higher in tumor tissues than in tumor tissues.7.The expression of CSF1 R in adjacent tissues has a negative correlation with the methylation level of the CSF1 R promoter region.By further mining the data of other liver cancer patients in the TCGA database,it was found that the methylation levels of multiple methylation sites in the CSF1 R promoter region and the expression of CSF1 R showed a negative correlation.At the same time,the CpG methyl site in the vicinity of the CSF1 R promoter in 160 patients was analyzed,and the methylation level of the adjacent site and the expression of CSF1 R also showed a negative correlation.8.Correlation analysis between DNA methylation of CSF1 R gene promoter region and clinicopathological characteristics of liver cancer.Analysis of the correlation between clinicopathological features and DNA methylation level of the CSF1 R gene showed that the methylation level of the CSF1 R promoter region in adjacent cancer tissues of 160 patients were lower with diabetes.At the same time,there was a significant correlation between the methylation level of the CSF1 R promoter region in the adjacent tissues and the common clinical diagnostic target CA199(P = 0.004).Therefore,the methylation level of the CSF1 R promoter region in adjacent cancer tissues has significance as a biomarker for liver cancer diagnosis.In addition,there is also a significant correlation between the low methylation level of the CSF1 R promoter region in adjacent tissues and the clinicopathological features of multiple tumors,tumor size,and high tumor stage.It is the first time that we found the methylation level of the CSF1 R promoter region in hepatocellular carcinoma tissues was significantly higher than that in tumor tissues,and the mRNA and protein expression levels of CSF1 R in the tissues adjacent to cancer cells were significantly higher than those in tumor tissues.The expression in CSF1 R promoter region has a negative correlation with the methylation level of the CSF1 R promoter region.The methylation level of the CSF1 R promoter region in adjacent cancer tissues has potential diagnostic value for liver cancer. |
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