The Role Of ASIC1 In Tumors And Its Correlation With The Risk And Prognosis Of Human Tumors

Objective:The acid-sensitive ion channel ASIC1 is not only widely expressed in the central and peripheral nervous systems,but also expressed in non-excitatory cells.They play a vital role in various physiological processes（such as sour taste induction and mechanical induction）.The activity of ASIC1 is induced by an acidic microenvironment,which may cause many pathological effects.In some pathological conditions,ASIC1 can act as a mediator of injury.The main characteristic of malignant tumor tissues and damaged tissues is acidic extracellular p H.Therefore,compared with the surrounding normal tissues,the tumor microenvironment often shows local acidosis.Many reports indicate that acidosis can accelerate the invasion and metastasis of cancer,but the underlying molecular mechanism is still unclear.The previous research of our group found that the knockout of mouse Asic1a gene can down-regulate the polarization of M2 macrophages,and the specific regulation mechanism is still being explored.At the same time,more and more reports have shown that in many solid tumors,tumor-associated macrophages（TAMs）are an important part of the tumor microenvironment（TME）,and TAMs are generally considered to be very similar to M2macrophages,which can stimulate proliferation,angiogenesis,and provide an immune barrier for tumor cells to promote tumor occurrence and development.We preliminarily explored the effect of Asic1a gene knockout on mouse skin melanoma through the in vivo mouse melanoma model,and then we used the ASIC1a inhibitor to observe its effect on tumor growth and tumor burden.and observed its effect on tumor growth and tumor burden.In addition,in order to explore whether the human ASIC1 gene has the same clinical effect on human tumors and its underlying mechanism,this study first obtained the expression of ASIC1 gene in human melanoma tissue infiltrating macrophages and human melanoma cells by analyzing human melanoma single cell sequencing data（GEO database data set:GSE72056）.Secondly,we use existing databases such as TCGA（The Cancer Genome Atlas）and GEO（Gene Expression Omnibus）to perform pan-cancer analysis of the ASIC1 gene,and use a variety of bioinformatics methods to analyze the expression,clinical prognostic significance and mechanism of ASIC1 gene in tumors from 33 types of cancers to explore its role in anti-tumor immunity and provide new support for its use as a target for the treatment and prevention of cancer.Methods:Ⅰ.The effect of Asic1a gene on tumor growth in mice with melanoma1.Asic1a knockout inhibits tumor growth in mice with melanomaA mouse melanoma tumor model was established by subcutaneously injecting B16melanoma cells to explore the effect of mouse Asic1a knockout on the growth of mouse melanoma.2.ASIC1a inhibitor inhibits tumor growth in mice with melanomaThe WT mice were randomly divided into two groups and B16 cells were injected subcutaneously to establish a mouse melanoma model.The control group was intraperitoneally injected with sterile water,and the experimental group was intraperitoneally injected with ASIC1a inhibitor to observe the tumor growth of tumor-bearing mice.Ⅱ.Pan-cancer analysis of ASIC1 gene and human tumors1.ASIC1 gene expression analysis（1）Use the genome browser（UCSC）to obtain the position information of ASIC1 in the human genome,use NCBI to query the detailed information of ASIC1 gene,obtain its homologous gene information and view the protein conserved domains of different species.The ASIC1 phylogenetic tree was obtained by comparing the ASIC1 protein sequences of different species with the COBALT protein sequence multiple alignment tool.（2）Log in the Human Protein Atlas Database（HPA）to view the expression information of the ASIC1 gene in normal tissues and cells combined with HPA,GTEx and FANTOM5databases.（3）Obtain the expression difference map of ASIC1 gene in all tumors and normal tissues in the TCGA database through the tumor immunity database（TIMER2.0）.Use online tumor gene expression and survival analysis tool（GEPIA2）combined with TCGA database and GTEx database to analyze the differential expression box diagram of ASIC1gene in tumor tissue and normal tissue.（4）Use the GEPIA2 website to obtain the expression of the ASIC1 gene in all tumors at different pathological stages in the TCGA database.2.Correlation analysis of ASIC1 gene expression and survival prognosis of cancer patients.（1）Log in the tumor microarray database（Oncomine）to obtain the reported meta-analysis results of ASIC1 expression in each tumor.（2）Use the GEPIA2 online tool to analyze the correlation between the ASIC1 gene expression level and the survival prognosis of different tumor patients in the TCGA database,and obtain the overall survival and disease-free survival data.（3）Use the online survival analysis website（KM-plotter）to analyze the relationship between the ASIC1 gene expression level and the survival prognosis of the five cancer types in the GEO database.3.ASIC1 gene genetic variation analysis and immune infiltration analysis.（1）Query the genetic variation characteristics,protein structure,variation site and 3D structure of the ASIC1 gene in all tumors in the TCGA database through the tumor genomics website（c Bio Protal）,and then the relationship between ASIC1 gene variation and clinical survival prognosis of SKCM was obtained through this website.（2）Use the TIMER2.0 online tool to analyze the correlation between ASIC1 gene expression and the immune infiltration of all tumors in the TCGA database.4.ASIC1 gene protein interaction network and enrichment analysis of related genes（1）Use the protein interaction network analysis database（STRING）to construct a protein network that interacts with ASIC1.Use the GEPIA2 tool to screen out the top 100ASIC1 similar genes and perform pair-gene Pearson correlation analysis between the ASIC1gene and the selected gene.（2）Through the"cluster Profiler"package and"ggplot2"package in the R language software,the GO function and KEGG pathway enrichment analysis were performed on the two sets of gene data obtained,and the results were visualized.Results:1.The mouse melanoma model was successfully established,and the tumor growth and tumor weight of Asic1a^-/-mice were significantly reduced.The use of ASIC1a inhibitor can significantly inhibit tumor growth and tumor burden in mice with melanoma.2.Comprehensive data sets of HPA,GTEx and FANTOM5 show that ASIC1 is expressed in most normal human tissues and cells,and it is highly expressed in brain tissues.3.Analysis of human melanoma single-cell sequencing data shows that ASIC1 is expressed in tumor-infiltrating macrophages,melanoma cells and tumor-associated fibroblasts.In addition,ASIC1 is highly expressed in most tumors in the TCGA database,but only in BRCA（Breast invasive carcinoma）,KIRC（Kidney renal clear cell carcinoma）,KIRP（Kidney renal papillary cell carcinoma）and BLCA（Bladder Urothelial Carcinoma）,the expression level is lower than that of normal tissues.4.The expression levels of ASIC1 in ACC（Adrenocortical carcinoma）,BLCA（Bladder Urothelial Carcinoma）,KICH（Kidney Chromophobe）,KIRP and THCA（Thyroid carcinoma）tumors is significantly different in different stages,and the expression levels increased with the pathological progression of tumors.5.Meta-analysis of the difference of ASIC1 expression between tumor tissues and normal tissues by Oncomine database showed that ASIC1 expression is higher in brain and central nervous system cancer,leukemia and colorectal cancer compared with the normal group.6.Analyze the correlation between the expression level of ASIC1 gene and the overall survival（OS）and disease-free survival（DFS）of all 33 tumor types in the TCGA database.The results can be divided into high expression groups and low expression groups:high expression of ASIC1 was associated with poor overall survival and disease-free survival of ACC,KIRC,KIRP and UVM（Uveal Melanoma）,and low expression of ASIC1 was associated with poor overall survival of LGG（Brain Lower Grade Glioma）,LUSC（Lung squamous cell carcinoma）,and disease-free survival of LGG.7.KM-plotter analysis shows that ASIC1 expression is correlated with the survival prognosis of breast cancer,ovarian cancer,lung cancer,gastric cancer and liver cancer in GEO database.8.Analysis of ASIC1 genetic variation characteristics found that ASIC1 has the highest copy number amplification frequency in ACC,and the highest frequency of gene mutations in UCEC（Uterine Corpus Endometrial Carcinoma）and SKCM（Skin Cutaneous Melanoma）.The mutation site information shows that the main type of ASIC1 gene change in SKCM is missense mutations,leading to poor prognosis of OS,DSS（Disease-specific survival）and PFS（Progression Free Survival）.9.Through online database analysis based on a variety of statistical algorithms,ASIC1gene expression levels in all tumors in the TCGA database are positively correlated with tumor-associated fibroblast（CAF）immune infiltration levels,but only in LGG,there is a statistically negative correlation.The expression level of ASIC1 gene was negatively correlated with CD8⁺T cells in most tumors.10.The GO functional biological process analysis of ASIC1 binding and related genes using R language software shows that ASIC1 related genes are in the co-translational protein targeting membrane pathway,nuclear transcription m RNA catabolism process,protein targeting endoplasmic reticulum process,and endoplasmic reticulum protein localization.The KEGG pathway enrichment analysis shows that ASIC1 and its related genes mainly play a role in the ribosomal pathway and the coronavirus disease COVID-19 pathway.Conclusion:1.Asic1a gene plays an important role in the occurrence and development of tumors in melanoma mice,and may become a potential target for tumor therapy.2.The expression level of ASIC1 gene is significantly increased in most human tumors and is related to the prognosis of different tumor cases.3.In most tumors,the expression level of ASIC1 gene is positively correlated with the level of tumor-associated fibroblast（CAF）immune infiltration,and negatively correlated with the level of CD8⁺T cell infiltration.4.ASIC1 may plays a biological role in the process of ribosomal protein synthesis,and may have a potential impact on the expression of tumor genes.