| BackgroundLaryngeal cancer is one of the most common malignant tumors in the head and neck.The main clinical symptoms are:hoarseness,cough,swallowing and dyspnea,cervical lymph node metastasis.Laryngeal cancer,like other malignancies,is a life-threatening disease.Multi-gene,multi-factor interaction results in laryngeal cancer.Therefore,laryngeal cancer is also a major difficulty in the current medical field of cancer prevention and therapy.Studies have shown that early diagnosis and treatment can improve the survival rate of laryngeal cancer after surgery and reduce complications.However,about60%of patients with laryngeal cancer was found to have been sick until the third or fourth stage.In recent years,although the overall incidence was decreased,the survival rate of patients within 5 years is declining.Although the level of clinical treatment of laryngeal cancer is rapidly increased,the treatment effect is still not ideal.Moreover,the treatment brings many adverse reactions to patients.Researchers have found that some biological agents that exist in the human body can enhance the sensitivity of tumor cells to radiotherapy,such as tumor necrosis factor??TNF??,mainly produced by macrophages as an important inflammatory factor.On the one hand,TNF?can mediate cachexia,produce cytotoxicity effect on tumor cells and also can produce cytotoxicity,and induce?-2interferon producing IL-1,so that it has a variety of biological activity.Tumor necrosis factor in tumor tissue secreted by autocrine,which induced CXL12 and VEGF production indirectly inducing tumor growth and proliferation.Moreover,TNF?is also the strongest anti-tumor cytokines.It plays a key role in inhibiting tumor progression by inhibiting apoptosis or killing tumor cells,as well as enhancing immunity through multiple signaling pathways.Toll-like receptor?TLR?belongs to the pattern recognition receptor family,so far in the human genome 10 Toll-like receptors have been found.TLRs are bridges that link adaptive immune responses and natural immunity,sensing different stimulations in the response of different organisms,recruiting specific adapter proteins,thereby activating the signal cascade and triggering a corresponding immune response.TLR4 is the earliest discovered pathogen recognition receptor in the TLR family,whose signal affects the innate immune response caused by most microorganisms.The TLR4 molecule has a repeating sequence of 16-28 multiple leucine at the N-terminus to identify PAMPs and contains the Toll/IL-1 receptor domain at the C-terminus.The primary role is to activate the downstream signaling pathway.Cancer is one of the major diseases that endanger human health.Individual differences between patients is a major challenge in the current clinical treatment.High-throughput sequencing technology can detect genetic variation of chromosome structure variation,copy number variation and sequence information variation,and can also analyze epigenetic status such as DNA methylation and histone modification,and can also analyze gene expression regulation combining with RNA-seq,ChIP-seq and other related techniques.High-throughput sequencing can get a large number of multi-dimensional data at one time to do comprehensive and systematic studies at the molecular level in cancer.With scientific statistical analysis,we can find tumor-related genome mutation or modification information to develop new technology and means for cancer prevention,diagnosis and treatment.Aim of our studyIn this study,the gene expression profiles of laryngeal HEp-2 cells were used as analytical materials.We used the gene annotation tool GATHERR online software?http://gather.genome.duke.edu/?to help interpreter and analyze the function of GO?Gene Ontology?in laryngeal carcinoma HEp-2 cells.We used Graphic Network Display and Analysis Editing Software JEPETTO?Java Enrichment of Pathways Extended To Topolgy??Version 1.3.1?to do signal pathway analysis of KEGG?Kyoto Encyclopedia of Genes and Genomes,Kyoto Database of Genome and Genome Encyclopedia?.The interaction map of the related gene expression proteins was drawn using the STRINGC?Search Tool for the Retrieval of Interacting Genes/Proteins?online software?http://string.embl.del/?and the key genes were screened out.This study can elucidate the molecular mechanism of TNF?on HEp-2 cells and the related signaling pathways.Methods1,HEp-2 cell cultureHEp-2 cells were seeded on 6-well cell culture plates.After the cells were adhered,the culture medium was discarded,and treated with TNF??100 ng/mL?or phosphate buffer saline?PBS?,and incubated in a 5%CO2 incubator at 37°C for 24 h.We set the experimental group and control group which were marked separately.2,Differential expression analysisIn this study,Trimmomatic was used to remove low-quality RNA-seq data from FASTQ files in HEp-2 cells.Data were mapped to the analytical tool HISATA for analysis of m RNA and LncRNA.In this process,a maximum of two values were allowed to mismatch.All other parameters were used the default values to map the data.StringTie v1.3.1c was used to quantify the FPKM expression matrix.Before the subsequent data analysis,FPKM value was added a value?0.01?.All statistical analyzes were performed using R language software,using threshold changes greater than 1.5 to get up-regulated differentially expressed genes?DEGs?.R language software was also used for DEGs clustering analysis and hotspot analysis.3,GO and KEGG pathway analysisIn this study,we compared the up-regulation and down-regulated genes of HEp-2cells by GO function enrichment analysis?P<0.05?using the DAVID online software?DAVID 6.8;https://david.ncifcrf.gov/?.After that,we sorted out the differentially expressed genes of HEp-2 cells and analyzed the KEGG pathway by using the online bio-tool KEGG Mapper 83.0?http://www.kegg.jp/kegg/mapper.html?to explore the difference expression genes involved in HEp-2 cells of the biological pathway.4,Protein to protein network constructionIn this study,HEp-2 differentially expressed genes were introduced into the STRING online search tool?https://string-db.org/?to get interaction network diagram of the genes.After the deletion of the interaction between the protein and the other protein,the PPI data of the differentially expressed genes of the STRING database were derived and the text format was introduced into Cytoscape software.The plug-in GenetiScape was used to calculate the network and the topological characteristics of each node.5,Western blottingThe expression of protein in HEp-2 cells treated with or without TNF?was analyzed by Western blot analysis.With the same amount of tissue protein mixed with SDS loading buffer for protein electrophoresis,the transfer,milk blocking,incubation of primary antibody,secondary antibody,exposure,development,fixing,the final result was examined by gray Quantitative analysis of quantitative analysis of expression differences.Research results1.Compared with adjacent tissues,TNF? was highly expressed in human laryngeal squamous cell carcinoma.HEp-2 cells were treated with TNF??100 ng / m L?,and the changes of gene expression between TNF? and control group were compared.The results showed that 2811 m RNAs were up-regulated and 1128 m RNAs were downregulated.2.RNA-seq data was obtained using NGS and significant DEGs were identified.The TNF? treatment was distinguished from the control group using the hot spots showing the expression of DEGs and the cluster analysis.3.Through GO analysis in the up-regulated DEGs,we established a protein interaction network map.The results showed that TNF? treatment accelerated the HEp-2 cell cycle and proliferation by up-regulating the key genes.4.The NF-?B,MAPK and PI3K-Akt pathways associated with cell survival,cell cycle and proliferation were up-regulated by GO enrichment analysis and KEGG signaling pathway analysis.NF-?B was the downstream of MAPK and PI3K-Akt signaling pathway.5.TLR4 and NF-?B1 expression levels were significantly increased in TNF? treatment group.MYD88,IRAK1 and TAB as TLR4 downstream receptor genes were significantly up-regulated.Moreover,TLR4 was also highly expressed in human laryngeal squamous cell carcinoma compared with paracancerous tissue.6.In order to further confirm our results from RNA-seq,we used immunoblotting to identify TLR4 protein expression levels in HEp-2 cells with or without TNF?treatment.The results showed that TNF??100 ng / m L?treatment for 24 h significantly increased TLR4 protein expression.7.Through Lnc RNA differential expression analysis,we found that 8 Lnc RNAs were upregulated,and 6 LncRNAs were down-regulated.ConclusionsThis study demonstrated that TNF? treatment upregulated m RNA and Lnc RNA expression of many key genes involved in cell cycle and DNA replication.In addition,TLR4,NF-?B,MAPK and PI3K-Akt signaling pathways also increased,and these increases may enhance TNF?-induced HEp-2 cell proliferation and growth.Our results suggest that TNF? may play a key role in the development of laryngeal cancer. |
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