LncRNA MIR99AHG Regulates Tumor-infiltrating Immune Cells In Head And Neck Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinoma(HNSCC)is the sixth most common malignant tumor worldwide.HNSCC has a high degree of malignancy,is prone to local recurrence and metastasis,and has a low 5-year survival rate with a poor prognosis.Therefore,improving the prognosis and increasing the survival rate of HNSCC is an urgent clinical issue.Long non-coding RNA(lnc RNA)is an RNA that does not have the ability to encode proteins.Specifically expressed lnc RNA can affect tumors’ occurrence,development,and prognosis.lnc RNA MIR99 AHG can affect the occurrence and development of Oral Squamous Cell Carcinoma(OSCC)and Tongue Squamous Cell Carcinoma(TSCC),and serves as a specific biomarker with crucial clinical significance for the prognosis and diagnosis of HNSCC.Competitive endogenous RNA(ce RNA)is a vast and sophisticated regulatory network,in which the mutual regulation pattern between lnc RNA and mi RNA and their downstream target genes is closely related to the occurrence and development of tumors,and has become a hot spot in the field of tumor research.Previous studies have focused on tumor cells,but studies have demonstrated that the mechanism of tumor changes is not just limited to tumor cells.The interaction between the tumor microenvironment and tumor cells is of great significance.Cancer-associated fibroblast(CAF)is the primary mesenchymal cell in the tumor microenvironment,which regulates the biological behavior of tumor cells by secreting growth factors and exosomes,and other factors.Therefore,CAFs may serve as potential tumor treatment and prognosis improvement targets.Tumor-infiltrating immune cells,as an essential component of the tumor microenvironment,are widely present in various stages of tumor occurrence and development,and can either inhibit or promote tumor progression.Exploring the correlation between lnc RNA MIR99 AHG and tumor-infiltrating immune cells will provide a solid foundation for subsequent in-depth studies.This study aims to explore and construct the possible ce RNA regulatory network of lnc RNA MIR99 AHG,which provides a new biomarker for the potential diagnosis and prognosis of HNSCC;And this study aims to examine the effect of CAFs regulating lnc RNA MIR99 AHG on tumor-infiltrating immune cells,which provides new therapeutic targets for the targeted therapy of HNSCC.Materials and methods: The expression profile and related clinical data of lnc RNA MIR99 AHG associated with HNSCC were obtained from the TCGA database and clinical tumor samples.The mi RNA and m RNA data related to HNSCC prognosis were obtained from the Xiantao Academic website.The mi RNA data predicted to be associated with HNSCC were obtained from the Lnc Base website.Cytoscape software was used for visualization analysis,and the Metascape website was used for functional enrichment analysis of screened RNAs.Cytoscape plug-in Cyto Hubba to obtain and identify Hub triple regulatory network and make visual data integration.Xiantao Academic website for differential expression analysis,prognosis analysis,and molecular correlation analysis of screened ce RNA regulatory network genes.The Human Protein Atlas database detects the expression of Autophagy Related 9B(ATG9B)in the tongue tissue.q RT-PCR and immunohistochemical staining to detect the expression of ATG9 B,respectively.Xiantao Academic website for the analysis of ATG9 B and its prognosis and Metascape website for the functional enrichment analysis of the top 200 genes associated with ATG9 B.We detected the expression of ATG9 B after overexpression of lnc RNA MIR99 AHG by q RTPCR and Western blot.We detected the expression of mi R-4775 and mi R-7854-3p after overexpression of lnc RNA MIR99 AHG by q RT-PCR.CAFs and Normal Fibroblasts(NFs)for primary culture and identification.q RT-PCR to detect the expression of MIR99 AHG in tongue cancer cells co-cultured with CAFs or NFs.Correlation analysis was conducted between lnc RNA MIR99 AHG in HNSCC and tumor-infiltrating immune cells.Results: According to TCGA database and clinical tumor samples data,it was found that lnc RNA MIR99 AHG was a low expression gene in HNSCC,and its high expression was positively correlated with prognosis.The bioinformatics analysis establishes the lnc RNA MIR99AHG-mi R-4775/mi R-7854-3p-ATG9 B ce RNA regulatory network.High expression of ATG9 B in tongue tissue was found in the HPA database.And ATG9 B was highly expressed in head and neck normal tissues and low in HNSCC tissues by q RTPCR and immunohistochemistry.q RT-PCR and Western blot showed that overexpression of lnc RNA MIR99 AHG can upregulate the expression of ATG9 B m RNA and protein.q RT-PCR showed that overexpression of lnc RNA MIR99 AHG could downregulate of mi R-4775 and mi R-7854-3p.Western blot and immunocytochemistry showed high expression of α-SMA in CAFs.q RT-PCR results showed that the expression of lnc RNA MIR99 AHG was significantly decreased in tongue cancer cells co-cultured with CAFs compared with the control group.Bioinformatics analysis found that lnc RNA MIR99 AHG positively correlated with T cells,CD8+ T cells,B cells,and NK cells,while negatively associated with Th1,Th2,neutrophils,and Tgd.Conclusion:(1)lnc RNA MIR99 AHG can function as a ce RNA,providing a new biomarker for the potential diagnosis and clinical prognosis of HNSCC.(2)CAFs affect the expression of tumor-infiltrating immune cells by regulating the expression of lnc RNA MIR99 AHG in HNSCC.