Mechanism Of Over-expressed Proline 4-hydroxylase Subunit Member α In HNSCC

BackgroundSquamous cell carcinoma of the head and neck(HNSCC)ranks eighth in the global cancer incidence of malignancies,accounting for about 90% of head and neck cancers.Current treatment methods do not significantly prolong the survival time of patients,mainly related to the characteristics of local invasion and metastasis of the tumor.The treatment of squamous cell carcinoma of the head and neck still lacks highly effective therapeutic drugs,so it remains a huge challenge for mankind.Therefore,the prognostic markers and specific molecular mechanisms of head and neck squamous cell carcinoma and tumor metastasis and invasion still need to be explored.Tumor metastasis is related to the tumor microenvironment,and studies have shown that collagen,as an important component of the tumor microenvironment,participates in the process of tumor metastasis.The rate-limiting step in collagen synthesis is posttranslational modification,and the important enzyme is prolyl hydroxylase.In this study,the effect of collagen proline 4-hydroxylase α members(C-P4HAs)on the prognosis of HNSCC was analyzed,and the impact on the occurrence,development and mechanism of HNSCC was further explored,aiming to provide a basis for exploring new biotargeted therapies for HNSCC.Methods1.The TCGA dataset in UALCAN was used to analyze the tumor types with relatively high expression of P4 HA gene,and then the best cut-off value was determined according to the expression of P4 HA gene in head and neck squamous cell carcinoma,and the sample was divided into high and low expression groups using the best cut-off value,and the Kaplan-Meier curve was drawn to analyze the relationship between gene expression and survival status to determine the tumor types with significant differences in the survival of patients with head and neck squamous cell carcinoma2.The effects of single and co-knockdown of P4 HA on the malignant phenotype of Cal27 cells were observed using small interfering RNA knockdown P4HA1,P4HA2,and P4HA3,and the changes of malignant phenotypes after knocking down and coknocking down of collagen P4 HA family genes were compared,and the impact on the survival prognosis of HNSCC patients was observed.3.To investigate the reasons why collagen P4 HA family genes influence the malignant phenotype of head and neck squamous cell carcinoma.Cal27 cells were treated with collagen prolyl 4-hydroxylase activity inhibitor EDHB or pythi DC to observe malignant phenotypic changes in tumor cells.4.In order to study the molecular interactions and signaling pathways of collagen P4 HA family genes in head and neck squamous cell carcinoma,we used the String database to analyze the molecules interacting with the collagen P4 HA family genes,and then enriched the molecules interacting with P4 HA with the DAVID database to analyze the signaling pathways that may be involved.5.The TCGA database was used to analyze genes that were positively and negatively correlated with collagen P4 HA family genes,and Kaplan-Meier survival curves were drawn according to P4HA-related gene expression in patients with head and neck squamous cell tumors.To investigate whether the survival prognosis of P4 HArelated genes in HNSCC patients is affected by P4 HA genes.Patients with high expression of P4HA-related genes were screened from patients with squamous cell carcinoma of the head and neck,and then divided into two groups according to whether the genes in the P4 HA family were highly expressed,and Kaplan-Meier survival prognosis analysis was performed.6.In order to explore the relationship between and collagen P4 HA family genes,changes in the malignant phenotype of cells were observed after knocking down in head and neck squamous cell carcinoma.7.Use gelatin enzymatic method to verify whether the activity of matrix metalloproteinase levels in Cal27 is affected after LLGL2 knockdown;Immunofluorescence experimental methods were used to verify the changes in cytoskeletal proteins in Cal27 after LLGL2 knockdownResults1.We found that all collagen P4 HA family genes were significantly up-regulated in KIRC,LUSC,STAD,LUAD,ESCA,HNSCC and BRCA.In head and neck squamous cell carcinoma,the high expression of collagen P4 HA family genes has a significant impact on the prognosis of patients.2.Knockdown of collagen P4 HA family gene significantly inhibited the migration,invasion and proliferation of Cal27 cells.At the same time,co-knocking down the gene of collagen P4 HA family can obviously inhibit the malignant expression of Cal27 cells.3.EDHB and pythi DC treatment did not reduce the level of collagen P4 HA family gene protein,but significantly reduced the pepsin-resistant type I collagen.Inhibitor treatment of Cal27 cells significantly reduced tumor migration,invasion and clone formation.4.The signal pathways related to P4 HA family genes mainly include PI3K-Akt signal pathway,local adhesion and extracellular matrix-receptor interaction pathway.5..The genes positively associated with P4 HA were ITGA5 and LOXL2,and the negatively correlated genes were SAMD10、ERBB3、LLGL2、RBM47、FUT6 和GPR110.The survival analysis of P4HA-related gene expression in patients with head and neck squamous cell carcinoma showed that the expression of ITGA5,ERBB3,LLGL2 and FUT6 genes had a significant effect on the prognosis of patients with head and neck squamous cell carcinoma.High expression of P4 HA family genes increases the poor prognosis of tumor patients with high expression of LLGL2 to a certain extent.6.In order to explore the relationship between and collagen P4 HA family genes,changes in the malignant phenotype of cells were observed after knocking down in head and neck squamous cell carcinoma.7.Use gelatin enzymatic method to verify whether the activity of matrix metalloproteinase levels in Cal27 is affected after LLGL2 knockdown;Immunofluorescence experimental methods were used to verify the changes in cytoskeletal proteins in Cal27 after LLGL2 knockdown.Conclusions1.In head and neck squamous cell carcinoma,the knock-down of collagen P4 HA family genes alone and together can significantly inhibit the malignant phenotype of the tumor.2.P4 HA mainly affects the malignant phenotype of head and neck squamous cell carcinoma through prolyl 4-hydroxylase activity.3.LLGL2 can inhibit the malignant phenotype of head and neck squamous cell carcinoma.4.In head and neck squamous cell carcinoma,LLGL2 may affect the migration and invasion of head and neck squamous cell carcinoma by affecting cytoskeletal aggregation,and then affect the metastasis of head and neck squamous cell carcinoma,playing the role of tumor suppressor gene.