| Part 1 Systematic analysis of EMT-related acid-base regulation genes in head and neck squamous cell carcinomaObjective: The treatment effect of head and neck squamous cell carcinoma(HNSCC)under the multidisciplinary treatment mode is still unsatisfactory.The acid-base balance regulation of malignant tumors is in a disordered state,which can form a reversal of the intracellular and extracellular p H gradients opposite to normal tissues.p H gradient reversal can promote the progression of malignant biological behaviors such as epithelial-mesenchymal transition(EMT)of tumors.In this chapter,the bioinformatics method is used to systematically analyze the p H regulation genes related to EMT in HNSCC,and to screen out the reliable p H regulation genes related to EMT in HNSCC.Methods:(1)Using the GSVA to score the variation of EMT key gene set in HNSCC samples of TCGA database,the samples were clustered and grouped based on the scores for survival analysis.(2)According to the variation score of EMT-related gene set,differentially expressed genes(DEGs)analysis was performed among the groups to screen the genes related to p H regulation.(3)Use the HNSCC single-cell sequencing data set in the GEO database to detect the expression of these p H-regulated genes in cancer cells.Results:(1)The patients with higher scores by EMT-related gene set variation analysis in HNSCC tissues had poorer prognosis.(2)Some p H-regulated genes were differentially expressed in the EMT-related gene set variation high and low score groups in HNSCC tissues.(3)SLC1A3,SLC4A7,SLC37A4,SLC39A4 and other p H regulation genes were highly expressed in HNSCC.Conclusions:(1)The prognosis of HNSCC patients with high variation of EMT-related gene sets is poor.(2)Some p H regulation genes were differentially expressed in HNSCC samples with high and low EMT-related gene set variation.Part 2 SLC4A7 promotes the migration,invasion and epithelial-mesenchymal transition of head and neck squamous cell carcinoma cellsObjective: In the previous chapter,we have screened EMT-related p H-regulated genes and pay our attention on SLC4A7.In HNSCC,the expression of SLC4A7 and its exact function and regulatory mechanism remain unknown.This chapter mainly explores the effect of SLC4A7 on the malignant biological behavior of HNSCC through in vivo and in vitro experiments.Methods:(1)The regulation effect of SLC4A7 on HNSCC was explored by bioinformatics method.(2)The protein expression level of SLC4A7 in DOK cells and 6 head and neck squamous cell lines was detected by WB.(3)Fadu and HN8 cells were infected with lentiviral sh RNA vectors were used to construct stable SLC4A7 knockdown cell lines,and CAL27 cells infected with vectors as overexpression ones.The knockdown or overexpression efficiency of SLC4A7 was detected by WB.(4)The effects of SLC4A7 on the malignant biological behavior of HNSCC were determined by CCK8,colony formation,wound healing experiments and Transwell experiments,and the expression of EMT-related markers were detected by RT-q PCR,WB and immunofluorescence.(5)Fadu cell with SLC4A7 stable knockdown by lentivirus-infected was used to establish subcutaneous tumor formation and tail vein injection lung metastatic cancer models in nude mice.Results:(1)High expression of SLC4A7 in HNSCC cells indicated highly malignant biological behavior of tumor cells.(2)The expression of SLC4A7 in the stable knockdown group of Fadu and HN8 cells decreased significantly,while the expression of SLC4A7 in the overexpressed stable CAL27 cells increased significantly.(3)CCK8,colony formation experiments and subcutaneous tumor formation experiments in nude mice showed that there was no significant difference in the proliferation ability of tumor cells in each group.(4)In the cells with stable knockdown or overexpression of SLC4A7,wound healing and Transwell experiments showed the cells with high expression of SLC4A7 have stronger ability to migrate and invade.(5)The expression of markers of mesenchymal state and transcription factor Slug were increased in group with higher expression of SLC4A7.(6)Nude mice by SLC4A7-knockdown Fadu cell injected had fewer lung metastatic nodules than the control group.Conclusions:(1)The expression level of SLC4A7 has no significant effect on the proliferation of HNSCC cells.(2)High expression of SLC4A7 promotes migration,invasion and metastasis in vivo of HNSCC cells.(3)High expression of SLC4A7 promotes epithelial-mesenchymal transition in HNSCC.Part 3 The mechanism of SLC4A7 promoting the metastasis and epithelial-mesenchymal transition of head and neck squamous cell carcinoma cellsObjective: In the second chapter,we confirmed that SLC4A7 promotes the malignant biological behavior of HNSCC in vivo and in vitro.In this part,we will explore the regulatory mechanism in detailed.Methods:(1)High-throughput sequencing compared the DEGs between the control and SLC4A7 knockdown Fadu cells,and enrichment analysis of pathways based on the DEGs found the PI3K/AKT/m TOR signaling pathway was highly enriched.(2)WB was used to detect the expression of PI3K/AKT/m TOR pathway proteins and their phosphorylation levels in the constructed stable cells.(3)Using GDC-0980,a small molecule inhibitor of the PI3K/AKT/m TOR signaling pathway,to treat CAL27 cells with stable overexpression of SLC4A7,and explored whether GDC-0980 can reverse the malignant phenotype promoted by SLC4A7 in HNSCC.Results:(1)Pathway enrichment was performed on the DEGs obtained by high-throughput sequencing,and the PI3K/AKT/m TOR signaling pathway was found to be highly enriched.(2)In HNSCC,the phosphorylation of PI3K/AKT/m TOR signaling pathway proteins were decreased in Fadu and HN8 cells with SLC4A7 knockdown,but were highly expressed in CAL27 cells with SLC4A7 overexpression.(3)Compared with the control group,SLC4A7 overexpressed CAL27 cells with GDC-0980 treatment had decreased migration and invasion abilities,and the expression of EMT markers and transcription factor Slug decreased significantly,and also,the phosphorylation levels of PI3K/AKT/m TOR signaling pathway proteins reduced.Conclusion:(1)SLC4A7 activates PI3K/AKT/m TOR signaling pathway.(2)GDC-0980,a small molecule inhibitor of PI3K/AKT/m TOR signaling pathway,can inhibit the promotion of migration and invasion of HNSCC mediated by SLC4A7.(3)GDC-0980 can inhibit the promotion of epithelial-mesenchymal transition mediated by SLC4A7 in HNSCC.Part 4 Expression and clinical significance of SLC4A7 in head and neck squamous cell carcinomaObjective: In the previous part of this study,we have shown through in vivo and in vitro experiments that SLC4A7 can promote the migration and invasion of HNSCC,as well as epithelial-mesenchymal transition.In this chapter,we discuss the expression and clinical value of SLC4A7 in HNSCC.Methods:(1)The difference in m RNA expression of SLC4A7 was compared in HNSCC and adjacent tissues in TCGA database,and the prognosis value of SLC4A7 was evaluated in GEO database.(2)To verify the expression of SLC4A7 in HNSCC samples and non-cancerous epithelial tissues and its correlation with clinical data by immunohistochemistry.Results:(1)In the cancer sample of the TCGA database,the m RNA level of SLC4A7 was higher than that in the adjacent tissues,and immunohistochemistry also showed that the expression of SLC4A7 protein was significantly increased in the HNSCC tissues.(2)In HNSCC,the high expression of SLC4A7 was associated with lymph node metastasis,but not with age,pathological grade and clinical stage.(3)In the two HNSCC data sets in the GEO database,patients with high expression of SLC4A7 had a poor prognosis,and in the TCGA database,patients with high expression of SLC4A7 had shorter disease-free survival time.Conclusions:(1)SLC4A7 is highly expressed in HNSCC tissues.(2)High expression of SLC4A7 in HNSCC tissues is associated with poor prognosis of patients.Figures:14,Tables:15,Text references:121,Review references:201... 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