Bioinformatic Analysis Of The Mechanism By Which Metformin Enhances Chemosensitivity Of Head And Neck Squamous Cell Carcinoma Cells

Objective: Metformin is one of the first-line drugs for clinical treatment of type II diabetes,and recent studies have found that metformin can inhibit the development of multiple malignant tumors.When metformin is combined with chemotherapeutic drugs to treat head and neck squamous cell carcinoma,it can effectively enhance the efficacy of chemotherapy.In this study,transcriptome sequencing and bioinformatics methods were used to elucidate the related molecular mechanism of metformin on the inhibitory effect and sensitizing chemotherapeutic efficacy of head and neck squamous cell carcinoma cells,in the hope of proposing a novel chemotherapeutic therapy with high efficiency and low side effects,which will provide a new opportunity for clinical treatment of head and neck squamous cell carcinoma.Methods: 1.Effects of metformin on biological behaviors of head and neck squamous carcinoma cells: the proliferation ability of head and neck squamous carcinoma cells after treatment with different concentrations of metformin(1 mm,5 mm,10 mm,20 mm,30 mm,40 mm,50 mm,60 mm)were detected by cytotoxicity assay(CCK-8assay),and the treatment time and the optimal concentration range of metformin were selected for subsequent experiments;Cell scratch assay to verify the effect of different concentrations of metformin on the migration ability of head and neck squamous carcinoma cells;Apoptosis assay to verify the effect of graded concentrations of metformin(10 mm,20 mm,30 mm,40 mm)on the apoptosis of human tongue squamous carcinoma cell Cal27,hypopharyngeal carcinoma cell Fa Du.2.Analysis of differentially expressed genes in head and neck squamous cell carcinoma cells treated with metformin: Cal27 were selected for transcriptome sequencing and bioinformatics analysis after being pretreated with metformin at appropriate experimental concentrations.3.Mechanism study of metformin enhanced the sensitivity of head and neck squamous cancer cells to chemotherapy: the sequencing data were intersected with the data set of post chemotherapy resistance in GEO(Gene Expression Omnibus)database,and the related genes that metformin enhanced the sensitivity of head and neck squamous cancer cells to chemotherapy were screened out,and the action targets of metformin were predicted after functional enrichment analysis of these genes.Real-time quantitative PCR was used to validate the sequencing results and the reliability of the key genes screened.The CCK-8 cytotoxicity assay was used to test the effect of metformin combined with cisplatin on the proliferation ability of Cal27 and Fa Du.4.Statistical analysis: the experiments were all repeated three times,the statistical analysis was completed using SPSS 21.0,and the data between the two groups were analyzed by independent samples t-test for differences with a statistical significance of P<0.05.Results: 1.CCK-8 cytotoxicity assay showed that the proliferation and viability of Cal27 and Fa Du significantly decreased in a dose-dependent manner after metformin treatment.The scratch assay results showed that metformin significantly inhibited the migration ability of head and neck squamous cell carcinoma cells,and the wound healing ability of head and neck squamous cell carcinoma cells was significantly reduced,both in a dose-dependent manner(P<0.05).Apoptosis assay showed that metformin induced cell apoptosis in Cal27 and Fa Du,and the apoptosis rate was positively correlated with the concentration of metformin(P<0.05).2.Through enrichment analysis of Cal27 transcriptome sequencing data after metformin treatment,the downregulated part of the differentially expressed genes was enriched in the DNA replication signaling pathway(P<0.05),and the upregulated part was enriched in the cytokine receptor interaction signaling pathway(P<0.05).3.The results of Cal27 transcriptome sequencing were intersected with the differentially expressed genes of head and neck squamous carcinoma patients before and after chemotherapy extracted from the GEO database,after bioinformatics analysis and screening of flap endonuclease 1 as a potential key gene for metformin to enhance the sensitivity of head and neck squamous carcinoma cells to chemotherapy.Real-time PCR analysis of the expression of flap endonuclease 1 and other genes related to base excision repair pathway,such as PCNA,Polβ and PARP1 after metformin treatment of Cal27,showed that the relevant genes in this class were all downregulated to various degrees,consistent with the transcriptome sequencing results.The results of the CCK-8cytotoxicity assay showed that the combination of cisplatin and metformin significantly inhibited the proliferation ability of Cal27 and Fa Du compared with cisplatin alone(P<0.05).Conclusion: Metformin can significantly inhibit the proliferation and migration of Cal27 and Fa Du,and induce apoptosis of these cells.Moreover,metformin exerts the effect of chemosensitization by weakening the base excision repair function of tumor cells and destroying the self-repair ability of damaged DNA.This study revealed the molecular mechanism of the inhibitory effect of metformin on head and neck squamous cell carcinoma cells and the efficacy of sensitized chemotherapy,which proposed a new chemotherapy therapy with high efficiency and low side effects.And this study provides a new opportunity for the treatment of patients with head and neck squamous cell carcinoma.