Bioinformatic analysis of the mechanism by which metformin enhances chemosensitivity of head and neck squamous cell carcinoma cells

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（HNSCC）, it can effectively enhance the efficacy of chemotherapy. The aim of this study was to define the signaling pathways regulated by metformin in HNSCC, and the underlying mechanisms by which metformin sensitizes HNSCC chemotherapy. We used RNA sequencing (RNA-seq) to examine metformin regulated genes in the cal27, human tongue squamous carcinoma cell line, using RT-PCR to determine alterations in gene expression. Data from RNA-seq analysis showed that gene products involved in DNA replication, cell cycle, IL-17 signaling pathway, mismatch repair were all regulated by metformin. Our results suggest that metformin regulates multiple pathways associated with tumor growth and progression in HNSCC. CAL27 cells were plated in 10 cm dishes at a density of ~ 300,000 cells/dish in high glucose DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin，and allowed to attach for one day. The cells were then treated for 48 hours with either PBS or 30 mM metformin. Total RNA was extracted from treated cells using the Trizol reagent and further purified using the Qiagen miRNeasy Micro Kit and assessed with Bioanalyzer 4200. Then, next-generation libraries were prepared using the VAHTS mRNA-seq v2 Library prep kit. Sequence runs were performed on an Illumina HiSeq X 10 utilizing a 150-bp RNA-Seq protocol.