MiRNA-31-5p Promotes Oral Tumor Progression By Modulating The AK2-AIF Signaling Axis

Objective This study aims to clarify the mechanism and pathogenesis of miR-31-5p in oral squamous cell carcinoma,to find the downstream targets of miR-31-5p and the molecular mechanism under the action of H₂O₂.Methods Bioinformatics analysis revealed the expression level of miR-31-5p in head and neck squamous cell carcinoma（HNSC）tissues was higher compared with adjacent normal tissues,and the clone formation assay was performed to verify the conclusion.To explore the specific mechanism of miR-31-5p in oral tumors,weighted correlation network analysis（WGCNA）of HNSC-related dataset（GSE6631）in gene expression omnibus（GEO）database was used to get the most tumor-related genes,followed by functional enrichment to identify closely related processes in HNSC.At the same time,the possible target genes of miR-31-5p were predicted in Target Scan and verified by luciferase reporter assay.Immunoprecipitation assay was used to verify the protein AK2（adenosine kinase 2）could bound to AIF（apoptosis-inducing factor）.In order to further confirm the regulatory relationship of miR-31-5p and AK2 on AIF in oral cancer cells,inhibition of miR-31-5p or overexpression of AK2 was performed in AIF knockdown tumor cells,and changes of clone formation were observed.Results Analysis of TCGA data set of head and neck squamous carcinoma indicated that the expression level of miR-31-5p in cancer tissues was significantly higher than that in adjacent tissues.The conclusion of bioinformatics was verified by clone formation assay.It was found that clone formation was significantly increased in OSCC3 cells transfected with miR-31-5p mimic,while the ability of OSCC3 cells transfected with miR-31-5p inhibitor tended to be slower than that of negative control cells.Clinical correlation results suggested that miR-31-5p was significantly interrelated in tumor stage and TNM stage.It was further suggested that miR-31-5p might play an important role in the development of head and neck cancer.Analysis of GSE6631 data showed that head and neck cancer-related genes were enriched in biological functions such as internal apoptosis signaling pathway,epithelial cell proliferation and NAD/NADP binding activity.The downstream target genes of miR-31-5p were predicted,which were compared with the above functions,biological function,cell localization and molecular function information,and AK2 was finally selected as the possible target gene of miR-31-5p for experimental verification.The results of the luciferase reporter assay showed that miR-31-5p directly targeted the 3’-UTR of AK2and inhibited its translation,thus promoting the progression of oral cancer.In addition,we found that the expression of miR-31-5p was significantly decreased under H₂O₂condition,while H₂O₂could also promote the expression of AK2,thus inhibiting the proliferation process of tumor cells.Under H₂O₂treatment,overexpressing miR-31-5p or knocking down AK2 could effectively reduce the apoptosis process of tumor cells.When stimulated by H₂O₂,the expression of miR-31-5p was decreased,but the interaction between AK2 and AIF was increased,thus promoting OSCC3 cell death.However,when miR-31-5p mimic was transferred into OSCC3 cells under H₂O₂stimulation,the binding of AK2 and AIF was weakened.Rescue recovery experiments targeting miR-31-5p regulation of AIF and AK2 regulation could observe phenotypic recovery formed by cloning,indicating that AIF was a downstream molecule of miR-31-5p and AK2 regulation signals.Conclusion Our results suggest that miR-31-5p/AK2/AIF signaling axis plays an important regulatory role in the evolvement of oral cancer under oxidative stress conditions,providing another potential therapeutic mechanism for targeted treatment of oral cancer.