Identification Of Novel Biomarkers For Adenoid Cystic Carcinoma And Study On The Regulatory Role Of MiR-331-3p In Its Occurrence And Development In Adenoid Cystic Carcinoma

Objective: This study aimed to screen and identify novel biomarkers for adenoid cystic carcinoma(ACC),investigate the regulatory behavior and targeting relationship of mi R-331-3p with these biomarkers,explore its impact on ACC cell proliferation,apoptosis,migration,invasion abilities,and its regulation of tumor chemokines.Methods:(1)Bioinformatics analysis was conducted on ACC-related datasets(GSE59701 and GSE88804,including 19 normal samples and 20 tumor samples)to identify differentially expressed genes(DEGs).Key DEGs were selected,and functional enrichment analysis was performed to elucidate their associations with cellular pathways,transcription factors,disease regulatory genes,and the tumor immune microenvironment.(2)The targeting relationship between mi R-331-3p and BCL6/ITGA9 was validated through a dual-luciferase reporter gene assay.Transfection models of SACC-83 cancer cells with mi R-331-3p mimics and pc DNA3.1 BCL6 were established,and RT-q PCR was used to measure the m RNA expression levels of mi R-331-3p,BCL6,and ITGA9 in each group.Western Blot was employed to assess protein expression levels.(3)Cell proliferation was evaluated using a CCK-8 assay,apoptotic status was determined by flow cytometry,and migration and invasion capabilities were assessed via scratch and Transwell assays.An ACC-M cell transfection model was established to measure CXCL9,CXCL10,and CXCL11 m RNA expression levels in various groups using RT-q PCR.Results:(1)A total of 711 DEGs were identified in both disease and normal groups,and three key genes,GABBR1,ITGA9,and MLKL,were selected.These genes showed strong correlations with immune cell content.The DEGs were primarily enriched in cellular pathways related to the cell cycle,taste transduction mechanisms,and glycerolipid metabolism.Two key genes were significantly enriched in transcription factor cisbp＿＿M0564.The disease regulatory gene MYB was significantly positively correlated with GABBR1 in ACC,while TP53 was significantly negatively correlated with MLKL.(2)mi R-331-3p exhibited a targeting relationship with BCL6 and ITGA9.Compared to the mi R-331-3p mimics +pc DNA3.1 NC group,the mi R-331-3p mimics + pc DNA3.1 BCL6 co-transfection group showed reduced ITGA9 m RNA and protein expression in SACC-83 cells.(3)Cell proliferation,migration,and invasion abilities of SACC-83 cells were significantly reduced,while apoptosis levels were significantly increased.mi R-331-3p and ITGA9 negatively regulated the gene expression levels of CXCL9,CXCL10,and CXCL11,while BCL6 positively regulated their expression.Conclusion:(1)GABBR1,ITGA9,and MLKL play roles in the occurrence and development of ACC,with GABBR1 and MLKL potentially regulating ACC through MYB and TP53,respectively.The relationship between ITGA9 and ECM and PI3K-Akt may also impact migration and invasion of ACC.(2)mi R-331-3p can target regulate the BCL6/ITGA9 axis.(3)We proved that mi R-331-3p can influence the proliferation,apoptosis,migration,and invasion abilities of ACC cells through the regulation of the BCL6/ITGA9 axis,and mi R-331-3p,ITGA9,and BCL6 can impact the secretion of chemokines at the first time.