Effects Of Autophagy Regulated By SIRT1/miR-182-5p On Biological Behavior Of Gastric Cancer Cells And Its Mechanism

Objective GC（Gastric cancer）is one of the most common malignant tumors.Previous studies have shown that SIRT1/miR-182-5p is involved in GC development,but the role of SIRT1/mi R-182-5p and its specific mechanism in GC have not been fully elucidated.This study aims to investigate the effects of SIRT1/miR-182-5p on cell proliferation and metastasis via autophagy and its molecular mechanism in order to find the potential new targets and theoretical basis for the pathogenesis and clinical treatment in GC.Methods Immunohistochemical staining was used to detect the expression of SIRT1 in90 pairs of GC tissues and adjacent tissues from Tissue Chip,western blot was used to detect the expression of SIRT1 in 16 pairs of GC tissues and adjacent tissues from clinical paitients.Fluorescence in situ hybridization was used to detect the expression of miR-182-5p in 87 pairs of GC tissues and paracancer tissues from Tissue Chip,real-time quantitative polymerase chain reaction was used to detect the expression of miR-182-5p in 16 pairs of GC tissues and adjacent tissues from clinical paitients,normal gastric mucosal epithelial cells GES-1 and GC cells BGC-823,AGS,SGC-7901.Double luciferase reporter gene assay and chromatin immunoprecipitation were detected the relationship between SIRT1 and miR-182-5p.Fluorescent labeling assay and western blot were used to detect autophagy.Cell proliferation,migration and invasion were detected by CCK-8 assay,scratch-wound assay and Transwell^TM invasion assay,respectively.Apoptosis assay were used to detect the percentage of apoptotic and dead cells,The effects of miR-182-5p on tumor volume and weight was performed in vivo.Results Compared with adjacent tissues and GES-1 cells,SIRT1 and miR-182-5p were significantly upregulated in GC tissues and GC cells（P<0.05）,and SIRT1 can bind to the promoter region of miR-182-5p to regulate the expression of mi R-182-5p.Overexpression of miR-182-5p promoted cell proliferation and metastasis(F_{proliferation}=31.72,F_migration=60.14 F_invasion=84.41,P<0.05)and decreased the percentage of apoptotic and dead cells（F=52.72,P<0.05）.Downregulation of miR-182-5p had the opposite effects（P<0.05）.Overexpression of miR-182-5p enhanced cell autophagy（F=43.94,P<0.05）,and the rate of LC3B-II/I and the expression of Beclin1 were increased(F_LC3B-Ⅱ/Ⅰ=60.48,F_Beclin1=37.56,P<0.05),and downregulation of mi R-182-5p significantly decreased the level of autophagy（P<0.05）.Moreover,CEBPA and PPARG were the target genes of miR-182-5p,SIRT1/miR-182-5p may regulate ATG7 through downstream target genes CEBPA and PPARG to enhance autophagy,thereby promoting proliferation and metastasis.Furthermore,we also found that downregulation of miR-182-5p significantly reduced tumor volume and weight in vivo(F_volume=77.33,F_weight=103.10,P<0.05).Conclusions SIRT1 and miR-182-5p are significantly upregulated in GC,and SIRT1can bind to the promoter region of miR-182-5p and regulate the expression of miR-182-5p.SIRT1/miR-182-5p probably promotes the proliferation and metastasis through the downstream target genes CEBPA and PPARG regulating ATG7 to enhance autophagy.