The Role And Mechanism Of DRAM1 In Proliferation And Migration Of Gastric Cancer

Objective:To detect the differential expression of DRAM1 in gastric tissues and tissues adjacent to cancer,and between normal gastric epithelial cells and gastric cancer cell lines.To understand the impact of DRAM1 on migration and proliferation of gastric cancer cells and the potential mechanism.To hopefully offer a fresh idea and target for future diagnosis and treatment of gastric cancer.Methods:(1)Online public databases were used to evaluate the expression level of DRAM1 in gastric cancer and normal gastric tissues.Both the m RNA and protein levels of DRAM1 were detected respectively using quantitative real-time polymerase chain reaction(qRT-PCR)and Western blotting,in order to compare the differential expression between gastric cancer tissues and adjacent nontumorous tissues.(2)The DRAM1 levels in normal gastric epithelial cells and in gastric cancer cell lines were assessed by means of qRT-PCR and Western blotting.The small interfering RNA(si DRAM1)was transfected into SGC-7901 cells which showed high expression of DRAM1,while the plasmid(pcDNA3.1-DRAM1)was transfected into HGC-27 cells where DRAM1 expression was relatively low,so that the model cells with low/high DRAM1 expression were built.The efficiency of knockdown or overexpression was examined by Western blotting.(3)The effect of DRAM1 on the proliferative potential of gastric cancer cells was evaluated by Cell Counting Kit-8 assay and cell clone formation assay,after DRAM1 knockdown or overexpression.Wound healing assay together with Transwell assay were performed to observe the changes in migration of SGC-7901 and HGC-27 cells after DRAM1 knockdown/overexpression.Western blotting assay was used to detect EMT(Epithelial-Mesenchymal Transition),cell proliferation and migration related proteins,and Wnt/β-catenin pathway proteins to explore the regulatory role of DRAM1 in the proliferation and migration of gastric cancer cells.Results:(1)qRT-PCR and Western blotting results indicated that in comparison to the adjacent tissues,the expression of DRAM1 was generally increased in gastric cancer tissues.Western blotting experiments found that the protein level of DRAM1 in gastric carcinoma cells was significantly higher than that in normal gastric epithelial cells,with the highest expression in SGC-7901 cells while a relatively low expression in HGC-27 cells.(2)The consistent results of CCK-8 assay and cell clone formation assay revealed the decrease in proliferation of SGC-7901 cells after transfected with si-DRAM1.The migration of SGC-7901 cells was also significantly inhibited after DRAM1 knockdown,as shown by the results of wound healing and Transwell migration assay.In addition,Western blotting results showed that the expression of E-cadherin,an epithelial marker,in SGC-7901 cells was upregulated due to knockdown of DRAM1,while the levels of Vimentin and N-cadherin,both are interstitial markers,were decreased,indicating that the downregulation of DRAM1 inhibited EMT.Matrix metalloproteinases MMP2,MMP9 and PCNA(proliferating nuclear antigen)were significantly decreased,indicating that the proliferation and migration of cells were restrained.The expression of β-catenin,Cyclin D1,p-GSK3βand C-Myc were downregulated,with no significant variation in GSK3β,showing that the Wnt/β-catenin signaling pathway was inhibited.(3)The cell function experiments revealed that,after transfection of HGC-27 cells with the overexpressed plasmid pcDNA3.1-DRAM1,the proliferation and migration ability of gastric cancer cells were significantly enhanced.Western blotting results consistently showed that EMT,cell proliferation and migration,and Wnt/β-catenin signaling pathway protein levels were changed accordingly.Conclusion:DRAM1 was up-regulated in gastric cancer tissues and cells in comparison with para-carcinoma tissues and normal gastric epithelial cells.In vitro gastric cancer cells,DRAM1 overexpression strengthened the cell proliferation as well as migration,and facilitated EMT.Knockdown of DRAM1 inhibited proliferation,migration,and EMT of gastric cancer cells.Mechanistically,DRAM1 may promote the occurrence and progression of gastric cancer via Wnt/β-catenin signaling pathway.