The Role And Mechanism Of CircDDX17 Inhibits Gastric Cancer Progression

Objective:Gastric cancer(GC)is one of the most common cancer in China,high incidence rate and mortality endanger people’s health.Circ RNA is a novel RNAs which play an important role on GC.Circ DDX17(hsa＿circ＿0063331)identified the differentially expression between tumor tissus and normal tissue.Analyzed the regulatory role of circ DDX17 on the biological behaviors of gastric cancer cells such as proliferation,apoptosis,migration and EMT,aiming to reveal the mechanism of circ DDX17 role in GC and development and to explore its value as a therapeutic target for GC.Method:1.Predicted differentially expressed circ RNA in GC tissues and cells.q RT-PCR to detect the differential expression of circ RNA in GC tissues and GC cells line to obtain differentially expressed circ DDX17.The intracellular localization in GC cells of circ DDX17 was detected by FISH and nuclear plasma isolation.q RT-PCR to detected the circ DDX17 expression in GC patients,and correlation with pathological data,such as tumor size,lymph node metastasis,and clinical staging.2.GC cell overexpression or knockdown circ DDX17,colony formation assay and CCK8 assay were performed to detect cell proliferation,flow cytometry to detect apoptosis,Transwell assay and wound healing assay to detect cell metastasis.In vivo,the expression of serum tumor-associated cytokines TGF-β,TNF-α and IL-1 in nude mice was detected by ELISA.IHC to detected the expression of E-cadherin,N-cadherin,MMP2,MMP9 and PCNA.Western blot and q RT-PCR to analysis the expression of proteins and m RNAs related with apoptosis,proliferation,metastasis and EMT.3.Database analysis of circ DDX17 target mi RNAs,q RT-PCR to detect mi RNAs expression after circ DDX17 overexpression/knockdown in GC cells.Luciferase assay to detect binding sites between circ DDX17 and mi RNAs.q RT-PCR to detect mi RNA expression in GC tissues,analyze the correlation with tumor size,lymph node metastasis,and clinical staging.q RT-PCR detect the expression of mi RNA in GC cells and normal gastric mucosal cells.4.GC cells overexpression or knockdown mi R-1208/mi R-1279,co-transfected mi R-1208/mi R-1279 mimics + pcic R-circ DDX17,co-transfected mi R-1208/mi R-1279 inhibitor + si-circ DDX17,colony formation assay,CCK8 assay to detect cell proliferation.Cell migration ability was detected by wound healing assay and Transwell assay;apoptosis was detected by flow cytometry;apoptosis,proliferation,migration,and EMT related proteins detected by Western blot.5.Database predicted mi R-1208 and mi R-1279 target genes,Western blot and q RTPCR detected gene expression after overexpression/knockdown of mi R-1208/mi R-1279,then obtained target gene FKBP5.q RT-PCR detected its expression difference in GC tissues and normal tissues,and analyzed the association between FKBP5 and clinicopathological data.The expression of FKBP5 in GC cell lines and normal gastric mucosa cells was detected by Western blot.6.GC cells overexpression or knockdown FKBP5,co-transfected mi R-1208/mi R-1279 mimics+p FKBP5,co-transfected mi R-1208/mi R-1279 inhibitor+si-FKBP5.Cell proliferation was detected by colony formation assay and CCK8 assay;migration ability was detected by cell wound healing assay and Transwell assay,flow cytometry was performed to detect apoptosis,Western blot was performed to detect the proteins of apoptosis,proliferation,migration and EMT.IP assay for FKBP5-interacting protein,Western blot for the effect of FKBP5 on proteins of pathway.7.To analyze the coding sequence of circ DDX17,construct eukaryotic expression vector,and observe amino acid expression and intracellular localization by Western blot and confocal laser after transfected in GC cells.The prokaryotic expression vector was constructed,and the expression was induced by sensing bacteria,and the amino acid expression was examined by Western blot.To construct circ DDX17-63 aa expressing GC cell line,colony formation assay,CCK8 assay to detect cell proliferation,wound healing assay and Transwell assay to detect cell migration;Western blot to detect the proteins of cell apoptosis,proliferation,migration and EMT.Results:1.Circ DDX17 lowly expressed in both GC tissues and GC cells,analysis the expression level was negatively correlated with tumor size and lymph node metastasis.The transcript of circ DDX17 m RNA was ddx17,which located on chromosome 22 and cyclized from exons 2-5,with a length of 451 bp.Its structure was verified by PCR.Resistance to RNase R digestion,and distributed in both nucleus and cytoplasm,mainly in the cytoplasm.2.Colony formation,CCK8 assay,apoptosis assay,Transwell assay,wound healing assay and Western blot results showed that overexpression of circ DDX17 promoted GC cells apoptosis and inhibited cell proliferation,migration and EMT.Conversely knockdown circ DDX17 inhibited GC cell apoptosis and promoted cell proliferation,migration and EMT.In vivo,overexpression of circ DDX17 inhibited subcutaneous tumor tissue growth,decreased serum TGF-β and TNF-α expression,and suppressed cell proliferation,metastasis and EMT-related proteins and m RNA expression.3.mi R-1208 and mi R-1279 as target mi RNAs of circ DDX17.mi R-1208 and mi R-1279 were highly expressed in GC tissues and GC cells,mi R-1208 expression level correlated with lymph node metastasis,and mi R-1279 expression level correlated with tumor size.Overexpression mi R-1208/mi R-1279 inhibited gastric cancer cell apoptosis and promoted cell proliferation,migration and EMT.Conversely,knockdown mi R-1208/mi R-1279 promoted gastric cancer cell apoptosis and inhibited cell proliferation,migration and EMT.In addition,complementation assay showed that mi R-1208 and mi R-1279 reversed the effect of circ DDX17 on the biological function in GC cells.4.FKBP5 as mi R-1208 and mi R-1279 target gene.FKBP5 was lowly expressed in GC tissues and GC cells,and the expression level was negatively correlated with tumor size.Overexpression FKBP5 promoted apoptosis and inhibited cell proliferation,migration and EMT.Knockdown FKBP5 inhibited apoptosis and promoted cell proliferation,migration and EMT.Complementation experiments indicated that FKBP5 could reverse the effects of mi R-1208 and mi R-1279 on the biological behavior in GC cells.5.Circ DDX17 encode 63 amino acids and is mainly localized in the cytoplasm.Transfection circ DDX17-63 aa in GC cells promoted apoptosis and inhibited cell proliferation,migration and EMT.ConclusionCirc DDX17,which is lowly expressed in GC tissues and GC cells and negatively correlated with tumor size and lymph node metastasis.Through mi R-1208/mi R-1279/FKBP5 axis and encoding circ DDX17-63 aa to inhibit gastric cancer cell proliferation,migration and EMT,promote apoptosis.It is promising to be a target for gastric cancer treatment.