CircRNA RTN4 Inhibits Progression Of Gastric Cancer By Regulating MiR-424-5p/LATS2 Pathway

Background:Gastric cancer is one of the most common malignant tumors of the digestive tract that causes patient deaths,and its incidence is still on the rise.Studies have shown that circular RNA(circ RNA)is one of the key regulatory factors in the occurrence and development of gastric cancer.Among them,circ RNA RTN4(circRTN4)shows significant expression abnormalities in gastric cancer.However,the specific biological role and molecular mechanism of circRTN4 in gastric cancer have not been elucidated.Objective: This study aims to explore the function of circRTN4 in gastric cancer and its mechanism of action in affecting the progression of gastric cancer,in order to provide new targets for the treatment of gastric cancer.Methods:1.Bioinformatics tools were used to analyze the expression level of circRTN4 in the GSE93541 dataset.QPCR was used to analyze the expression abundance of circRTN4 in the serum,tissue,and cells of gastric cancer patients.Kaplan-Meier survival curves were used to evaluate the relationship between circRTN4 and patient prognosis.The chi-square test was used to analyze the correlation between circRTN4 and clinical information of patients.2.Cell transfection experiments were performed in gastric cancer cell lines HGC-27 and AGS to construct a circRTN4 overexpression cell model.CCK-8 and EDU experiments were used to detect cell proliferation after overexpression of circRTN4.Transwell experiments were used to detect the effect of circRTN4 overexpression on the invasion and migration of HGC-27 and AGS cells.Tumor sphere experiments were used to detect changes in the tumor sphere-forming ability of HGC-27 and AGS cells after overexpression of circRTN4.Western Blot was used to detect the expression of MMP2 and OCT4 proteins in gastric cancer cells after overexpression of circRTN4.3.Bioinformatics analysis software was used to predict miRNAs that can interact with circRTN4 and their downstream target genes.Dual-luciferase reporter experiments,RNA pull-down,and RIP experiments were used to study the interaction between molecules.QPCR was used to detect the expression levels of miR-424-5p and LATS2 in gastric cancer tissues.Kaplan-Meier curves were used to analyze the relationship between the expression of miR-424-5p and LATS2 and patient prognosis.Spearman correlation coefficient was used to analyze the correlation between the expression of circRTN4,miR-424-5p,and LATS2 in gastric cancer tissues.4.Cells were divided into pLCDH group,pLCDH-circRTN4 group,pLCDH-circRTN4+miR-424-5p group,and pLCDH-circRTN4+si-LATS2 group.CCK-8and EDU experiments were used to detect the proliferation of cells in each experimental group,Transwell experiments were used to detect the invasion and migration of each experimental group,and tumor sphere formation experiments were used to detect the tumor sphere-forming ability of HGC-27 and AGS cells in each intervention group.In addition,Western Blot was used to detect the expression of MMP2 and OCT4 proteins in gastric cancer cells.Results:1.Compared with the control group,circRTN4 was significantly downregulated in the serum,tissue,and cells of gastric cancer patients(P<0.05).2.In HGC-27 and AGS cell lines,the cell proliferation level,tumor sphere-forming ability,invasion and migration ability of the pLCDH-circRTN4 transfection group were significantly lower than those of the pLCDH group(P<0.05).In addition,overexpression of circRTN4 could significantly reduce the protein content of MMP2 and OCT4(P<0.05).3.Online prediction results showed that circRTN4 has a binding site for miR-424-5p,and the downstream target gene of miR-424-5p is LATS2.QPCR experiments showed that miR-424-5p was highly expressed in gastric cancer tissues(P<0.05).Kaplan-Meier curve analysis showed that patients with high expression of miR-424-5p had a significantly worse prognosis than those with low expression of miR-424-5p(P<0.05),while low expression of LATS2 was associated with poor prognosis in patients(P<0.05).Spearman analysis showed that the expression levels of circRTN4 and LATS2 were positively correlated(R2=0.309),and there was a significant negative correlation between the expression of miR-424-5p and LATS2(R2=0.0009)in gastric cancer tissues.4.In HGC-27 and AGS cell lines,compared with the pLCDH-circRTN4 group,the cell proliferation level,EDU positive staining rate,invasion and migration,and tumor sphere-forming ability of the pLCDH-circRTN4+miR-424-5p group and pLCDH-circRTN4+si-LATS2 group were significantly increased(P<0.05).Conclusion:1.CircRTN4 is downregulated in the serum,tissue,and cells of gastric cancer patients;2.Overexpression of circRTN4 can inhibit the proliferation,invasion,migration,and tumor sphere-forming ability of gastric cancer cells,and inhibit the stemness of gastric cancer cells;3.CircRTN4 regulates LATS2 expression by acting as a molecular sponge for miR-424-5p.