Circular RNA Hsa_circ₀000467 Promotes The Development Of Gastric Cancer By Competitively Binding To MiR-326-3p

Objectives To investigate the expression of circRNA hsa_circ₀000467 in gastric cancer tissues and cell lines.To explore the mechanism of hsa_circ₀000467 promoting the development of gastric cancer by competitively binding to miR-326-3p.Methods Hsa_circ₀000467 was selected as the research object according to the results of microarray assay analysis.The expression of hsa_circ₀000467 in gastric cancer tissues and cell lines was detected by qRT-PCR.The biological functions of hsa_circ₀000467 were explored by CCK8 assays,invasion assays and cell cycle assays.The bioinformatics analysis and dual-luciferase reporter assays were used to identify the binding site of hsa_circ₀000467 to the downstream miR-326-3p.In addition,the regulation of miR-326-3p by hsa_circ₀000467 in gastric cancer was examined in vitro.Results In a study of 30 patients with primary gastric adenocarcinoma who did not receive chemotherapy or radiotherapy,we found that hsa_circ₀000467 was elevated in gastric cancer tissues compared with the corresponding adjacent tissues?P<0.05?and correlated with gastric cancer histological grade?P=0.0224?.In addition,the expression level of hsa_circ₀000467 in two gastric cancer cell lines?BGC-823,SGC-7901?was dramatically higher than in normal human gastric mucosal cell line GES-1?P<0.001?.We constructed stable reduced expression of hsa_circ₀000467 in human gastric cancer cell lines BGC-823 and SGC-7901 by using RNA interference technology.The cell proliferation assays explained that the cell proliferation ability of si-hsa_circ₀000467 group was significantly lower than that si-NC group at 48 and 72 hours after cell inoculation?P<0.05;P<0.01;P<0.05?;Transwell assays results indicated that the number of invasive cells of si_hsa_circ₀000467 group was significantly lower than the control group 24 hours after seeding the cells?P<0.01;P<0.01?;Cell cycle assays showed that the number of cells entering the G2/M phase in the si-hsa_circ₀000467 group was lower than that in the si-NC group?P<0.01;P<0.001?.In further experiments,we predicted the miRNAs downstream of hsa circ 0000467 by bioinformatics analysis,and the dual-luciferase reporter assays showed that over-expressing miR-326-3p inhibited the luciferase activity of wild type reporter for hsa_circ₀000467,but not the mutant reporter for hsa_circ₀000467?P<0.05?.Finally,we divided the BGC-823 and GGC-7901 cells into four different groups by transfection:si-NC,si-hsa_circ₀000467,miR-326-3p inhibitor and si-hsa_circ₀000467+miR-326-3p inhibitor,and performed rescue experiments of hsa_circ₀000467 and miR-326-3p.The CCK8 assays results showed that the cell proliferation rate of BGC-823 and GGC-7901 cells in si-hsa_circ₀000467+miR-326-3p inhibitor group was higher than that in si-hsa_circ₀000467 group?P<0.01;P<0.05?.The results of Transwell invasion assays showed that the number of invasive cells in si-hsa_circ₀000467+miR-326-3p inhibitor group was higher than that in si-hsa_circ₀000467 group?P<0.05;P<0.001?.Cell cycle assays showed that the number of cells entering to G2/M phase in si-hsa_circ₀000467+miR-326-3p inhibitor group was higher than that in si-hsa_circ₀000467 group?P<0.05;P<0.001?.Western blot analysis showed that the expression of Cyclin D1 in si-hsa_circ₀000467+miR-326-3p inhibitor group was higher than that in si-hsa_circ₀000467 group?P<0.001;P<0.001?.There was no significant difference between the groups with the expression of c-MYC.Conclusions In this study,we found that hsa_circ₀000467 was elevated in gastric cancer tissues and cell lines.After down-regulating hsa_circ₀000467,the proliferation and invasion ability of BGC-823 and SGC-7901 cells decreased,and the number of cells entering G2/M phase decreased,suggesting that hsa_circ₀000467 is involved in regulating the biological function of gastric cancer cells;In addition,hsa_circ₀000467 had binding sites with miR-326-3p,and inhibition of miR-326-3p reversed the decline in proliferation and invasion of BGC-823 and SGC-7901 cells caused by downregulation of has_circ₀000647,as well as the decrease in the number of cells entering the G2/M phase and the expression of Cyclin D1,suggesting that hsa_circ₀000467 affects the biological function of gastric cancer by binding to miR-326-3p.Hsa_circ₀000467 is expected to become a new molecular marker for the diagnosis of gastric cancer.Silencing hsa circ 0000467 may be the new direction for the treatment of gastric cancer.