Preliminary Study On The Role And The Mechanism Of Hsa＿circ＿0002473/miR-873-5p Axis In The Progression Of Human Gastric Cancer

Objective:To clarify the expression of hsa＿circ＿0002473 in human gastric cancer(GC),and the correlation between hsa＿circ＿0002473and the GC phenotype via the systemic experiments of human GC cell lines and the tumor-bearing NOD/SCID mice.The mechanism of hsa＿circ＿0002473 in the progression of gastric cancer was preliminarily discussed to provide laboratory data and theoretical basis for enriching molecular type and therapeutic targets of gastric cancer.Methods:Total of 8 pairs of human gastric cancer and adjacent tissues were collected,and the expression level of hsa＿circ＿0002473 was detected by q RT-PCR.Two human gastric cancer cell lines,MKN-45 and HGC-27,were used to identify the loop structure of hsa＿circ＿0002473 by Sanger sequencing and Rnase R digestion.The subcellular localization of hsa＿circ＿0002473 was detected by nucleoplasmic separation assay.In vitro,the expression level of hsa＿circ＿0002473 in human gastric cancer cell lines(MKN-45,HGC-27,MKN-74,SGC-7901,MGC-803,AGS)and normal gastric epithelial cell line(GES-1)was detected by q RT-PCR.Three gastric cancer cell lines,MKN-45,AGS and HGC-27,were used for subsequent experiments.Gastric cancer cell lines with transient knockdown and overexpression of hsa＿circ＿0002473 were further established,and the knockdown and overexpression efficiencies were detected by q RT-PCR.Incucyte S3 real-time dynamic cell imaging analysis system and scratch assay were used to investigate the effects of knockdown and overexpression of hsa＿circ＿0002473 on the proliferation and migration of gastric cancer cells.The effect of knockdown and overexpression of hsa＿circ＿0002473 on the colony formation ability of gastric cancer cells was analyzed by cell clone formation assay.In vivo,the tumor-bearing NOD/SCID mice models were built by using MKN-45 cells of si-hsa＿circ＿0002473 and si-NC group,respectively.The weight and tumor volume of the mice were monitored every week.The differences between the two groups were statistically analyzed.In the mechanism exploration,we used bioinformatics analysis to predict the mi RNA that hsa＿circ＿0002473 might combine with,and detected the correlation between their expression by q RT-PCR.The Circular RNA Interactome database was used to search the binding sites of hsa＿circ＿0002473 and the target mi RNA.The dual luciferase reporter gene assay was used to analyze the interaction between hsa＿circ＿0002473 and the target mirna.Finally,the expression of hsa＿circ＿0002473 and target mi RNA was detected by q RT-PCR in gastric cancer cell lines and human gastric cancer tissues,respectively.Results : Hsa＿circ＿0002473 had a circular structure in gastric cancer cells and was mainly located in the cytoplasm.The expression of Hsa＿circ＿0002473 in gastric cancer tissues was significantly higher than that in adjacent tissues(p<0.05).Compared with normal gastric mucosal cells,hsa＿circ＿0002473 was highly expressed in gastric cancer cell lines.We knocked down hsa＿circ＿0002473 in MKN-45 and HGC-27 cells,and overexpressed hsa＿circ＿0002473 in AGS cells.The effect of hsa＿circ＿0002473 on the proliferation,migration and colony formation of gastric cancer cells was analyzed by functional experiments.The results showed that when hsa＿circ＿0002473 was knocked down in MKN-45 and HGC-27 cells,the proliferation and migration ability of gastric cancer cells were weakened(p<0.05),and the number of cell clone formation was reduced(p<0.05).When hsa＿circ＿0002473 was overexpressed in AGS cells(p<0.001),the proliferation and migration ability of gastric cancer cells were enhanced(p<0.001),and the number of cell clone formation was increased(p<0.01).In vivo,the xenograft tumor volume of si-hsa＿circ＿0002473 group was significantly reduced compared with that in the control group,and the difference was statistically significant(p<0.001).Bioinformatics analysis predicted that the target mi RNA of hsa＿circ＿0002473 was mi R-873-5p,and q RT-PCR showed that the expression of mi R-873-5p increased after knockdown of hsa＿circ＿0002473(p<0.001).Circular RNA Interactome database analysis showed that there were specific binding sites between hsa＿circ＿0002473 and mi R-873-5p.Dual luciferase reporter assay showed that hsa＿circ＿0002473 directly bound to mi R-873-5p through this site(p<0.001).In gastric cancer cells,q RT-PCR showed that the expression of mi R-873-5p was decreased in GC tissues compared with adjacent tissues(p<0.05).Conclusion: Hsa＿circ＿0002473 is up-regulated in GC tissues and cell lines.Hsa＿circ＿0002473 promoted the GC progression by adsorbing mi R-873-5p.Hsa＿circ＿0002473 is a potential tumor marker and molecular therapeutic target for gastric cancer.