Circular RNA Hsa_circ₀002669 Inhibits Gastric Cancer Progression By Regulating The MiR-223-3p/ARID1A Axis

Background:Gastric cancer is a common heterogeneous disease in the world and the third leading cause of cancer-related deaths in the world.Gastric cancer has the highest incidence in East Asia,including China.To improve the level of early diagnosis of gastric cancer,finding effective biomarkers is particularly important for improving the survival rate of gastric cancer patients.Circular RNA is a type of single-stranded RNA molecule that doesn't have a 5 'end cap and a 3' end poly?A?tail,and it is connected by a covalent bond head to tail to form a covalent closed loop structure.This structure makes circular RNA not easily degraded by exonuclease RNaseR and is more stable than linear RNA.In addition,circular RNA also has the characteristics of rich variety,conserved sequence and specific expression of cells and tissues.More and more studies have found that a variety of circular RNAs are abnormally expressed in tumor tissues and are closely related to the occurrence and development of tumors.These characteristics make circular RNAs have unique advantages in tumor diagnosis and treatment and clinical intervention.In this paper,we screened the circular RNA in gastric cancer and adjacent tissues,and found the circular RNA hsa_circ₀002669 with large expression difference between adjacent and cancerous tissues.We further studied the role of this circular RNA in gastric cancer.And regulatory mechanisms to evaluate its potential value as a target for early diagnosis and treatment of gastric cancer.Objective:To explore the expression,role and molecular regulation mechanism of hsa_circ₀002669 in gastric cancer,provide a new marker for early diagnosis and treatment of gastric cancer.Methods and results:1.hsa_circ₀002669 is significantly down-regulated in gastric cancer and has high stability.1)The expression of hsa_circ₀002669 in gastric cancer tissues and cells was significantly down-regulated:we used qRT-PCR to detect the expression level of hsa_circ₀002669 in 46 pairs of gastric cancer and paracancerous tissues and 4 kinds of gastric cancer cells and gastric immortalized epithelial cells GES-1,The results showed that:hsa_circ₀002669 was down-regulated in?39/46?84.8%of gastric cancer tissues.Statistical analysis showed that:hsa_circ₀002669 was significantly down-regulated in gastric cancer tissues?p<0.05?,and was immortalized with gastric mucosa Compared with the epithelial cell GES-1,the expression of hsa_circ₀002669 in 4 kinds of gastric cancer cells was significantly reduced.We performed Sanger sequencing on the PCR amplification products,and the results showed that the PCR products contained the reverse splicing site of hsa_circ₀002669.Further,by designing diffusion primers?Divergent primers?and convergence primers?Convergent primers?for hsa_circ₀002669 and its source gene DOCK1,PCR amplification was performed using cDNA and gDNA as templates,and it was found that using Divergent primers can only amplify the hsa_circ₀002669,using genomic gDNA as a template,no amplified bands can be obtained.The above results exclude the possibility that the amplification product is a rearrangement of the genome,which proves that the PCR amplification product is hsa_circ₀002669.2)Compared with linear molecules,hsa_circ₀002669 has higher stability:we use RNase R to treat gastric cancer cell RNA,and qRT-PCR was used to detect the expression of hsa_circ₀002669 and linear molecule DOCK1.It was found that after RNaseR treatment,the expression of linear DOCK1 gene was significantly reduced,while the expression of hsa_circ₀002669 was not significantly changed Further,we treated gastric cancer cells with the transcription inhibitor Actinomycin D?ActD?for different times,and qRT-PCR detected the expression of hsa_circ₀002669 and DOCK1.The expression of DOCK1 can be gradually reduced,but the expression of hsa_circ₀002669 has not changed significantly.The above results indicate that the stability of hsa_circ₀002669 is significantly higher than that of linear DOCK1 gene.2.hsa_circ₀002669 inhibits the proliferation,migration and invasion of gastric cancer cellsWe transfected hsa_circ₀002669 overexpression plasmid?pLCDH-circ-2669?or interfering RNA?si-circ-2669?into BGC-823 and SGC-7901 cells,and used qRT-PCR to detect the overexpression and interference efficiency.Through the EdU assay,Scratch assay and Transwell assay to detect cell proliferation,migration and invasion ability.It was found that high expression of hsa_circ₀002669 can significantly inhibit the proliferation,migration and invasion of gastric cancer cells.Interfering with the expression of hsa_circ₀002669 can significantly increase the proliferation,migration and invasion ability of gastric cancer cells.3.hsa_circ₀002669 acts as a molecular sponge for miR-223-3p in gastric cancer cellsIn order to study the molecular mechanism of hsa_circ₀002669,we first detected the subcellular localization of hsa_circ₀002669,extracted RNA in the nucleus and cytoplasm and detected the expression of hsa_circ₀002669 in the nucleus and cytoplasm by qRT-PCR.As a result,we found that hsa_circ₀002669 mainly exists in the cytoplasm.Then,we used AGO2 antibody for RNA binding protein immunoprecipitation?RIP?detection,and found that hsa_circ₀002669 can be detected in the immunoprecipitate of AGO2.It is speculated that hsa_circ₀002669 plays a role by binding miRNA,and bioinformatics software is used to predict the possible binding of hsa circ 0002669.For miRNAs,we screened four miRNAs.We transfected gastric cancer cells with hsa circ₀002669 overexpression vectors.qRT-PCR detected the expression of four miRNAs and found that hsa_circ₀002669 can significantly down-regulate the expression of miR-223-3p.Further,we used dual luciferase activity experiment to detect whether hsa_circ₀002669 can directly bind to miR-223-3p.The results showed that hsa_circ₀002669 and miR-223-3p can directly bind.We further used Western Blot to examine the effect of hsa_circ₀002669 on the expression of the downstream target molecule ARID1A of miR-223-3p in gastric cancer cells.It was found that high expression of hsa_circ₀002669 can promote the expression level of ARID1A,and hsa_circ₀002669 interference can inhibit the expression of ARID1A.4.hsa_circ₀002669 regulates the proliferation and invasion and migration of gastric cancer cells through the miR-223-3p/ARID1A pathway In order to detect whether hsa_circ₀002669 functions as a "molecular sponge" of miR-223-3p,we did a recovery experiment.After co-transfecting hsa circ 0002669 interfering RNA?si-circ-2669?and miR-223-3p inhibitor in BGC-823 and SGC-7901 cell lines,we used EdU experiment,scratch experiment and Transwell experiment?with or without matrigel?The effects of hsa_circ₀002669 on the proliferation,migration and invasion of gastric cancer cells were examined.The results showed that miR-223-3p inhibitor can partially reverse the promotion of hsa_circ₀002669 interference on the proliferation,invasion and migration of gastric cancer cells.Further,we used Western Blot experiment to detect the expression of miR-223-3p downstream target molecule ARID1A,and found that miR-223-3p inhibitor can reverse the regulation effect ofhsa circ 0002669 siRNA on ARID1A.Conclusion:hsa_circ₀002669 is down-regulated in gastric cancer tissues and cells,and its silencing promotes the proliferation,migration,and invasion of gastric cancer cells.Mechanistically,hsa_circ₀002669 competitively binds to miR-223-3p and prevents miR-223-3p from inhibiting its target gene ARID1A,which ultimately leads to the proliferation,migration and invasion of gastric cancer cells.Therefore,hsa_circ₀002669 may provide potential biomarkers and therapeutic targets for the prognosis and treatment of gastric cancer patients...