Mechanism And Clinical Significance Of Hsa＿circ＿0052012 In Promoting The Progression Of Gastric Cancer

Objective:Circular RNAs(circ RNAs)are a new type of endogenous non-coding RNAs,which are structurally stable due to the covalent closed loop structure.They are widely enriched in the cytoplasm of cells and can perform a variety of functions.Currently,many studies at home and abroad have revealed the close relationship between circ RNAs and tumors.Gastric cancer is one of the most common malignant tumors,and it is the fifth most common cancer and the third main cause of cancer death.It is of great research value to search for the early biological markers and effective diagnosis and treatment methods of gastric cancer.This paper aims to explore the circ RNA hsa＿circ＿0052012 with research value,and research its clinical significance in the diagnosis and progression of gastric cancer.Methods:1.Five pairs of human gastric cancer tissues and adjacent tissues were collected for examination.High-throughput sequencing technology was used to detect whole genome sequence for analyzing all circ RNAs and miRNAs in the sample tissues,and circ RNAs with different expressions were analyzed and drawn into circ RNA profiles.2.Tissue specimens after radical gastrectomy were collected clinically,including gastric and paracancerous tissues,peripheral blood of gastric cancer patients and healthy subjects.The sample size was expanded by q RT-PCR technology to detect whether the circ RNA expression trend in the samples was consistent with the circ RNA expression trend obtained by sequencing,so as to analyze and study the circ RNA,and verify the analysis again in gastric cancer cells and human normal gastric mucosa cells in vitro.3.Verifying the circular structure and characteristics of hsa＿circ＿0052012,the circular structure was analyzed by Sanger sequencing,RNase assay and agarose gel electrophoresis assay,and then the expression position of hsa＿circ＿0052012 in gastric cancer cells was detected by intracellular fluorescence assay.The relationship between hsa＿circ＿0052012 and clinicopathological characteristics was analyzed.4.SiRNA was designed to knockdown hsa＿circ＿0052012 to silence its expression in gastric cancer cells,and si-NC group was set as meaningless fragment control.The effect of hsa＿circ＿0052012 on the function of gastric cancer was analyzed by plate cloning,cell scratch,cell invasion and flow cytometry.Tumor bearing test in nude mice confirmed whether hsa＿circ＿0052012 has biological effect in vivo.5.MiR-1231 has binding sites with EGFR.After hsa＿circ＿0052012 silenced,miR-1231 was upregulated,EGFR was down-regulated and EGFR protein expression was reduced(P<0.05),EGFR was down-regulated and EGFR protein expression was up-regulated after miR-1231 inhibition(P<0.05).6.Hsa＿circ＿0052012 regulates the EGFR/PI3K/ Akt /m TOR axis,and upregulated hsa＿circ＿0052012 promotes tumor progression through the EGFR/PI3K/Akt /m TOR axis.Results:1.Hsa＿circ＿0052012 is located in the genome CHR19 :50902107-50902741.Fluorescence detection mainly exists in the cytoplasm of gastric cancer cells,with a covalently closed loop structure.2.The expression level of Hsa＿circ＿0052012 in gastric cancer tissues was significantly higher than that in adjacent tissues(P<0.001);The serum expression level of Hsa＿circ＿0052012 in patients with gastric cancer was significantly higher than that in healthy subjects(P<0.01),and the expression level of hsa＿circ＿0052012 in serum of gastric cancer patients before surgery was significantly higher than that in serum one week after surgery(P<0.01).In clinicopathological analysis,the high expression of hsa＿circ＿0052012 was only associated with tumor stage(P<0.001).3.The expression levels of Hsa＿circ＿0052012 in gastric cancer cell lines were higher than those in normal gastric mucosa cells(P<0.05).After silencing hsa＿circ＿0052012 with si RNA,the proliferation,migration and invasion levels of hsa＿circ＿0052012 cells were significantly decreased(P<0.05)and the cell cycle progression of gastric cancer was inhibited(P<0.05).In the subcutaneous tumor-bearing experiment in nude mice,hsa＿circ＿0052012 was silenced by si RNA,and the tumor growth rate was slowed down(P<0.05).4.Hsa＿circ＿0052012 binding sites with miR-1231,both of which are abundant in the cytoplasm.Hsa＿circ＿0052012 can sponge miR-1231,and after silencing Hsa＿circ＿0052012,miR-1231 is upregulated.The levels of tumor proliferation,migration and invasion were significantly decreased(P<0.05),miR-1231 inhibited tumor proliferation,migration and invasion(P<0.05).Downregulation of miR-1231 reversed the changes in cell proliferation,migration,and invasion induced by hsa＿circ＿0052012 knockdown(P<0.05),the addition of miR-1231-mimics enhanced its tumor inhibition effect(P<0.05).5.MiR-1231 has binding sites with EGFR.Hsa＿circ＿0052012 being silenced,miR-1231 was upregulated,EGFR was down-regulated and EGFR protein expression was reduced(P<0.05),EGFR was down-regulated and EGFR protein expression was up-regulated after miR-1231 inhibition(P<0.05).6.Hsa＿circ＿0052012 regulates the EGFR/PI3K/ Akt /m TOR axis,and upregulated hsa＿circ＿0052012 promotes tumor progression through the EGFR/PI3K/Akt /m TOR axis.Conclusion:1.Hsa＿circ＿0052012 has a stable circ RNA structure and is enriched in the cytoplasm.Hsa＿circ＿0052012 was highly expressed in tissues,serums and gastric cancer cells of gastric cancer patients,and the highly expressed hsa＿circ＿0052012was correlated with tumor staging.2.Silencing hsa＿circ＿0052012 inhibited tumor proliferation,migration and invasion,and hsa＿circ＿0052012 promoted gastric cancer progression through the hsa＿circ＿0052012/ miR-1231/EGFR/PI3K/ Akt /m TOR axis.3.Hsa＿circ＿0052012 can be used as a marker for gastric cancer,and detection and silencing of Hsa＿circ＿0052012 can be a new research direction for the diagnosis and treatment of gastric cancer.