Expression Of CircRNA＿23113 In Lung Adenocarcinoma And Its Effect On Proliferation And Migration Of Lung Adenocarcinoma Cells

Background and Objective:Circular RNA(circular RNA,circ RNA)is a new class of covalently closed RNA molecule with no or only partial coding function,which can exist stably in mammals.Circ RNA have been confirmed express and induce the cancer in a variety of tumors by more and more studies.In the world,lung cancer is one of the most common malignant tumors harmful to human health,with the highest morbidity and fatality rate.During these years,the incidence of lung adenocarcinoma is increasing year by year.Although molecular targeted therapy has achieved good clinical results,the overall 5-year survival rate of lung adenocarcinoma patients is still hovering at 20% due to the low positive rate in the mutant population.Therefore,it is urgent to find new biomarkers and therapeutic targets for early diagnosis of lung adenocarcinoma.In the previous work of this study,low expression of circ RNA was found in clinical samples of lung adenocarcinoma.we studied the expression of circ RNA＿23113 in lung adenocarcinoma and its effect on the proliferation and migration of lung adenocarcinoma cells,in order to identify a new molecular target of lung adenocarcinoma and provide new ideas for clinical diagnosis and treatment.Methods:1.60 pairs of lung adenocarcinoma tissue and adjacent tissue samples were selected from patients with lung adenocarcinoma undergoing surgery at the Affiliated People’s Hospital of Jiangsu University from January 2019 to January 2020.None of the patients received preoperative radiotherapy or chemotherapy.Intraoperative and postoperative pathology suggested lung adenocarcinoma.The 5 m L peripheral blood samples were collected from 45 patients with lung adenocarcinoma before treatment and 45 healthy outpatients.After 3000r/min centrifugation for 5 minutes,the serum was collected in 1.5 m L clean enzyme-free tube and stored in refrigerator at-80℃.All specimens were obtained with the consent of the patients and their families and approved by the Ethics Committee of the people’s Hospital affiliated to Jiangsu University.2.High-throughput sequencing was performed on 5 pairs of clinical lung adenocarcinoma and paracancerous tissues.Circ RNA,with significant difference in expression between lung adenocarcinoma and paracancerous tissues was selected and circular RNA circ RNA＿23113 with significant difference was selected as the purpose of the study.The cyclization structure of circ RNA＿23113 was verified by gel electrophoresis,RNA enzyme experiment and Sanger sequencing,and the cyclization site was found.3.The expression of circ RNA＿23113 was verified by q RT-PCR in 60 pairs of fresh lung adenocarcinoma tissues,45 unoperated lung adenocarcinoma patients and 45 healthy patients in outpatient physical examination.The SPSS statistical analysis software was used to analyze and process the data to determine whether there were differences or not.4.Expression of circ RNA＿23113 in A549,H1299,H1975,which blong to lung adenocarcinoma cell lines,and normal bronchial epithelial cell BEAS-2B was detected by q RT-PCR.A549 and H1299 with lower expression content were selected for the subsequent experiments.The over-expression plasmid of circ RNA＿23113 was further constructed,and its effects on cell proliferation and migration were analyzed by CCK-8 assay,clone formation,cell scratch and transwell assay.5.Through circ Interactome database to predict circ RNA＿23113 corresponding micro RNA,Target Scan Human database to predict micro RNA corresponding target gene m RNA;according to various score parameters to comprehensively select mi R-661 and DKK3 with high matching degree as the research object of follow-up competitive mechanism.6.The over-expression plasmid of circ RNA＿23113 was constructed.OEcirc RNA＿23113 was transfected into lung adenocarcinoma cell lines to increase the content of circ RNA＿23113,and OE-NC was used as control group.Real-time fluorescence quantitative PCR was used to detect the expression of mi R-661 in OE-circ RNA＿23113 group compared with OE-NC group after over-expression plasmid transfection.Western blot was used to detect the expression changes of DKK3 and Wnt/β-catenin signaling pathway related downstream molecules β-catenin,cyclin D1 and c-myc protein after over-expression plasmid transfection.7.mi R-661 mimic was used to transfect lung adenocarcinoma cell lines to increase the expression of mi R-661,and its empty vector mi R-NC was used as control.The experimental groups were divided into OE-NC+mimic NC group,OE-circ RNA＿23113+mimic NC group,OE-circ RNA＿23113+mi R-661 mimic group and OE-NC+mi R-661 mimic group.The effects of these groups on cell proliferation and migration were analyzed by CCK-8 method,clone formation,cell scratch and transwell experiment and the protein expression levels of DKK3 and β-catenin,cyclin D1 and c-myc related to Wnt/β-catenin signal pathway.Results:1.The expression of circ RNA＿23113 was significantly low in lung adenocarcinoma tissue,peripheral blood serum and lung adenocarcinoma cells.2.Over-expression of circ RNA＿23113 inhibited the proliferation and migration of A549 and H1299 cells.At the same time,the results showed that the expression level of DKK3 protein increased and the expression levels of β-catenin,cyclin D1 and c-myc decreased after transfection with circ RNA＿23113 over-expression plasmid.3.Over-expression of mi R-661 could reverse the changes of cell proliferation and migration caused by the increase of circ RNA＿23113 content.4.Over-expression of mi R-661 could reverse the increase of DKK3 protein expression and decrease of β-catenin,cyclin D1 and c-myc protein expression caused by the increase of circ RNA＿23113 content.Conclusions:1.circ RNA＿23113 is poorly expressed in lung adenocarcinoma.circ RNA＿23113 is expected to be a molecular marker for the diagnosis of lung adenocarcinoma.2.Over-expression of circ RNA＿23113 can inhibit the proliferation and migration of lung adenocarcinoma cells.circ RNA＿23113 is expected to be a potential therapeutic target for lung adenocarcinoma.3.circ RNA＿23113,as a competitive endogenous RNA(ce RNA)in lung adenocarcinoma,can combine competitively with mi R-661,regulate the expression of DKK3 and the inactivate of Wnt/β-catenin signaling pathway to change the proliferation and migration ability of lung adenocarcinoma cells.