Clinical Significance And Biological Mechanism Of Circrna Hsa-circ-0002360 In Lung Adenocarcinoma

Objective:Circular RNA is a special non-coding RNA with a closed ring structure.With the development of sequencing technology,more and more circRNAs have been discovered,and their various functional studies have followed in recent years.At present,circRNAs have become the focus of tumor research.The incidence and mortality of lung cancer are high,and it is urgent to find effective treatment and diagnosis methods.The further exploration of the relationship between circRNAs and lung adenocarcinoma is gradually put on the agenda.The purpose of this study is to explore the clinical significance of hsa_circ0002360 in lung adenocarcinoma,so as to provide new ideas and research basis for the diagnosis and treatment of lung adenocarcinoma in the future.Methods:1.From January 2018 to September 2019,122 cases of lung adenocarcinoma tissues were collected from the Affiliated People's Hospital of Jiangsu University.In addition,5ml peripheral blood samples were collected from 50 patients and healthy volunteers.2.Hsa_circ₀002360 was selected as the study object by high-throughput sequencing in different lung tissues.PCR,agarose gel electrophoresis and Sanger sequencing were used to identify the structure of circRNA hsa_circ₀002360.3.The expression levels of hsa_circ₀002360 in lung adenocarcinoma tissues,adjacent tissues and peripheral blood serum were detected by qRT-PCR.Paired T-test and independent sample t-test were used to analyze the expression of has_circ₀0002360 in lung tissues,serum and the relationship of the has_circ₀002360 expression and clinical characteristics.4.Miranda database data were used to predict microRNA matching hsa_circ₀002360 and microRNA matching mRNA.The differences were analyzed in TCGA data and hsa_circ₀002360,miR-762 and mRNA PODXL were screened out as follow-up research objects.The targeting effects of hsa_circ₀002360,miR-762 and PODXLwere further confirmed by luciferase reporter method.5.qRT-PCR was used to detect the expression of hsa_circ₀002360 in human bronchial epithelial cells and lung adenocarcinoma cell lines.The cell lines with higher expression were selected for subsequent experiments.Lung adenocarcinoma cell lines transfected with Si-circ₀002360 was used to down-regulate the expression of hsa_circ₀002360 and Si-NC as the control group.Lung adenocarcinoma cell lines were transfected with miR-762 inhibitor or miR-762 mimic to down-regulate or upregulate the expression of miR-762.Respectively,their empty vector miR-NC was used as normal control.The experimental groups were Si-circ₀002360 group,Si-NC group,Si-circ₀002360 + miR-762 inhibitor group,miR-762 inhibitor group,miR-762 mimic group and miR-NC group.CCK8,colony formation experiment,transwell,wound healing assays were used to investigate the effects of hsa_circ₀002360 on the proliferation,migration and invasion of lung adenocarcinoma cells.Western blot was used to detect the expression of mRNA PODXL and epithelial-to-mesenchymal transition related proteins after the intervention of the above groups.Results:1.The expression of hsa_circ₀002360 was significantly higher in the LAC tissue than in the adjacent tissue?2.84 ± 0.12 vs 1.46 ± 0.08,P < 0.01?,so was it in the serum of the lung adenocarcinoma patients than in the normal controls?2.59 ± 0.17 vs 1.21 ± 0.11,P < 0.01?.But it's expression remarkably lower in the serum of the patients after surgery than before?1.58 ± 0.09 vs 2.65 ± 0.13,P < 0.01?.The expression of RNA was correlated with lymph node metastasis?P =0.004?and tumor differentiation?P =0.02?,but not with age,gender,and smoking history?P > 0.05?in lung adenocarcinoma patients2.Compared with human bronchial epithelial cells,the expression of hsa_circ₀002360 in A549 and H1975 were significantly higher.Compared with the control group,the proliferation,migration and migration level of lung adenocarcinoma cells were significantly decreased after down-regulation of hsa_circ₀002360?P<0.01?.3.The dual-luciluciase experiment assay reports showed that the inhibition rate of luciferase activity in hsa_circ₀002360-WT?wild type?+ miR-762 group was higher than that in hsa_circ₀002360-MUT?mutant type?+ miR-762 group.The inhibition rate of PODXL-WT?wild type?+ miR-762 group was higher than that in PODXLMUT?mutant type?.4.Overexpression of miR-762 inhibited the proliferation,migration and invasion of human lung adenocarcinoma cells.Downregulation of miR-762 could reverse the proliferation and migration reduction of human lung adenocarcinoma cell lines due to the downregulation of hsa_circ₀002360.The differences were statistically significant?P<0.05?.Conclusion:1.Hsa_circ₀002360 is highly expressed lung adenocarcinoma patients,and its expression level is related to tumor cell differentiation and lymph node metastasis.Hsa_circ₀002360 is expected to be a molecular marker for the diagnosis of lung adenocarcinoma.2.Silence of hsa_circ₀002360 can inhibit the proliferation,migration and invasion of lung adenocarcinoma cells.Hsa_circ₀002360 is expected to be a potential target for lung adenocarcinoma.3.In terms of mechanism,hsa_circ₀002360 can promote the progression of lung adenocarcinoma through the mechanism of ceRNA.The existence of hsa_circ₀002360/ miR-762 /PODXL axis in lung adenocarcinoma can promote the progression of lung adenocarcinoma.