| Objective:The incidence rate of lung cancer is the highest in the world.Among them,non-small-cell lung cancer(NSCLC),accounting for almost 85%of lung cancer,is the most common.Highly expressed ITGB1 is involved in the proliferation,migration and invasion of NSCLC.MiR-493-5p,as a tumor suppressor miRNA,is negatively correlated with poor prognosis in NSCLC.Long non coding RNA(lncRNA),NR2F1-AS1,used as ceRNA,could regulate the expression of downstream target by binding with miRNA,and affect the development of tumour.However,in NSCLC,whether NR2F1-AS1 modulates the expression of ITGB1 by sponging miR-493-5p and then affects the process of NSCLC has not been much studied.Chapter 1:Correlation between the expression of NR2F1-AS1 and clinicopathological features in NSCLCMethods:First,we collected 73 cases of NSCLC clinical samples and adjacent normal tissues,and analyzed the expression of NR2F1-AS1 by RT-qPCR;then,we used NSCLC cell lines(H1299,H522,A549,H460,and SK-MES-1)and lung epithelial cells(BEAS-2B)to detect the levels of NR2F1-AS1 at the cell level;at the same time,we took the median levels of NR2F1-AS1 in NSCLC tissues as the cut-off value,and NSCLC patients were divided into low NR2F1-AS1 group(n=36)or high NR2F1-AS1 group(n=37).Pearson correlation coefficient analysis was used to analyze the correlation between NR2F1-AS1 and tumor size,TNM stage and lymph node metastasis in NSCLC patients.Results:Compared with adjacent normal tissues,NR2F1-AS1 was highly expressed in clinical samples of NSCLC.And compared with normal lung epithelial cells(BEAS-2B),NR2F1-AS1 was also highly expressed in NSCLC cells.The clinical characteristics of 73 patients with NSCLC were analyzed.The results showed that NR2F1-AS1 expression was correlated with tumor size(P=0.005),tumor(TNM)stage(P=0.038)and lymph node metastasis(P=0.025).Conclusion:NR2F1-AS1 is highly expressed in NSCLC tissues and cells,and it is associated with poor prognosis.Chapter 2:Effects of NR2F1-AS1 on proliferation,invasion and migration of NSCLCMethods:First of all,we transfected NSCLC cell lines H460 and A549 with si-NP2F1-AS1#1,si-NP2F1-AS11#2,si-NP2F1-AS1#3 into NSCLC cells,respectively.Then,we collected NSCLC cells after transfection for 0,24,48 and 72 hours,and CCK-8 was used to detect cell proliferation after NR2F1-AS1 knockdown;And flow cytometry were used to evaluate NSCLC cell apoptosis,and Transwell assay was performed to detect NSCLC cell migration and invasion.Results:RT-qPCR showed that when si-NP2F1-AS1#1,si-NP2F1-AS1#2,or si-NR2F1-AS1#3 were transferred into H460 and A549 cells,the silencing efficiency of si-NP2F1-AS1#1 was the highest.Then,CCK-8 assay showed that the cell activity was decreased after knockdown of NR2F1-AS1,and the decrease was more obvious with the prolongation of time.Flow cytometry showed that NR2F1-AS1 knockdown increased apoptosis.Trans well assay showed that NR2F1-AS1 knockdown inhibited cell migration and invasion.Conclusion:NR2F1-AS1 knockdown can inhibit the proliferation,invasion and migration of NSCLC,indicating that NR2F1-AS1 promotes the proliferation and metastasis of NSCLC.Chapter 3:The effect of NR2F1-AS1 targeting miR-493 on NSCLCMethods:In order to further understand the mechanism of NR2F1-AS1 regulating NSCLC,we analyzed the intracellular localization of NR2F1-AS1 by subcellular localization.The combination of NR2F1-AS1 and miR-493-5p was predicted by StarBase3.0 software.Therefore,we transfected NSCLC cells with miR-493-5p inhibitor to detect the expression of miR-493-5p;then,dual-luciferase reporter experiment and RIP assay were used to verify the binding of NR2F1-AS 1 with miR-493-5p.Finally,we detected the expression of miR-493-5p in 73 cases of NSCLC,and analyzed the relationship between NR2F1-AS1 and miR-493-5p.Results:NR2F1-AS1 was mainly located in cytoplasm.After transfection of miR-493-5p inhibitor,the expression of miR-493-5p in H460 and A549 cells was decreased.The luciferase activity was reduced after transfection with the wild-type NR2F1-AS1(wt-NR2F1-AS1)and miR-493-5p mimic by dual-luciferase reporter assay.However,the luciferase activity of mutant NR2F1-AS1(mut-NR2F1-AS1)combined with miR-493-5p did not decrease,indicating that miR-493-5p could directly bind to NR2F1-AS1 in H460 and A549 cells.The same results were observed in RIP experiment.Finally,we analyzed the clinical samples of NSCLC and found that miR-493-5p was lowly expressed in NSCLC,and there was a negative correlation between NR2F1-AS1 and miR-493-5p,indicating that NR2F1-AS1 negatively regulated miR-493-5p.Conclusion:1.NR2F1-AS1 binds directly with miR-493-5p;2.NR2F1-AS1 regulates miR-493-5p and mediates NSCLC process.Chapter 4:Effect of NR2F1-AS1 on NSCLC via miR-493-5p regulation of ITGB1Methods:The expression of ITGB1 mRNA was detected by RT-qPCR in 73 pairs of NSCLC and paracancerous tissues,and the relationship between NR2F1-AS1 and ITGB1 was analyzed by Pearson correlation coefficient.Then,we transfected si-NP2F1-AS1 and/or miR-493-5p inhibitor(ITGB1 overexpression plasmids)into H460 and A549 cells,H460 and A549 cell proliferation,apoptosis,migration and invasion were assessed by CCK-8,flow cytometry and Transwell assay,respectively.Finally,we established tumor formation mice models of NSCLC knockdown by subcutaneous injection,then we detected the size,weight and volume of the tumor,and evaluated the expression of NR2F1-AS1,miR-493-5p and ITGB1 in the tumor.Results:The expression of ITGB1 mRNA in NSCLC tissues was significantly increased compared with adjacent normal tissues,and there was a significant positive correlation between NR2F1-AS1 and ITGB1 mRNA levels by Pearson correlation coefficient analysis.Then,we found that miR-493-5p inhibitor or overexpression of ITGB1 could reverse the decrease of proliferation,migration and invasion,and increase of apoptosis induced by NR2F1-AS1 knockdown.Finally,in vivo experiments also showed that knockdown of NR2F1-AS1 decreased ITGB1 and increased the expression of miR-493-5p,thus inhibiting the growth of tumor.Conclusion:1.NR2F1-AS1 targets miR-493-5p mediated inhibition of malignant phenotype of NSCLC cells;2.NR2F1-AS1,as a ceRNA,regulates miR-493-5p/ITGB1 axis and promotes the malignant phenotype of NSCLC cells;3.NR2F1-AS1 promotes NSCLC tumor growth via miR-493-5p/ITGB1 axis in vivo. |
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