Linc01419调控miR-34a-5p/E2F3轴促进膀胱癌细胞增殖与侵袭

Linc01419 regulates miR-34a-5p/E2F3 axis to promote proliferation and invasion of bladder cancer cells

目的:探讨长链非编码RNA Linc01419对膀胱癌细胞增殖和侵袭的影响及作用机制。方法:通过UALCAN软件(https://ualcan.path.uab.edu/)分析TCGA数据库中Linc01419在膀胱癌组织与正常膀胱组织中的表达差异;采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)检测Linc01419在不同膀胱癌细胞系、22例经病理证实为膀胱癌的手术患者的肿瘤组织及癌旁正常膀胱组织中的表达水平;应用细胞增殖/毒性检测(cell counting kit-8,CCK-8)实验和Transwell小室侵袭实验检测敲低Linc01419表达对膀胱癌细胞增殖与侵袭的影响;采用双荧光素酶报告基因检测法分析Linc01419与miR-34a-5p及miR-34a-5p与E2F3之间的靶向调控关系。结果:UALCAN数据库分析显示相较正常膀胱组织,Linc01419在膀胱癌组织中显著高表达(P<0.001);RT-qPCR分析结果显示相较癌旁正常膀胱组织,Linc01419在22例膀胱癌组织中表达明显上调(P<0.001),与UALCAN数据库分析结果一致;Linc01419在4株膀胱癌细胞中的表达明显高于正常膀胱上皮细胞(P<0.01);敲低Linc01419表达,可显著抑制膀胱癌细胞的增殖、侵袭及N-cadherin、PCNA蛋白的表达,而显著促进E-cadherin的蛋白表达(P<0.05);miR-34a-5p过表达对膀胱癌细胞具有类似的抑制作用;双荧光素酶报告基因实验、RIP及Pull-down实验证实Linc01419可靶向结合miR-34a-5p,而后者进一步介导了对E2F3的靶向调控;抑制miR-34a-5p表达,可显著削弱Linc01419沉默对膀胱癌细胞生物学行为及E-cadherin、N-cadherin、PCNA、E2F3表达的影响。结论:Linc01419在膀胱癌中异常高表达,其通过调控miR-34a-5p/E2F3轴促进膀胱癌细胞增殖和侵袭,很可能是膀胱癌发生、发展过程中的一个重要环节。

Objective:To investigate the effect and the molecular mechanism of long non-coding RNA Linc01419 in facilitating the proliferation and invasion of bladder cancer cells.Methods:Differential expression of Linc01419 between bladder cancer tissues and normal bladder tissues was determined using the online software UALCAN(https://ualcan.path.uab.edu/).The data for this analysis was retrieved from the TCGA database.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)analysis was used to investigate the differential expression of Linc01419 in several types of bladder cancer cell lines,and in bladder cancer tissues and adjacent normal bladder tissues from 22 patients who received surgery and were pathologically diagnosed with bladder carcinoma.To determine the effect of Linc01419 on proliferation and invasion of bladder cancer cells,cell counting kit-8(CCK-8)and Transwell assays were performed.Dual-luciferase reporter assay was applied to verify the targeting relation among Linc01419,miR-34a-5p and E2F3.Results:The UALCAN database analysis revealed that Linc01419 was highly expressed in bladder cancer tissues when compared to normal bladder tissues(P<0.001).Consistent with the results of UALCAN database analysis,Linc01419 was confirmed to be up-regulated in 22 bladder cancer tissues as compared with adjacent normal bladder tissues(P<0.001).Moreover,Linc01419 expression was higher in four types of bladder cancer cells than in normal bladder epithelial cells(P<0.01).Silencing of Linc01419 led to a significant reduction in the proliferation and invasion of bladder cancer cells.Additionally,it resulted in a notable decrease in the expression of N-cadherin and PCNA proteins,while there was a significant increase in the expression of E-cadherin(P<0.05).Overexpression of miR-34a-5p also exerted similar inhibitory effects on bladder cancer cells.Dual-luciferase reporter gene assay,RIP as well as Pull-down assays verified that Linc01419 can target and bind to miR-34a-5p,which further mediated the targeted regulation of E2F3.The suppression of miR-34a-5p expression substantially mitigated the influence of Linc01419 silencing on the biological characteristics of bladder cancer cells,as well as the expression levels of E-cadherin,N-cadherin,PCNA,and E2F3.Conclusion:Linc01419 is aberrantly overexpressed in bladder cancer and can promote bladder cancer cell proliferation and invasion by targeting the miR-34a-5p/E2F3 pathway,which indicates that abnormal expression of Linc01419 may act as an important procedure in carcinogenesis and development of bladder cancer.