基于TGF-β2/Smad2信号通路探讨长链非编码RNA NEAT1对膀胱癌T24细胞生物学行为的影响

Effect of long non-coding RNA NEAT1 on biological behavior of bladder cancer T24 cells was investigated based on TGF-β2/Smad2 signaling pathway

目的:探讨长链非编码RNA(lncRNA)NEAT1调控TGF-β2/Smad2信号通路对膀胱癌(BC)T24细胞上皮间质转化(EMT)和生物学行为的影响及潜在分子机制。方法:收集2015年3月~2018年5月于我院接受手术治疗的128例BC患者癌组织及距离癌组织超过2cm处的正常组织,qRT-PCR检测lncRNA NEAT1在BC及相应的正常组织、不同BC细胞系中的表达情况;以T24为空白对照,构建lncRNA NEAT1沉默细胞系T24-siNEAT1及阴性对照T24-NC,分别采用MTT、平板克隆实验及Transwell实验检测lncRNA NEAT1对T24细胞增殖和侵袭能力;免疫荧光实验检测E-cadherin、Vimentin表达;生物信息学网站starBase预测可与lncRNA NEAT1互补结合的miRNA,根据Targetscan网站预测相应miRNA可靶向结合的基因;Western blot检测TGF-β2/Smad2信号通路蛋白和E-cadherin、Vimentin表达情况。结果:qRT-PCR结果显示,BC组织和癌细胞中lncRNA NEAT1表达水平明显高于正常组织和正常细胞(P<0.05);与空白对照组和T24-NC组相比,T24-siNEAT1组细胞OD 492值明显下降,细胞克隆数、通过matrigel基质胶的数量明显减少,E-cadherin表达明显升高、Vimentin明显降低(P<0.05);starBase网站预测结果显示,lncRNA NEAT1可与miR-200a-3p互补结合,Targetscan网站预测分析显示,miR-200a-3p与TGF-β2存在靶向结合位点;qRT-PCR和Western blot结果显示T24-siNEAT1组中miR-200a-3p、TGF-β2及p-Smad2表达明显低于空白对照组与T24-NC阴性对照组(P<0.05);空白对照组和T24-NC组中上述指标的表达量无统计学意义(P>0.05)。结论:lncRNA NEAT1在BC组织及细胞中高表达,高表达lncRNA NEAT1可能通过上调miR-200a-3p水平强化TGF-β2/Smad2信号通路,促进EMT过程,影响BC进展。

Objective:To study the effect of TGF-β2/Smad2 signaling pathways regulated by long-chain noncoding RNA(lncRNA)NEAT1 on epithelial-mesenchymaltrasition(EMT)and biological behaviour in bladder cancer(BC)cell T24 and and its potential mechanism.Methods:BC tissues and normal tissues more than 2 cm away from the cancer tissues were collected form 128 patients with BC for surgical treatment in our hospital from March 2015 to May 2018.lncRNA NEAT1 expression was measured by qRT-PCR in BC and corresponding adjacent tissues,as well as different BC cell lines.T24 was used as blank control,lncRNA NEAT1 silenced cell line T24-siNEAT1 and negative control T24-NC were constructed.Effect of lncRNA NEAT1 on T24 cell proliferation and invasion ability were tested using MTT,flat-plate cloning test and Transwell test,respectively;expressions of E-cadherin and Vimentin were detected by immunofluorescence assay;bioinformatics website starBase was used topredict lncRNA NEAT1 that could bind to complementary miRNA,furthermore,according to the website Targetscan,target binding genes of corresponding miRNA were predicted;expression of TGF-β2/Smad2 signaling pathway protein and E-cadherin and Vimentin were detected by Western blot.Results:qRT-PCR results showed that expression levels of lncRNA NEAT1 in BC tissues and cancer cells were significantly higher than that of normal tissues and normal cells(P<0.05);compared with T24-NC group,OD 492 value of cells in T24-siNEAT1 group decreased significantly,numbers of cell clones and matrigel matrix gels decreased expressions of E-cadherin was increased and Vimentin decreased(P<0.05).Results of starBase website showed that lncRNA NEAT1 was complementary with miR-200a-3p,and predictive analysis on website Targetscan showed that there was a targeted binding site for miR-200a-3p and TGF-β2;qRT-PCR and Western blot results showed that expressions of miR-200a-3p,TGF-β2 and p-Smad2 in T24-siNEAT1 group were significantly lower than those in blank control group and T24-NC negative control group(P<0.05);expressions of the above indicators in blank control group and T24-NC group were not statistically significant(P>0.05).Conclusion:lncRNA NEAT1 is highly expressed in BC tissues and cells,and high expression of lncRNA NEAT1 may affect the development of BC by up-regulating level of miR-200a-3p,enhancing TGF-β2/Smad2 signaling pathway,and promoting EMT process.