摘要目的 通过RNA免疫共沉淀寻找长链非编码RNA(lncRNA)核富集转录体1(NEAT1)相关的RNA结合蛋白,验证这些RNA结合蛋白参与NEAT1调节肝癌相关基因核内不均一核糖核蛋白A2(hnRNPA2)表达的分子机制,观察NEAT1调节hnRNP A2的表达对人肝细胞癌的进展的影响.方法 查询Starbase 2.0数据,寻找与NEAT1相互作用密切的RNA结合蛋白,通过RNA免疫共沉淀明确能同时与NEAT1和NEAT1所调控基因mRNA相互作用的RNA结合蛋白,RNA pull-down明确RNA结合蛋白与NEAT1作用靶点片段,使用转染试剂Lipofectamine 2000和siRNA NEAT1(终质量浓度100 nmol/L)敲低HepG2细胞NEAT1表达,Western blot、免疫组织化学检测NEAT1对hnRNP A2蛋白的调节,使用逆转录病毒STP-重组逆转录病毒载体作为载体建立hnRNP A2过表达模型并应用于NEAT1低表达的HepG2细胞模型中,细胞计数试剂盒(CCK-8)增殖实验(10 μl/2 000个细胞)和Transwell小室研究NEAT1低表达的HepG2细胞(2×105个/孔)过表达hnRNP A2后与对照组的增殖、侵袭功能.HepG2经NEAT1敲低后2×106/0.2 ml腋下注射建立裸鼠皮下成瘤模型,与对照组(si-NC)比较肿瘤体积,hnRNP A2表达.结果 U2AF65能够与NEAT1和hnRNP A2 mRNA相互作用(富集度分别为IgG的82.659倍和53.527倍),NEAT1敲低降低了hnRNP A2 mRNA转录和蛋白表达(P＜0.01),这种调控作用可能与NEAT1-U2AF65复合物相关,hnRNPA2过表达可以促进NEAT1敲低的HepG2细胞增殖和侵袭功能(P＜0.01).结论 NEAT1通过调节hnRNP A2蛋白影响了肝细胞癌细胞的增殖和侵袭能力.

abstractsObjective To explore the molecular mechanism of long noncoding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1)-heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) regulation in hepatocellular carcinoma (HCC) progression,and study the involvement of RNA binding protein by RNA immunoprecipitation (RIP).Methods Selected RNA binding proteins (Starbase 2.0) were verified by RIP assay.Targeting sites were verified by RNA pull-down assay.Western blotting assay was performed to detect hnRNP A2 expression after si-RNA (100 nmol/L) NEAT1 or U2AF65 treatment.Cells were stably transduced with STP-puro retroviral vectors expressing T7-tagged hnRNP A2 cDNA to overexpress hnRNP A2.Proliferation and invasion of each group were detected by CCK-8 count assay (10 μl/2 000 cells) and Transwell assay (2 × 105 cells/well).The axillary fossae of male athymic nude mice aged 4-6 weeks were then bilaterally inoculated with 2 × 106 stably overexpressing si-NEAT1 HepG2 cells or control cells per site to explore NEAT1-knockdown in vivo.Results U2AF65 interacted with NEAT1 and hnRNP A2 mRNA (enrichment fold changes compared with IgG group:82.659 for NEAT1 and 53.527 for hnRNP A2 mRNA).NEAT1-knockdown led to down-regulation of hnRNP A2 (P ＜ 0.01).This regulation might be associated with the NEAT1-U2AF65 protein complex.Overexpression of hnRNP A2 rescued the proliferation and invasion of HCC cells that expressed low levels of NEAT1 (P ＜ 0.01).Conclusion Long noncoding RNA NEAT1 is an oncogene that,when down-regulated,inhibits HCC cell proliferation and invasion as well as HCC tumor growth in xenograft models.These effects strongly correlated with altered hnRNP A2 expression,which is also regulated by NEAT1.