lncRNA NEAT1调控肝癌干细胞扩增及自我更新的机制研究

Study on the mechanism of lncRNA NEAT1 regulating the amplification and self-renewal of liver cancer stem cells

目的探讨lncRNA NEAT1对肝癌干细胞(liver cancer stem cells,LCSCs)扩增及自我更新能力的调控作用及机制。方法培养人肝癌细胞系HCCLM3,利用低黏附成球培养富集LCSCs,qRT-PCR检测LCSCs和HCCLM3中NEAT1表达差异;过表达NEAT1慢病毒感染LCSCs,qRT-PCR检测感染效率;流式细胞术、克隆形成实验、细胞成球实验分别检测过表达NEAT1对LCSCs的扩增、克隆形成及球体形成能力;Western blotting检测Hippo信号通路关键分子LATS1和YAP蛋白磷酸化水平。结果 LCSCs中NEAT1表达水平显著高于HCCLM3细胞(P<0.05);感染过表达NEAT1慢病毒的LCSCs中NEAT1表达水平显著高于对照感染(P<0.001),过表达NEAT1模型构建成功;过表达NEAT1使EpCAM^+和CD133^+的LCSCs细胞亚群比例明显增多(P<0.05);过表达NEAT1促进细胞成球能力和克隆形成能力(P<0.05);过表达NEAT1降低YAP和LATS1蛋白磷酸化水平(P<0.01)。结论 lncRNA NEAT1在LCSCs中高表达,过表达NEAT1促进LCSCs的扩增与自我更新功能,可能通过活化Hippo信号通路来实现。

Objective To investigate the regulation and mechanism of lncRNA NEAT1 on the amplification and self-renewal of liver cancer stem cells(LCSCs).Methods Human liver cancer cell line HCCLM3 was cultured, LCSCs were enriched by low-adhesion pellet culture, and NEAT1 expression differences in LCSCs and HCCLM3 were detected by qRT-PCR. Over-expressed NEAT1 lentivirus infected LCSCs, and the infection efficiency was detected by qRT-PCR. Flow cytometry, clone formation experiment and cell pellet formation experiment were used to detect the amplification, clone formation and sphericity of LCSCs induced by overexpression of NEAT1. Phosphorylation levels of key molecules of Hippo signaling pathway LATS1 and YAP proteins were detected by Western blotting.Results The expression level of NEAT1 in LCSCs was significantly higher than that in HCCLM3 cells(P<0.05). The expression level of NEAT1 in LCSCs infected with overexpression of NEAT1 lentivirus was significantly higher than that in control infection(P<0.001). Overexpression of NEAT1 significantly increased the proportion of LCSCs subsets of EpCAM^+ and CD133^+(P<0.05). Overexpression of NEAT1 promoted pellet formation and clone formation(P<0.05). Overexpression of NEAT1 decreased the phosphorylation levels of YAP and LATS1(P<0.01). Conclusion LncRNA NEAT1 is highly expressed in LCSCs, and overexpression of lncRNA NEAT1 promotes the amplification and self-renewal of LCSCs, possibly by activating the Hippo signaling pathway.