LncRNA-HOTAIR表观遗传调控miR-34a的表达影响肝癌细胞侵袭迁移及转移的作用及分子机制

Invasion and metastasis are the main causes of high mortality in hepatocellular carcinoma (HCC). Several studies indicated that long non-coding RNA-HOTAIR and microRNA involved in the invasion and metastasis of HCC. However, whether there is a regulatory relationship between lncRNA and microRNA, and to clarify the mechanism between them, it is a key role in the treatment of HCC invasion and metastasis targeting different non-coding RNA. It has been found that HOTAIR combined with PRC2 (EZH2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) in HOXD locus to form silencing markers and inhibits HOXD gene expression. In addition, inhibition of EZH2 in pancreatic ductal adenocarcinoma cells led to the re-expression of miR-34a. We therefore hypothesize that HOTAIR may recruit and bind to PRC2 then epigenetically regulate miR-34a, which controls the target c-met, thus contributing to influencing invasion and metastasis in HCC. Here, we try to illuminate HOTAIR crosstalk with miR-34a influencing invasion and metastasis during epigenetic regulation by RNA immunoprecipitation, luciferase reporter assays and ChiP in HCC cells, HCC specimens and xenograft mouse models. It will open a new avenue in HOTAIR-miR-34a-oriented therapy against HCC.

侵袭转移是肝癌高死亡率的主要原因。文献报道lncRNA-HOTAIR和microRNA参与肝癌的侵袭转移，然而lncRNA和microRNA之间是否存在调控关系以及明确两者之间的作用机制，对于针对不同非编码RNA联合靶向治疗肝癌侵袭转移具有关键性作用。研究发现HOTAIR结合PRC2（EZH2）催化HOXD基因形成H3K27me3沉默标志物进而抑制其表达；并且在胰腺癌中抑制EZH2的活性可重新表达miR-34a。因此，我们推测HOTAIR可能募集并结合 PRC2表观遗传调控miR-34a的表达，靶向c-met基因影响肝癌的侵袭转移。在此，利用人肝癌细胞、肝癌组织样本和异种移植小鼠模型通过RIP、荧光素酶报告基因和ChIP等方法试图阐明HOTAIR与miR-34a的交联调控可通过表观遗传的调节影响肝癌的侵袭转移，它将为HOTAIR-miR-34a定向治疗肝癌打开一个新途径。

长链非编码RNA-HOTAIR在肝癌细胞中高表达，并作为调节肿瘤侵袭和转移的重要分子引起越来越多的关注。目前，肝癌检测的临床生物标志物尚不明确，但多变量分析显示，HOTAIR是预测肝癌患者总体生存期的独立预后因素，这表明HOTAIR可能是肝癌进展的潜在生物标志物。因此，阐明HOTAIR调控肝癌细胞增殖，侵袭转移，凋亡等方面的分子机制对于肝癌的早期预防，诊断以及靶向治疗具有借鉴意义。研究发现，与肝正常细胞相比，五种肝癌细胞系中，HepG2细胞中HOTAIR的基础表达量最高(4.2-fold)，其次是SNU-387（3.5-fold）。过表达HOTAIR组SNU-387细胞的增殖速率较CON组加快，细胞倍增时间缩短。sh-HOTAIR组中HepG2，SNU-387细胞的增殖速率较sh-NC组减慢，倍增时间延长。过表达HOTAIR组SNU-387细胞的侵袭迁移能力较CON组增强；sh-HOTAIR组HepG2，SNU-387细胞的侵袭迁移能力较sh-NC组减弱。与CON组相比，过表达HOTAIR后，SNU-387细胞中MMP2和MMP9在蛋白和mRNA转录水平均明显上升；上皮细胞标志物E-cadherin的表达量在蛋白和mRNA转录水平均明显降低；间质标志物N-cadherin在蛋白水平明显上调，mRNA转录水平无明显变化；Snail2在蛋白和mRNA转录水平均明显上升。Snail2核内表达量增加，荧光强度增强。敲低组结果与此相反。sh-HOTAIR组中HepG2和SNU-387细胞的线粒体膜电势较sh-NC组明显降低，细胞凋亡率明显增加。线粒体促凋亡蛋白Bak 表达量增加；线粒体抗凋亡蛋白Bcl-2，Mcl-1表达量降低。与sh-NC组相比，sh-HOTAIR组中HepG2和SNU-387细胞中可观察到核染色质凝集，核浓缩，甚至核碎裂等异常核形态。TCGA数据库分析HOTAIR和E-cadherin（CDH1）mRNA转录水平没有明显的负向调控关系；HOTAIR和Snail2的mRNA转录水平没有明显的正向调控关系。总之， HOTAIR促进肝癌细胞增殖，缩短其倍增时间，调控Snail2的核内表达量，介导EMT的发生并通过基质金属蛋白酶MMP2和MMP9增加肝癌细胞侵袭转移的能力。敲低HOTAIR可促进线粒体诱导的细胞凋亡的发生。本实验为肝癌的靶向治疗做初步探索。