长链非编码RNA HOXD-AS1调控HOXD3表达介导结直肠癌转移的功能及分子机制研究

Long noncoding RNAs (lncRNAs) are emerging as critical epigenetic and transcriptional regulators of gene expression and biological events. Thus investigation of lncRNAs' role on tumorigenesis and metastasis has become one of the developing trends. We found that HOXD-AS1 was down-regulated in colorectal carcinoma（CRC）tissues and cell lines，especially in metastatic tumor samples. The inhibition of HOXD-AS1 expression increased CRC cell growth，anchorage-independent growth，migration and invasion. Taken together，these findings suggested that HOXD-AS1 expression dysregulation contributing to the development and /or progression of CRC. In the upcoming study，we are going to investigate the clinicopathological and prognostic significance of HOXD-AS1 expression in a large cohort of CRC and normal mucosal samples by Realtime RT-PCR，LNA-ISH and FISH. Then we will focus on HOXD-AS1’role on cell proliferation， invasion and migration using in vitro and in vivo assays. Finally，we will identify one or several potential downstream target genes of HOXD-AS1. In summary，by adopting RIP，RNA-Protein Pull Down，gene microarray and bioinformatics， we will elucidated the potential signaling pathway， HOXD-AS1～HOXD3～target genes， and its implication in CRC metastasis. Our results will certainly provide important clues for understanding the key roles of lncRNA functional networks in CRC treatments and successfully built CRC control efforts.

lncRNA与肿瘤的发生、演进密切相关，是目前肿瘤转移调控机制研究的前沿和热点。本课题组前期实验结果表明lncRNA HOXD-AS1在结直肠癌中异常低表达，外源性下调HOXD-AS1，可提升HOXD3基因在结直肠细胞内的表达水平，从而增强直肠癌细胞的体外生长、侵袭能力。在此基础上，本项目拟采用Realtime RT-PCR、原位杂交等技术，大样本量精确评价HOXD-AS1与HOXD3的表达相关性及转移相关临床意义。并通过细胞体内、外功能实验，证实HOXD-AS1可通过表达调控HOXD3参与结直肠癌生物学过程，再运用RIP、RNA-Protein Pull Down、基因芯片及生物信息学分析技术，在转录后层面阐明HOXD-AS1与HOXD3基因作用的分子机制，并初步阐明“HOXD-AS1～HOXD3～下游分子”信号调控网络，为结直肠癌转移的预防和治疗提供新分子靶点及策略。

背景：长链非编码RNA与肿瘤的发生、演进密切相关。近年来研究发现lncRNA HOXD-AS1在多种肿瘤中表达失调。然而，HOXD-AS1在结直肠癌中的表达水平、细胞定位、确切功能及作用机制尚不清楚。.方法：Real-time PCR和原位杂交技术用于检测HOXD-AS1在结直肠癌组织标本和细胞系中的表达情况。其次在体内、体外水平，分别通过表达获得及敲除功能实验检测HOXD-AS1在结直肠癌细胞系中的生物学作用。最后，通过RNA-pulldown、RIP和CHIP等实验，探讨HOXD-AS1在结直肠癌中的作用机制。.结果：HOXD-AS1定位于结直肠癌细胞核内，多数结直肠癌标本和细胞株的核内HOXD-AS1均低表达。HOXD-AS1核内异常低表达与患者预后不良密切相关。HOXD-AS1下调可促进结直肠癌细胞体外增殖和迁移，及体内成瘤及转移能力。HOXD-AS1通过招募PRC2复合体诱导HOXD3启动子区H3K27me3蓄积，从而抑制HOXD3转录。并发现，HOXD3可作为转录激活因子结合于ITGB3启动子区，激活整合素β3转录，进而活化下游MAPK / AKT信号通路活性。.结论：HOXD-AS1～HOXD3～整合素β3表达调控轴通过表观遗传、转录调控机制，参与抑制结直肠癌的发生及演进。