HOXA11-AS通过靶向miR-146a-5p调节HAb18G在NSCLC转移中的作用及机制研究

No study of antisense HOXA11 (HOXA11-AS), a long non-coding RNA (LncRNA) has been available in non-small cell lung cancer (NSCLC). In our previous study, we verified the targeting correlation between HOXA11-AS and miR-146a-5p. We also found that in NSCLC tissues, the expression of HOXA11-AS was upregulated, whereas miR-146a-5p level was downregulated and its potential target gene HAb18G was overexpressed. Furthermore, the expression of HOXA11-AS, miR-146a-5p and HAb18G was all correlated to the metastasis of NSCLC. Additionally, in previous in vitro experiments, knock-down of HOXA11-AS suppressed the proliferation and migration of NSCLC cells. Therefore, we hypothesize that HOXA11-AS may regulate HAb18G expression via targeting miR-146a-5p and thus, enhance the invasion and metastasis of NSCLC cells. To test this hypothesis, we plan to perform a series of experiments, including real time RT-qPCR, in situ hybridization, luciferase reporter, RNA binding protein immunoprecipitation (RIP), RNA pull-down, chromatin immunoprecipitation (ChIP), RNA interference, western blot, invasion and metastasis assays and animal models in this application. The effect and mechanism of HOXA11-AS on metastasis of NSCLC will be investigated from molecule, cell, tissue and animal levels. The overall objectives are to investigate the clinical significance and relationships of HOXA11-AS, miR-146a-5p and HAb18G in NSCLC; to study the effect and molecular mechanism of HOXA11-AS-miR-146a-5p-HAb18G regulation on the invasion and metastasis of NSCLC. Focusing on the new insight of HOXA11-AS-miR-146a-5p-HAb18G axis, this study will reveal the molecular mechanism of NSCLC metastasis, also provide a novel target for clinical prevention and therapeutic strategy of NSCLC metastasis.

LncRNA HOXA11-AS在NSCLC中的研究未见报道。前期我们证实了HOXA11-AS与miR-146a-5p的靶向关系，并发现NSCLC中HOXA11-AS表达增高，miR-146a-5p下调，同时miR-146a-5p潜在靶标HAb18G表达增高，三者均与转移相关；体外敲除HOXA11-AS可减缓NSCLC增殖及迁移。据此提出假说：HOXA11-AS可通过靶向miR-146a-5p调控HAb18G而影响NSCLC侵袭转移潜能。为验证此假说，我们采用RT-qPCR、原位杂交、RIP、RNA pull-down、ChIP、RNAi、WB、侵袭转移及动物实验等手段，从分子、细胞、组织及动物水平探讨HOXA11-AS的临床意义、对侵袭转移的作用及其分子机制。本研究以HOXA11-AS靶向miRNA这一新视点揭示NSCLC的分子转移机制，为临床预防和控制NSCLC转移提供新的分子靶点。

本课题前期证实了HOXA11-AS与miR-146a-5p的靶向关系，发现非小细胞肺癌（non-small cell lung cancer，NSCLC）中HOXA11-AS表达增高，miR-146a-5p下调，miR-146a-5p潜在靶标HAb18G表达增高，三者均与转移相关；体外敲除HOXA11-AS可减缓NSCLC细胞增殖及迁移。为进一步阐明HOXA11-AS在NSCLC中的作用机制，本课题的研究内容围绕“HOXA11-AS可通过靶向miR-146a-5p调控HAb18G而影响NSCLC侵袭转移潜能的分子机制”开展，针对HOXA11-AS和miR-146a-5p、HAb18G的表达水平和体内外的生物学功能及机制进行研究。本研究联合了分子生物学实验技术和计算生物学相关分析的方法，具体包括逆转录-定量聚合酶链反应（RT-qPCR）、免疫组化技术、细胞培养技术、基因敲除、双荧光素酶报告实验、鸡胚模型的建立、公共数据库挖掘等，探究HOXA11-AS与miR-146a-5p和HAb18G在NSCLC的靶向调控关系及在NSCLC转移中的作用。研究发现，HOXA11-AS与miR-146a-5p在NSCLC组织中的表达呈负相关、它们之间存在靶向关系，miR-146a-5p与HAb18G在NSCLC中也具有靶向调控关系。HOXA11-AS高表达与NSCLC的临床分期及淋巴结转移存在正相关。同时，HOXA11-AS敲除后，细胞增殖减慢，迁移能力降低，细胞凋亡增加；miR-146a-5p表达上调和HAb18G表达下调也有明显的抑制NSCLC肿瘤细胞活性和增殖、促进肿瘤细胞凋亡作用。体内活体成瘤实验提示HOXA11-AS敲除和miR-146a-5p过表达均可抑制NSCLC成瘤与血管生成。此外，我们也对相关的数个HOX家族基因进行了检测和分析，发现HOXA13在肺腺癌中高表达，通过调控一系列基因的表达促进肺腺癌的发生发展。HOXA1为致癌基因，在NSCLC中高表达且具有诊断价值，与肿瘤分期有关，miR-181b和miR-181d在肺癌组织中低表达，与HOXA1负相关，结合通路分析结果我们推测在肺鳞癌中HOXA1可能是miR-181b-5p或miR-181d-5p的直接靶标，通过调节各种通路，特别是p53信号通路在NSCLC中发挥重要作用。