长链非编码RNA RP3-337D23.3促进非吸烟者肺腺癌侵袭转移的机制研究

By screening with microarray and validation with quantitative PCR, we identified RP3-337D23.3 as a first lung adenocarcinoma in never smokers (LACINS) associated long non-coding RNA (lncRNA). Our previous work revealed that: RP3-337D23.3 was significantly over-expressed in LACINS compared with adjacent normal lung tissue and shRNA-mediated silence of RP3-337D23.3 suppressed proliferation, invasion, and migration ability of LACINS cell lines. Furthermore, both bioinformatics and in vitro studies demonstrated that AKAP12, a novel lung adenocarcinoma suppressor gene, was a potential down-stream target of RP3-337D23.3. The expression of AKAP12 might be epigenetically regulated by RP3-337D23.3. In this proposal, firstly, we will systematically assess the translational value of RP3-337D23.3 by characterization the expression profile and prognostic value of RP3-337D23.3 in a large set of LACINS samples.Secondly, we plan to clarify the functional role of RP3-337D23.3 in the invasion and metastasis of LACINS. To achieve this goal, we design to over-express and knock-down RP3-337D23.3 both in vivo and in vitro (in two separated LACINS cell lines) and evaluate the impact of RP3-337D23.3 on malignant biological behaviors, including the abilities of proliferation, migration and invasion. Finally, we aim to explore how RP3-337D23.3 exerts its regulatory function. Highlighted by the relationship between AKAP12 gene and RP3-337D23.3, we design to investigate the mutual interaction between AKAP12 and RP3-337D23.3 via RIP, CHIP, and resecue experiments. To date, no data is available about LACINS specific lncRNA at home or abroad, therefore, our study is the original source of innovation and can provide new potential biomarkers for the prevention and treatment of LACINS.

RP3-337D23.3（简称RP）是我们应用芯片技术筛选获得的非吸烟者肺腺癌（LACINS）相关、差异高表达于肿瘤组织、功能未知的长链非编码RNA。已发现：RP在20例LACINS 标本中均呈现高表达且与淋巴结转移正相关；沉默RP可显著抑制LACINS细胞侵袭迁移能力；生物信息学及实验初步确定RP可能通过表观遗传机制调控肺癌转移抑制基因AKAP12表达，提示RP可通过调控AKAP12促进LACINS侵袭转移。本研究拟以大样本临床评价RP与LACINS转移的相关性；以两种LACINS细胞株为工具，采用过表达与沉默策略，结合体外实验及小鼠肺癌转移模型，论证RP对LACINS侵袭转移的影响；结合RIP、CHIP等方法及拯救实验，阐明RP通过调控AKAP12促进LACINS侵袭转移的分子机制。有关RP促进LACINS侵袭转移的作用与机制国内外未见报道，本研究将可能为LACINS防治提供新靶标。

非吸烟者肺腺癌的发病率在逐渐升高，而其发病机制与分子生物学特征尚未被研究。在国家自然科学基金面上项目的资助下，本项目通过lncRNA与mRNA芯片，在5例女性非吸烟肺腺癌患者中系统地研究了lncRNA和mRNA的表达谱，发现众多差异表达的lncRNA与mRNA。我们发现、鉴定出了其中显著上调的lncRNA，将其命名为LUADT1。LUADT1在肺腺癌患者中表达水平显著上调，平均上调8.34倍；且LUADT1表达与肿瘤T分期显著相关（P=0.043）。CCK8和克隆实验证实，敲减LUADT1之后肺癌细胞的增殖能力被限制抑制；敲减LUADT1后肺癌细胞周期被阻滞在G1期，且p27表达显著下降。RIP实验证实LUADT1可与SUZ12绑定；CHIP实验发现p27启动子区存在SUZ12富集和H3K27me3；敲减LUADT1后，p27启动子区SUZ12富集减少，且H3K27me3水平也明显降低。敲减LUADT1后，肺癌细胞的成瘤和增殖能力被抑制。.本项目阐释了lncRNA LUADT1参与肺癌发生发展的分子生物学机制，证实LUADT1可作为潜在的肺癌治疗靶标；本研究为lncRNA与肺癌的研究提供了新的思路和依据，为肺癌防治提供了新的思路。