一个功能未知的长链非编码RNA——AC093850.2调控肺腺癌侵袭转移的分子机制研究

By microarray and transcriptome sequencing, we have identified an uncharacterized long non-coding RNA (lncRNA) AC093850.2,which is highly expressed in lung adenocarcinoma (LAC). Quantitative RT-PCR demonstrated that AC093850.2 was significantly over-expressed in LAC tissues compared with adjacent normal lung tissue and positively correlated with lymph node metastasis. AC093850.2 was also specifically highly expressed in LAC cell lines but not in other epithelium derived cancer cells or in lung saqumaous cell carcinoma. Silencing AC093850.2 by siRNA significantly suppressed the invasion and migration ability of LAC cell lines. Moreover, bioinformatic analysis and RIP-PCR experiment revealed that AC093850.2 could bind with an important chromosome remodeling complex, polycomb repressive complex 2 (PRC2), indicating the potential of epigenetic regulation. Microarray and in vitro validation suggested that silencing AC093850.2 significantly up-regulated the expression of a lung cancer metastasis suppressor gene , E-cadherin, which is also a target gene of PRC2. Based on above results, we therefore hypothesized that AC093850.2 might suppress the metastasis of LAC by recuiting PRC2 and subsequently decreasing the expression of E-cadherin. In this proposal, firstly, we plan to clarify the functional role of AC093850.2 in invasion and metastasis of LAC. To achieve this goal, we design to over-express and knock-down AC093850.2 both in vivo (in both passitive and initiative metastatic NOD/SCID mice model) and in vitro (in two separated LAC cell lines) and evaluate the impact of AC093850.2 on migration and invasion. Secondly, we aim to explore how AC093850.2 exerts its regulatory function. Highlighted by the relationship between E-cadherin gene and AC093850.2, we design to investigate the mutual interaction between E-cadherin and AC093850.2 via RIP, ChIP, ChIRP, and rescue experiments.Finally, we design to evaluate the expression level of AC093850.2, EZH2, and E-cadherin in a high through-put human LAC tissue microarray, as well as their correlation with clinical profiles. Our study is the original source of innovation and can provide new potential biomarkers for the prevention and treatment of LAC.

AC093850.2（简称AC）是我们前期应用芯片及二代测序技术筛选获得的一条特异高表达于肺腺癌、功能未知的长链非编码RNA。已发现AC在肺腺癌中高表达程度与淋巴结转移程度正相关；沉默AC可显著抑制肺腺癌细胞侵袭能力；生物信息学分析和实验提示AC与染色质修饰复合物PRC2相互结合；沉默AC后PRC2靶基因E-cad显著上调；临床标本中AC与E-cad表达水平高度负相关。因此我们提出AC招募PRC2调控E-cad等肺癌转移相关基因促进肺腺癌侵袭转移的科学假说。本研究拟采用过表达/沉默策略，结合体内体外实验，论证AC在肺腺癌侵袭转移中的作用；应用RIP、ChIP、ChIRP等技术及拯救实验，深入阐明AC招募PRC2调控E-cad的分子机制；最后大样本病例评价AC、PRC2及E-cad表达在肺腺癌诊疗中的临床价值。有关AC促进肺腺癌侵袭转移的作用和机制未见报道，本研究可望为肺腺癌防治提供新靶标。

背景：AC093850.2（即LINC01614，简称LI）是项目组前期结合肺腺癌表达谱芯片和RNA-seq筛选出的潜在促癌长非编码RNA，迄今尚无研究阐释其在肺腺癌中的功能和分子机制。.内容：①结合生信分析，应用流式分选及qRT-PCR、组织原位杂交染色评价LI在肺腺癌中的表达定位，并结合肺腺癌新鲜组织及组织芯片（TMA），系统评价肿瘤相关成纤维细胞（CAFs）浸润程度、LI表达与肺腺癌侵袭转移、预后的相关性；②分离原代CAFs细胞，在体内外分别探讨LI响肺腺癌侵袭转移、增殖等恶性行为的生物学功能；③结合转录组、代谢组学及生物信息分析，运用沉默/过表达策略及拯救实验阐明CAFs释放外泌体包裹LI传递至肿瘤细胞、通过影响氨基酸代谢促进肺腺癌恶性表型的机制；④应用RNA pull-down、RNA免疫沉淀、免疫共沉淀、染色质共沉淀等实验系统阐明LI调控肺腺癌细胞氨基酸代谢的分子机制。.结果：①肺腺癌中CAFs浸润水平越高，患者生存时间越短（P=0.0004）；②CCLE数据库显示LI几乎不表达于所有肺腺癌细胞系，流式分选及PCR验证显示LI仅高表达于CAFs；TMA原位杂交显示LI表达与淋巴结转移显著正相关（P＜0.001），表达水平越高预后越差（P=0.0018）；③ 沉默CAFs中LI可逆转CAFs在体外对A549细胞株增殖、侵袭迁移的影响；在A549中过表达LI可促进其增殖、侵袭迁移；沉默CAFs中LI后可显著抑制NO-SCID小鼠及斑马鱼模型的体内被动转移；④CAFs可通过释放包裹LI的外泌体进入肿瘤细胞，增加肿瘤细胞摄取L-Gln，从而促进肺癌增殖、侵袭与转移；⑤LI由外泌体传递至A549细胞后，在胞质内可与ANXA2、p65形成三元复合物，激活p65磷酸化促进其入核，进一步转录激活氨基酸转运受体SNAT2、LAT1表达，从而增加肿瘤细胞摄取L-Gln。.结论及意义：本课题首次鉴定了一个肺腺癌CAFs相关、肺腺癌促癌lncRNA—LINC01614，阐明了“CAFs细胞释放包裹LINC01614的外泌体，传递至肿瘤胞质内通过绑定ANXA2及p65、促使p65磷酸化入核、进一步转录激活氨基酸转运受体SNAT2及LAT1、增加肺腺癌L-Gln摄取、从而促进肺腺癌恶性进展”的分子机制。本研究具有源头创新性，可为肺腺癌防治提供潜在新靶标。