一个功能未知的长链非编码RNA PIK3CD-AS2调控非小细胞肺癌侵袭转移的机制研究

Using microarray analysis and big data from TCGA project, we identified a novel long noncoding RNA (lncRNA), PIK3CD-AS2 (PI), with unknown function that is highly expressed in non-small cell lung cancer (NSCLC) tissues. Our previous work has demonstrated that PI is significantly overexpressed in in NSCLC tissues, positively correlated with lymph node metastasis, and negatively correlated with prognosis. Knockdown of PI significantly inhibited migration and invasion of NSCLC cells. By mining of bid data and experiment, we found that ECT2 could be the downstream target of PI and we hypothesized that PI could promote the invasion and metastasis of NSCLC by epigenetic activation of ECT2 via binding to WDR5, a core subunit of histone 3 lysine 4 methyltransferase. Here, we plan to validate our hypothesis by assess the relationship between PI expression level and NSCLC metastasis; investigate the influence of PI on invasion ability of NSCLC both in vivo and in vitro in over-expression/knockdown manner. The rescue experiment along with chromatin immunoprecipitation, RNA immunoprecipitation, and RNA pull down assays were designed to underlying molecular mechanism by which PI epigenetically regulates ECT2 expression and promotes NSCLC invasion. There is no report about PI function so far and our study offers a new therapeutic target for the diagnosis and treatment of NSCLC.

PIK3CD-AS2（简称PI）是前期应用芯片和TCGA大数据挖掘筛选到的一条在人非小细胞肺癌（简称肺癌）组织中差异高表达、功能未知的长非编码RNA。前期发现PI在肺癌组织中显著高表达，与淋巴结转移程度正相关，且表达越高预后越差；沉默PI后可显著抑制肺癌细胞株侵袭转移能力；进一步预实验和生物信息学分析提示癌基因ECT2是PI潜在下游靶点，PI可能通过结合甲基转移酶核心亚基WDR5表观上调ECT2表达、促进肺癌侵袭转移。故我们提出PI招募WDR5表观上调ECT2促进肺癌侵袭转移的科学假说。本研究拟临床评价PI与肺癌侵袭转移的关系；结合过表达/沉默策略，在细胞和动物水平共同评价PI调控肺癌侵袭转移的生物学功能；设计拯救实验，应用ChIP、RIP等技术，阐明PI通过表观调控ECT2表达、促进肺癌侵袭转移的分子机制。有关PI促进肺癌侵袭转移的功能和机制尚未见报道，本研究将可为肺癌防治提供新靶标。

PIK3CD-AS2是项目组前期应用芯片和TCGA大数据挖掘筛选到的一条在人非小细胞肺癌组织中差异高表达、功能未知的长非编码RNA。通过TCGA、GEO数据库分析和肺腺癌组织芯片验证，结果表明PIK3CD-AS2在肺腺癌组织中显著高表达，与肿瘤大小、病理分级相关，且表达越高预后越差。沉默PIK3CD-AS2后可显著抑制肺腺癌细胞株增殖、侵袭和迁移，促进凋亡；抑制裸鼠皮下荷瘤动物模型肿瘤生长。PIK3CD-AS2全长958nt，FISH实验提示其大多定位在细胞浆，少量分布在细胞核。进一步采用RNA-seq检测PIK3CD-AS2沉默后A549细胞差异表达基因，Go和KEGG pathway分析，核酸、蛋白及拯救实验验证，提示PIK3CD-AS2通过调控p53信号通路影响肺腺癌恶性进展。RNA pull down、RIP、co-IP和western blot实验表明PIK3CD-AS2与YBX1蛋白相互结合，且抑制YBX1泛素化降解，稳定蛋白表达。YBX1是p53蛋白负性调控因子，通过与p53直接结合抑制p53下游靶基因转录。功能实验结果显示，过表达/沉默YBX1可以拯救PIK3CD-AS2导致的肺腺癌细胞恶性表型。进一步在人来源的p53野生型肺腺癌PDX模型中验证PIK3CD-AS2/YBX1/p53轴作用机制，发现沉默PIK3CD-AS2后，肿瘤生长减慢，体积变小，凋亡增加，YBX1表达减少，p53表达增多。在TCGA数据库中筛选p53野生型及突变型肺癌患者，比较PIK3CD-AS2表达水平可见，p53突变型患者PIK3CD-AS2表达更高，且高表达PIK3CD-AS2与患者预后相关。有关PIK3CD-AS2促进肺腺癌恶性进展的功能和机制尚未见报道，本研究将可为肺癌防治提供新靶标。