拷贝数变异相关环状RNA circP4HB促进肺腺癌恶性进展的功能与机制研究

Aberrant expression of noncoding RNA caused by genomic alterations has rarely been explored, especially for circular RNA. In the current proposal, by integrating high-throughput ribosomal RNA-depletion sequencing, microarray, and copy number data of lung adenocarcinoma from TCGA, we filter out a copy number variation associated circular RNA, circP4HB. We have found circP4HB is significantly over-expressed in lung adenocarcinoma; circP4HB expression is positively correlated with P4HB gene copy number; high circP4HB expression indicates lymph node metastasis and poor survival of lung adenocarcinoma patients. Silence of circP4HB significantly inhibits cell proliferation and invasion, induces apoptosis, and blocks cell cycle at G1 phase. MS2 pull down followed by mass spectrum discovers circP4HB could bind with a RNA binding protein, FMRP. Intriguingly, target mRNAs were significantly enriched among the differentially expressed genes after circP4HB silence. Based on the abovementioned evidence, we hypothesize “circP4HB might promote malignant progression of lung adenocarcinoma by binding with FMRP”. This study was initially aimed to identify key regulators during lung adenocarcinoma malignant progression, and found an uncharacterized circRNA with critical biological function. This study if of potential novelty and might provide new insight into the diagnosis and treatment of lung adenocarcinoma.

肿瘤基因组变异导致的ncRNA异常表达较少被关注，尤其是环状RNA。申请人通过高通量测序与芯片，结合TCGA拷贝数数据，筛选出一个在肺腺癌中显著高表达的拷贝数变异相关环状RNA，circP4HB。已发现，circP4HB表达与宿主基因拷贝数显著正相关，且circP4HB表达与淋巴结转移、TNM分期、预后相关，敲减circP4HB显著抑制肺腺癌细胞增殖、侵袭能力，并诱导凋亡和细胞周期阻滞。Pull down联合质谱发现circP4HB可以绑定FMRP蛋白，敲减circP4HB后转录组测序发现FMRP蛋白的靶基因被显著富集。因此提出“circP4HB可能通过绑定FMRP蛋白促进肺腺癌恶性进展”的科学假说。本课题从基因组变异角度入手，寻找肺腺癌恶性进展过程中的关键分子，并发现了circP4HB这一尚未被报道、具有重要生物学功能的环状RNA；因此具有源头创新性，将为肺腺癌诊疗提供新的线索。

申请人通过高通量测序与芯片，结合TCGA拷贝数数据，筛选出一个在肺腺癌中显著高表达的拷贝数变异相关环状RNA，circP4HB(hsa_circ_0046263)。结合临床与预后信息，circP4HB表达与宿主基因拷贝数显著正相关且circP4HB表达与淋巴结转移和TNM分期相关，同时具有高表达circP4HB的肺腺癌病人通常具有较短的生存时间。敲减circP4HB显著抑制肺腺癌细胞增殖、迁移和侵袭能力。机制实验上，核质分离实验发现circP4HB主要分布在细胞质中， RNA pull-down联合质谱分析以及RIP实验说明，circP4HB结合丙酮酸激酶异构体2（PKM2）。PKM2是有氧糖酵解过程的关键酶，体外实验证明circP4HB可以在转录水平调节PKM2的表达以及调节PKM2的酶活性，并且促进有氧糖酵解过程。通过联合培养不同circP4HB表达水平的腺癌细胞与THP1细胞说明circP4HB可以通过调节PKM2影响巨噬细胞的极化。综上，circP4HB在肺腺癌中作为癌基因可以作为潜在的治疗靶点。本项目资助发表SCI文章4篇，培养博士后1名，博士生5名（其中4人已毕业），硕士生2名。本项目获得直接经费58万元，支出53.3703万元，结余4.6297万元，支出符合预算。