m6A甲基化介导新发现lncRNA-KCNK15-AS1结合TLN1调控胰腺癌侵袭与转移的作用与机制研究

Long non-coding RNA (lncRNA) plays an important role in the regulation of pancreatic cancer progression. m6A methylation was found to regulate the function of microRNA. which has not been reported in pancreatic cancer related lncRNA. Our results indicated that the expression of a novel lncRNA-KCNK15-AS1 was low in human pancreatic cancer tissue samples, while the methylation degree of m6A was significantly higher than that of normal pancreatic duct epithelial cells. Overexpression of KCNK15-AS1 could suppress the proliferation and migration of pancreatic cancer cells and epithelial mesenchymal transition. Furthermore, KCNK15-AS1 binding proteins TLN1 was screened by RNA pull down-mass spectrometry, but the molecular mechanism of pancreatic cancer invasion and metastasis regulated by KCNK15-AS1 remains unclear. Accordingly, we hypothesize that increased KCNK15-AS1 m6A methylation in pancreatic cancer results in its low expression, and KCNK15-AS1 regulated pancreatic cancer invasion and metastasis through its binding protein TLN1. In order to verify this hypothesis, the project is intended to be carried out by means of clinical pathology, RNA binding protein immunoprecipitation, UV cross-linked RNA binding protein immunoprecipitation, bioinformatics and animal intravital fluorescence imaging,at different levels of molecular, cellular, tissue and animal to investigate the molecular mechanism of KCNK15-AS1 m6A methylation and its binding proteins TLN1 regulating pancreatic cancer invasion and metastasis. This study will reveal the mechanism of pancreatic cancer invasion and metastasis from the new viewpoint of m6A methylation of lncRNA and provide a novel and valuable target for pancreatic cancer treatment.

长链非编码RNA（lncRNA）在调控胰腺癌的发生发展中起重要作用。m6A甲基化可在microRNA中调控其功能，但其调控胰腺癌相关lncRNA未见报道。课题组首次发现lncRNA KCNK15-AS1在人胰腺癌组织样本中低表达，其m6A甲基化程度较正常胰腺显著增高，过表达KCNK15-AS1可抑制胰腺癌细胞增殖、侵袭及上皮间质转化，RNA pulldown质谱筛选到其结合蛋白TLN1，然而KCNK15-AS1调控胰腺癌侵袭转移的机制尚不清楚。我们据此提出假说：胰腺癌中m6A甲基化修饰程度增高导致KCNK15-AS1低表达，其通过结合TLN1调控胰腺癌侵袭转移。本项目拟进一步通过临床病理观察、RNA结合蛋白免疫沉淀及动物活体荧光成像等实验，从分子、细胞、组织及动物水平等多层次探讨KCNK15-AS1的m6A甲基化及TLN1调控胰腺癌侵袭转移具体机制，为胰腺癌治疗提供有价值的新靶点。

越来越多证据表明，表观转录修饰调节多个细胞过程。N6-甲基腺苷（m6A）甲基化修饰是最丰富的，并在癌症发病机制中具有关键作用。然而，长链非编码RNA（lncRNA）m6A甲基化修饰的机制和功能尚不清楚。胰腺癌恶性程度极高，到确诊时，身体其他部位往往已经发生转移。课题组利用lncRNA芯片检测发现lncRNA KCNK15-AS1在胰腺癌组织中表达下调，并且可以抑制胰腺癌细胞迁移和侵袭能力。此外，胰腺癌细胞中总RNA的m6A甲基化水平显著增加，而癌细胞中表达下调的m6A相关分子ALKBH5可使lncRNA KCNK15-AS1去甲基化并调节lncRNA介导的细胞运动。除此以外，lncRNA NT5E和linc01232在胰腺癌中也存在差异表达。综上所述，ALKBH5通过去甲基化lncRNA KCNK15-AS1抑制胰腺癌迁移和侵袭，证实了lncRNA m6A修饰在胰腺癌生发展过程的重要作用，亦为胰腺癌靶向治疗提供了潜在靶点。