Gao W, Gu Y, Li Z, Cai H, Peng Q, Tu M, Kondo Y, Shinjo K, Zhu Y, Zhang J, Sekido Y, Han B, Qian Z, Miao Y
Department of General Surgery, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China.
Department of Hepato-Biliary Surgery, The First People's Hospital of Changzhou, Changzhou, China.
Oncogene. 2015 Mar 26;34(13):1629-40. doi: 10.1038/onc.2014.101. Epub 2014 Apr 28.
Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive cancer with a poor prognosis. Although microRNA (miRNA) transcripts have a crucial role in carcinogenesis and development, little information is known regarding the aberrant DNA methylation of miRNAs in PDAC. Using methylated DNA immunoprecipitation-chip analysis, we found that miR-615-5p was hypermethylated in its putative promoter region, which silenced its expression in PDAC cell lines. In addition, the overexpression of miR-615-5p in pancreatic cancer cells suppressed cell proliferation, migration and invasion. Insulin-like growth factor 2 (IGF2) is an imprinted gene, and its abnormal expression contributes to tumor growth. Here, we identified IGF2 as a target of miR-615-5p using a luciferase reporter assay. IGF2 upregulation in PDAC tissues was not correlated with a loss of imprinting but was inversely correlated with miR-615-5p downregulation. In addition, miR-615-5p suppressed pancreatic cancer cell proliferation, migration and invasion by directly targeting IGF2, and this effect could be reversed by co-transfection with IGF2. Furthermore, the stable overexpression of miR-615-5p inhibited tumor growth in vivo and was correlated with IGF2 expression. Using RNA sequencing, we further identified miR-615-5p as potentially targeting other genes, such as the proto-oncogene JUNB, and interfering with the insulin signaling pathway. Taken together, our results demonstrate that miR-615-5p was abnormally downregulated in PDAC cells due to promoter hypermethylation, which limited its inhibition of IGF2 and other target genes, thereby contributing to tumor growth, invasion and migration. These data demonstrate a novel and important role of miR-615-5p as a tumor suppressor in PDAC.
胰腺导管腺癌（PDAC）是一种侵袭性很强且预后较差的癌症。尽管微小RNA（miRNA）转录本在癌症发生和发展过程中起着关键作用，但关于PDAC中miRNA异常DNA甲基化的信息却知之甚少。通过甲基化DNA免疫沉淀芯片分析，我们发现miR-615-5p在其假定的启动子区域高度甲基化，这使其在PDAC细胞系中的表达沉默。此外，miR-615-5p在胰腺癌细胞中的过表达抑制了细胞增殖、迁移和侵袭。胰岛素样生长因子2（IGF2）是一个印记基因，其异常表达促进肿瘤生长。在此，我们通过荧光素酶报告基因实验确定IGF2是miR-615-5p的一个靶标。PDAC组织中IGF2的上调与印记缺失无关，但与miR-615-5p的下调呈负相关。此外，miR-615-5p通过直接靶向IGF2抑制胰腺癌细胞的增殖、迁移和侵袭，并且这种作用可通过与IGF2共转染而逆转。此外，miR-615-5p的稳定过表达在体内抑制肿瘤生长，且与IGF2表达相关。通过RNA测序，我们进一步确定miR-615-5p可能靶向其他基因，如原癌基因JUNB，并干扰胰岛素信号通路。综上所述，我们的结果表明，由于启动子高甲基化，miR-615-5p在PDAC细胞中异常下调，这限制了其对IGF2和其他靶基因的抑制作用，从而促进肿瘤生长、侵袭和迁移。这些数据证明了miR-615-5p作为PDAC肿瘤抑制因子的新的重要作用。
