Meg3 binding sites in mouse insulinoma cells

We have shown that increased ß-cell proliferation in functioning pancreatic neuroendocrine tumors (insulinomas) correlated with reduced expression of the long non-coding RNA Meg3 and increased expression of the oncogenic receptor c-Met. To investigate the target binding sites of Meg3 in and around the c-Met gene, we did ChIRP-Seq using biotinylated probes from the mouse Meg3 RNA sequence. This would help us better understand how Meg3 regulates ithe expression of c-Met to control ß-cell proliferation in insulinoma cells. Overall design: Glutaraldehyde cross-linked chromatin was prepared from 10 million cells of a mouse pancreatic islet ß-cell line (MIN6-4N) stably transfected with Vector or stably overexpressing Meg3. Chromatin isolation by RNA purification (ChIRP) as performed using 2 pools of odd-numbered or even-numbered biotinylated probes from the mouse Meg3 RNA sequence using standard protocols (Sigma). ChIRP-Seq libraries (input, odd probe and even probe) were prepared with Diagenode''s MicroPlex Library Preparation kit and IP-Star Compact protocol. ChIRP-Seq libraries were sequenced for single-end 50 bp, and sequences were analyzed (NIDDK genomics core facility and Genomatix).

我们已证实，功能性胰腺神经内分泌肿瘤（胰岛素瘤，insulinomas）中β细胞增殖的增强，与长链非编码RNA（long non-coding RNA）Meg3的表达下调以及致癌性受体c-Met的表达上调显著相关。为探究Meg3在c-Met基因及其侧翼区域的靶结合位点，我们采用针对小鼠Meg3 RNA序列设计的生物素标记探针开展了ChIRP-Seq实验，以期更清晰地阐明Meg3如何通过调控c-Met的表达，进而控制胰岛素瘤细胞内的β细胞增殖。整体实验设计：从分别稳定转染空载体或稳定过表达Meg3的小鼠胰岛β细胞系（MIN6-4N）的1000万细胞中，制备戊二醛交联的染色质。按照标准实验流程（Sigma提供），使用针对小鼠Meg3 RNA序列设计的奇数组与偶数组生物素标记探针池，完成染色质RNA纯化（ChIRP）实验。使用Diagenode公司的MicroPlex文库制备试剂盒与IP-Star Compact实验流程，构建ChIRP-Seq文库（包含输入组、奇数探针组与偶数探针组）。对上述ChIRP-Seq文库进行单端50bp测序，并由NIDDK基因组核心设施与Genomatix完成序列数据分析。