Section of Gastroenterology, Hepatology, and Nutrition, University of Chicago, Chicago, Illinois; and
Section of Gastroenterology, Hepatology, and Nutrition, University of Chicago, Chicago, Illinois; and.
Am J Physiol Gastrointest Liver Physiol. 2015 Feb 1;308(3):G179-87. doi: 10.1152/ajpgi.00208.2014. Epub 2014 Dec 4.
The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.
结直肠在局部腔环境、排泄功能和基因表达方面存在区域性差异。多顺反子 microRNA (miR)-143 和 miR-145 在结肠癌早期下调。我们想知道这些 microRNAs (miRNAs) 是否在近端结肠与远端结肠之间存在差异表达，从而导致蛋白质表达的区域性差异。通过实时 PCR 定量检测初级转录物和成熟的 miR-143 和 miR-145，通过 Western 印迹法测量推定靶标，并通过测序 bisulfite 处理的近端和远端正常结肠黏膜以及结肠癌中的 DNA 甲基化进行评估。用 miR-143 或 miR-145 转染后评估这些 miRNA 的推定靶标。成熟的 miR-143 和 miR-145 的平均表达在近端结肠分别高出 2.0 倍（P < 0.001）和 1.8 倍（P = 0.03）。这些 miRNA 的 DNA 甲基化或初级转录物表达没有位置差异。与 miR-143 和 miR-145 在近端结肠中的表达增加一致，这些 miRNA 的预测靶标，凋亡抑制剂 5（API5）、ERK5、K-RAS 和胰岛素受体底物 1（IRS-1），是细胞周期和存活调节剂，在近端结肠的表达水平低于远端结肠。用 miR-145 转染 HCA-7 结肠癌细胞下调 IRS-1，用 miR-143 转染 HT-29 结肠癌细胞降低 K-RAS 和 ERK5 的表达。总之，miR-143 和 miR-145 以及预测的靶蛋白 API5、ERK5、K-RAS 和 IRS-1 在结肠中表达存在区域性差异。我们推测，这些肿瘤抑制因子的差异可能导致正常结肠基因表达的区域性差异，并调节恶性易感性的特定部位差异。
