Figure 3.

ASOs against miR-21 Altered Global Gene Expression of Tumor Tissue, and DUSP8 Was a New Target of miR-21

Human CRC SW620 cells were injected subcutaneously into the right flank of BALB/c nude mice (n = 10 per group). Seven days later, the plasmid of p-miR-21-ASOs (100 μg) or p-Cont (100 μg) was locally given by subcutaneous injection into the tumor tissues of nude mice four times every 3 days. Six days after the last injection, tumor tissues were collected. Global gene expression was analyzed and matched using cDNA microarray chip. (A) Gene fold change of all upregulation and downregulation genes. (B) Prediction of 18 target genes using MIRDB (http://www.mirdb.org) and TargetScan (http://www.targetscan.org) and (C) calculation of fold change in expression from microarray chip. (D) Expression of five genes, which were upregulated, was determined using real-time PCR. (E) Human CRC SW620 cells were transiently transfected with p-miR-21-ASOs (2.5 μg) or p-Cont (2.5 μg) in vitro. Forty-eight hours later, cells were collected, and expression of the indicated genes was detected using real-time PCR. (F) TargetScan, RNA22-SHA (https://cm.jefferson.edu/rna22/), and MIRDB had intersection elements with microarray chip using Venn analysis. (G) Putative miR-21-5p-binding sites in the 3′-UTR of human DUSP8 and (H) the result of dual luciferase reporter assay were shown. The protein level of DUSP8 in tumor tissues was analyzed using (I) western blot and (J) immunohistochemical staining (200× and 400×). *p < 0.05 and **p < 0.01.