Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer

Abstract

Despite recent advances in cancer management, colorectal cancer (CRC) remains the third most common cancer and a major health-care problem worldwide. MicroRNAs have recently emerged as key regulators of cancer development and progression by targeting multiple cancer-related genes; however, such regulatory networks are not well characterized in CRC. Thus, the aim of this study was to perform global messenger RNA (mRNA) and microRNA expression profiling in the same CRC samples and adjacent normal tissues and to identify potential miRNA-mRNA regulatory networks. Our data revealed 1273 significantly upregulated and 1902 downregulated genes in CRC. Pathway analysis revealed significant enrichment in cell cycle, integrated cancer, Wnt (wingless-type MMTV integration site family member), matrix metalloproteinase, and TGF-β pathways in CRC. Pharmacological inhibition of Wnt (using XAV939 or IWP-2) or TGF-β (using SB-431542) pathways led to dose- and time-dependent inhibition of CRC cell growth. Similarly, our data revealed up- (42) and downregulated (61) microRNAs in the same matched samples. Using target prediction and bioinformatics, ~77% of the upregulated genes were predicted to be targeted by microRNAs found to be downregulated in CRC. We subsequently focused on EZH2 (enhancer of zeste homolog 2 ), which was found to be regulated by hsa-miR-26a-5p and several members of the let-7 (lethal-7) family in CRC. Significant inverse correlation between EZH2 and hsa-miR-26a-5p (R(2)=0.56, P=0.0001) and hsa-let-7b-5p (R(2)=0.19, P=0.02) expression was observed in the same samples, corroborating the belief of EZH2 being a bona fide target for these two miRNAs in CRC. Pharmacological inhibition of EZH2 led to significant reduction in trimethylated histone H3 on lysine 27 (H3K27) methylation, marked reduction in cell proliferation, and migration in vitro. Concordantly, small interfering RNA-mediated knockdown of EZH2 led to similar effects on CRC cell growth in vitro. Therefore, our data have revealed several hundred potential miRNA-mRNA regulatory networks in CRC and suggest targeting relevant networks as potential therapeutic strategy for CRC.

Figure 1

Differentially expressed genes in CRC. (a) Hierarchical clustering of 13 CRC and 13 adjacent normal tissue samples based on differentially expressed mRNA levels. Each column represents a sample and each row represents a transcript. Expression level of each gene in a single sample is depicted according to the color scale. (b) Pie chart illustrating the distribution of the top 20 pathway designations for the upregulated genes in colon cancer cells. The pie size corresponds to the number of matched entities. (c) Inhibition of Wnt pathways using XAV 939 and IWP-2 or TGF-β pathway using SB-431542 small-molecule inhibitors led to significant reduction in cell viability in HT115 colon cancer cells. Data are presented as mean±S.E., n=24. (d) Expression levels of selected genes (WNT2, MMP9, EZH2, and BMP3) based on the microarray data and validation of those genes using qRT-PCR (duplicate). **P<0.005; **P<0.0005

Figure 2

miRNA expression profiling in CRC. (a) Hierarchical clustering of 13 colon cancer and 13 normal tissue samples based on miRNA expression levels. Each column represents a sample and each row represents a transcript. Expression level of each miRNA in a single sample is depicted according to the color scale. (b) Pie chart illustrating the distribution of the top 20 pathway designations for predicted targets (TargetScan) for the downregulated miRNAs in colon cancer. The pie size corresponds to the number of matched entities. (c) Venn diagram depicting the overlap between the predicted gene targets for the downregulated miRNAs (based on TargetScan) versus the differentially upregulated genes in CRC identified in the current study. (d) Expression levels of selected miRNAs (hsa-miR-145-5p, hsa-miR-26a-5p, and hsa-miR-30-5p) based on microarray data and validation of those miRNAs using Taqman qRT-PCR (duplicate). **P<0.005; ***P<0.0005

Figure 3

Inhibition of EZH2 using DZNep mediates significant reduction in cell viability and in vitro migration in colon cancer cells. (a) HT115, HT-29, and SW620 cells were treated with the indicated dose of DZNep, and cell viability was measured on days 4 and 8 posttreatment using the alamarBlue assay. Data are presented as mean±S.E., n=8. (b) DZNep treatment (5 days) led to significant reduction in EZH2 protein expression in HT-29 and SW620 cells. Similarly, DZNep treatment led to substantial reduction in H3K273me in the colon cancer cells. *P<0.05, n=2. (c) DZNep treatment led to remarkable reduction in HT115 cell in vitro transwell migration. (d) Hierarchical clustering of HT115 treated with DZNep (5 μM) compared with controls based on mRNA expression levels. Each column represents one replica. Expression level of each gene in a single replica is depicted according to the color scale. (e) Pie chart illustrating the distribution of the top 20 pathway designations for the differentially expressed genes in HT115-DZNep versus control. The pie size corresponds to the number of matched entities

Figure 4

Regulation of EZH2 by hsa-miR-26a and hsa-let-7 family in CRC. (a) Schematic presentation showing miRNAs predicted to target EZH2 using TargetScan database. (b) EZH2 is predicted to be regulated by several members of the let-7 family, miR-26a-5p, and miR-363-3p, which were downregulated in colon cancer. (c) Schematic presentation depicting alignment of Let-7b-5p and miR-26a-5p mature sequence, and the putative binding sites within the 3′-UTR region of the EZH2 mRNA using TargetScan database. The exact positions of the interaction between EZH2 3′-UTR and both miRNA seed regions are indicated. Immunoblotting for EZH2 protein in HT115 transfected with the indicated pre-miRs at 48 h posttransfection. GAPDH was used as a loading control. (d) Inverse relationship between EZH2 and hsa-miR-26a-5p and hsa-let-7b-5p expression in 13 CRC and matched normal tissues. (e) siRNA-mediated knockdown of EZH2 (lower panel) or exogenous expression of hsa-let7-b-5p and hsa-miR-26a-5p (upper panel) led to significant reduction in cell viability in HT115 colon cancer cell. Data are presented as mean±S.E., n=12. *P<0.05; **P<0.005; ***P<0.0005