Purpose: Cancer stroma plays an important role in the progression of cancer. Although alterations in miRNA expression have been explored in various kinds of cancers, the expression of miRNAs in cancer stroma has not been explored in detail.

Experimental Design: Using a laser microdissection technique, we collected RNA samples specific for epithelium or stroma from 13 colorectal cancer tissues and four normal tissues, and miRNA microarray and gene expression microarray were carried out. The expression status of miRNAs was confirmed by reverse transcriptase PCR. Furthermore, we investigated whether miRNA expression status in stromal tissue could influence the clinicopathologic factors.

Results: Oncogenic miRNAs, including two miRNA clusters, miR-17-92a and miR-106b-25 cluster, were upregulated in cancer stromal tissues compared with normal stroma. Gene expression profiles from cDNA microarray analyses of the same stromal tissue samples revealed that putative targets of these miRNA clusters, predicted by Target Scan, such as TGFBR2, SMAD2, and BMP family genes, were significantly downregulated in cancer stromal tissue. Downregulated putative targets were also found to be involved in cytokine interaction and cellular adhesion. Importantly, expression of miR-25 and miR-92a in stromal tissues was associated with a variety of clinicopathologic factors.

Conclusions: Oncogenic miRNAs were highly expressed in cancer stroma. Although further validation is required, the finding that stromal miRNA expression levels were associated with clinicopathologic factors suggests the possibility that miRNAs in cancer stroma are crucially involved in cancer progression. Clin Cancer Res; 18(11); 3054–70. ©2012 AACR.

Translational Relevance

Cancer stroma plays a critical role in cancer progression. To investigate the role of miRNAs in colorectal cancer stroma, we carried out epithelial and stromal tissue-specific miRNA microarray analyses using a laser microdissection technique. We found that oncogenic miRNAs, including the miR-17-92a cluster and the miR-106b-25 cluster, which are known to be involved in cancer progression in epithelial tissue, were significantly upregulated in cancer stromal tissue compared with normal stroma. Gene expression profiles from cDNA microarray analyses of the same stromal tissue samples revealed that putative targets of these miRNA clusters, predicted by TargetScan, such as TGFBR2, SMAD2, and BMP family genes, were significantly downregulated in cancer stromal tissue. Furthermore, miRNA expression in colorectal cancer stroma was associated with a number of clinicopathologic factors. Although further validation is required, these findings suggest the possibility that miRNAs in stromal tissues are functionally associated with cancer progression.

Cancer tissues consist of cancer cells and surrounding stromal cells, including inflammatory cells, immunocompetent cells, endothelial cells, and fibroblasts. Cancer stroma interacts with cancer tissues directly or indirectly through cytokines, creating a niche for the cancer cells. Recent studies have focused on altered expression of oncogenes and tumor suppressor genes in stromal tissues. Kurose and colleagues reported that the downregulation of PTEN and p53 is a key step in breast cancer progression, and other reports have indicated that ablation of TGFBR2 in fibroblasts can lead to carcinogenesis in vivo (1–3).

With regard to clinical aspects, a number of studies have revealed the gene expression status of cancer stroma and its correlation to prognosis as well as clinicopathologic factors (4, 5). Particularly, Finak and colleagues analyzed global gene expression patterns in breast cancer stromal tissues and identified gene sets that potently influenced prognosis (6). They revealed that the aggressiveness of cancer could be defined by gene expression patterns in stromal tissue. Moreover, Fukino and colleagues revealed that cancer-specific LOH or allelic imbalance in stromal cells is more highly correlated with clinicopathologic features than that in epithelial cells (7). These findings suggest that cancer stromal tissues are actively involved in cancer progression.

miRNAs constitute a class of small (19–25 nucleotides) noncoding RNAs that function as posttranscriptional gene regulators by binding to their target mRNAs (8). Alterations in miRNA expression are reported in various kinds of human cancers, suggesting miRNAs function both as tumor suppressors and oncogenes in cancer development (9). Genetic alterations in cancer tissues are likely dependent largely on the expression status of miRNAs (9). We considered the possibility that gene expression in cancer stroma is also regulated by miRNAs expressed in cancer stroma.

Conventional gene expression analysis using bulk tumor samples could not reveal the gene and miRNA expression profiles in cancer stroma. In this study, using a laser microdissection (LMD) method, we collected epithelium-specific and stoma-specific RNAs, and investigated the miRNA and gene expression profiles. By analyzing many kinds of microarray data, including that of epithelium, stroma, normal, and cancer, we show how miRNAs in cancer stroma are involved in cancer progression.

Clinical samples

Tissues from 13 cases of colorectal cancer and 4 normal colorectal tissues (located more than 5 cm from the colorectal cancer) were obtained during surgery. All patients underwent resection of the primary tumor at Kyushu University Hospital at Beppu and affiliated hospitals between 1993 and 2006. Written informed consent was obtained from all patients, and the study protocol was approved by the local ethics committee. Detailed information is described in Supplementary Data.

Laser microdissection

Tissue samples were microdissected using the LMD system LMD6000 (Leica Laser Microdissection System; Leica Microsystems) as previously described (10). Detailed protocols are described in Supplementary Information.

To show the accuracy of LMD and RNA separation, we presented tissue section before (left) and after (right) LMD in 6 representative samples of colorectal cancer (Supplementary Fig. S1).

miRNA microarray

Total RNAs from epithelial and stromal tissues of cancer and normal samples were analyzed by miRNA microarray. Total RNA was extracted from tissue using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Concentrations and purities of the total RNAs were assessed with a spectrophotometer and RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies). OD260/OD280 ratios of 1.8 to 2.1 were accepted to be adequate for microarray. Total RNA (100 ng) was directly labeled with cyanine 3-CTP (Cy3), without fractionation or amplification, using an Agilent protocol that produces precise and accurate measurements spanning a linear dynamic range from 0.2 amol to 2 fmol of input miRNA. Each total RNA sample (100 ng) was competitively hybridized to a miRNA array (Agilent Microarray Design ID = 014947, Early Access version) containing 455 miRNAs [version 15 of the Sanger miRNA database (http://www.mirbase.org/)], according to the manufacturer's protocol (11). The intensity of each hybridization signal was evaluated using Extraction Software Version A.7.5.1 (Agilent Technologies), which used the locally weighted linear regression curve fit (LOWESS) normalization method (12).The robust multiarray average algorithm normalization method was also used for analysis in downregulated miRNAs in cancer stroma (13, 14). miRNA arrays have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database with accession code GSE35602.

cDNA microarray

We used the commercially available Human Whole Genome Oligo DNA Microarray Kit (Agilent Technologies). A list of genes on this cDNA microarray is available from http://www.chem.agilent.com. Cyanine (Cy)-labeled cRNA was prepared using T7 linear amplification as described in the Agilent Low RNA Input Fluorescent Linear Amplification Kit Manual (Agilent Technologies). Labeled cRNA was fragmented and hybridized to an oligonucleotide microarray (Whole Human Genome 4 × 44 K Agilent G4112F). Fluorescence intensities were determined with an Agilent DNA Microarray Scanner and were analyzed using G2567AA Feature Extraction Software Version A.7.5.1 (Agilent Technologies), which used the LOWESS normalization method (12). This microarray study followed MIAME guidelines issued by the Microarray Gene Expression Data group (15). Gene expression arrays have been deposited in the NCBI GEO database with accession code GSE35602.

Gene ontology analysis and miRNA target prediction

A total of 1,939 Gene set enrichment analyses of differentially expressed genes were carried out using Gene Codis version 2.0 (16, 17). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (18) was used for systematic analysis of gene functions. TargetScan (19, 20) Version 5.1 algorithm was used to predict putative targets of each miRNA. TargetScan focuses on the exact match to 7 bases or more (hexamer match in positions 2–7, plus an adenosine at a position on the 3′ side) of the miRNA seed sequence in the 3′-untranslated region of target messenger RNA. In our analysis, we included all targets that met TargetScan criteria without taking into account evolutionary conservation.

Quantitative real-time reverse transcriptase PCR

For miR-25 and miR-92a quantitative real-time reverse transcriptase PCR (qRT-PCR), cDNA was synthesized from 10 ng of total RNA using TaqMan miRNA hsa-miR-25- or 92a-specific primers (Applied Biosystems) and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). RT-PCR protocols are described in Supplementary Information.

Statistical analysis

Differences between groups were estimated using the χ2 test and Student t test. After expression signals were calculated by log2 transformation of the normalized data, differentially expressed miRNAs and genes were detected by using the fold-change value and Q value. We used the significance analysis of microarrays method in the “samr” package of the R language (http://www.r-project.org/). RT-PCR data were analyzed using JMP software (SAS Institute Inc.). All differences were considered statistically significant at the level of P < 0.05 or Q < 0.05.

The miR-17-92a cluster and miR-106b-25 cluster were upregulated in cancer stroma compared with normal stroma

To investigate the functions of miRNAs in cancer stroma, we carried out miRNA microarray analyses with samples of 13 cancer stromal tissue and 4 samples of normal stromal tissue. Those miRNAs that were significantly upregulated in cancer stromal tissues compared with normal stromal tissues are listed in Table 1 (fold change >1.5, and Q < 0.05). For example, oncogenic miRNAs, including miR-21, miR-221, and almost all components of the oncogenic miR-17-92a cluster and miR-106b-25 cluster except miR-19b were upregulated in cancer stromal tissue compared with normal stromal tissue (Table 1). Unsupervised hierarchical clustering of normal and cancerous stromal samples revealed that all components of the miR-17-92a cluster and the miR-106b-25 cluster (except for miR-92a) were classified in the same cluster (Supplementary Fig. S2A and S2B). MiR-92a was classified in another cluster, perhaps because miR-92a has a homolog (miR-92a-2) on chromosome X, besides miR-92a-1 in the miRNA cluster on chromosome 13 (21). Therefore, its expression could have diverged from the expression of other miRNAs in the cluster.

Table 1.

Upregulated miRNAs in colorectal cancer stromal tissue compared with normal stromal tissue

Systematic nameSignal in cancer stromal tissue (log2)Signal in normal stromal tissue (log2)Fold changeQ (%)
hsa-miR-214 7.3612 5.1633 4.5882 0.0000 
hsa-miR-21 13.7414 11.7651 3.9348 0.0000 
hsa-miR-455-3p 5.0351 3.4322 3.0376 0.0000 
hsa-miR-663 4.4278 2.7578 3.1820 0.0000 
hsa-miR-127-3p 3.7355 1.9303 3.4947 0.0000 
hsa-miR-92a 7.2865 5.7584 2.8842 0.0000 
hsa-miR-381 3.9035 2.5478 2.5593 0.0000 
hsa-miR-93 5.5042 4.0246 2.7886 0.0000 
hsa-miR-224 4.5767 2.2308 5.0840 0.0000 
hsa-miR-432 2.6401 1.3709 2.4102 0.0000 
hsa-miR-221 5.1187 3.6628 2.7433 0.0000 
hsa-miR-125b 7.7959 5.7433 4.1486 0.0000 
hsa-miR-17 6.3813 4.9449 2.7064 0.0000 
hsa-miR-337-5p 2.8150 1.4391 2.5952 0.0000 
hsa-miR-502-3p 1.8047 0.5097 2.4538 0.0000 
hsa-miR-300 3.6767 2.3557 2.4985 0.0000 
hsa-miR-128 2.5359 1.3263 2.3127 0.0000 
hsa-miR-532-5p 2.7469 1.5402 2.3080 0.0000 
hsa-miR-609 3.8840 2.8980 1.9806 0.0000 
hsa-miR-130b 4.1915 2.7904 2.6409 0.0000 
hsa-miR-18a 3.7635 1.3688 5.2588 0.0000 
hsa-miR-181d 3.1301 2.0477 2.1175 0.0000 
hsa-miR-362-5p 3.2330 2.0853 2.2156 0.0000 
hsa-miR-424 7.7230 5.9653 3.3816 0.0000 
hsa-miR-152 3.1990 1.6066 3.0156 0.0000 
hsa-miR-654-3p 4.3411 2.6129 3.3130 0.0000 
hsa-miR-149 3.8869 2.7086 2.2632 0.0000 
hsa-miR-7 4.7678 2.8344 3.8196 0.0000 
hsa-miR-485-3p 5.3006 4.2385 2.0880 0.0000 
hsa-miR-99b 3.9191 2.6203 2.4603 0.0000 
hsa-miR-933 2.7300 1.7161 2.0193 0.0000 
hsa-miR-615-3p 3.8335 2.6442 2.2804 0.0000 
hsa-miR-640 2.9456 1.4328 2.8535 0.0000 
hsa-miR-483-3p 6.2669 5.0075 2.3940 0.0000 
hsa-miR-605 3.8121 2.5917 2.3301 0.0000 
hsa-miR-135b 3.8255 0.7653 8.3408 0.0000 
hsa-miR-296-5p 1.9389 1.1457 1.7329 0.0000 
hsa-miR-647 3.8730 2.1903 3.2102 0.0000 
hsa-miR-1236 3.4103 1.8925 2.8635 0.0000 
hsa-miR-34b 4.2683 2.3956 3.6620 0.0000 
hsa-miR-592 2.5548 0.8012 3.3721 0.4839 
hsa-miR-25 5.9887 4.7911 2.2936 0.4839 
hsa-let-7i 8.6952 7.6400 2.0781 0.4839 
hsa-miR-20b 5.5041 4.2857 2.3269 0.4839 
hsa-miR-130a 7.3585 6.0856 2.4165 0.4839 
hsa-miR-199a-5p 7.4083 5.9666 2.7163 0.4839 
hsa-miR-1238 4.2775 3.5734 1.6291 0.4839 
hsa-miR-331-3p 6.6993 5.5074 2.2846 0.4839 
hsa-miR-24 9.5984 8.5492 2.0693 0.4839 
hsa-miR-20a 7.5321 6.3601 2.2532 0.4839 
hsa-miR-23a 10.0837 9.0655 2.0254 0.4839 
hsa-miR-199b-5p 6.7800 5.3520 2.6907 0.4839 
hsa-miR-181b 4.6692 3.7467 1.8955 0.4839 
hsa-miR-377 4.4965 3.2165 2.4283 0.4839 
hsa-miR-18b 2.5247 0.9391 3.0013 0.4839 
hsa-miR-539 2.0809 1.0446 2.0510 0.4839 
hsa-miR-22 9.7884 8.6262 2.2379 0.4839 
hsa-miR-223 8.8473 7.7969 2.0712 0.4839 
hsa-miR-491-3p 2.5471 0.9916 2.9393 0.4839 
hsa-miR-125a-5p 5.6168 4.5834 2.0469 0.4839 
hsa-miR-299-5p 3.1356 1.7790 2.5607 0.4839 
hsa-miR-483-5p 4.9531 3.9757 1.9689 0.4839 
hsa-miR-379 2.4551 0.4574 3.9938 0.4839 
hsa-miR-1234 5.0701 4.3136 1.6894 0.4839 
hsa-miR-99a 3.9888 2.3726 3.0657 0.4839 
hsa-miR-1225-3p 3.9937 3.3510 1.5613 0.4839 
hsa-miR-495 2.3389 0.7363 3.0369 0.4839 
hsa-miR-574-5p 7.1083 6.2371 1.8292 0.4839 
hsa-miR-125a-3p 3.2257 2.3094 1.8873 0.4839 
hsa-miR-326 1.9496 0.8913 2.0825 0.4839 
hsa-miR-1224-3p 4.8757 3.6868 2.2798 0.4839 
hsa-miR-1229 3.7613 2.5318 2.3448 0.4839 
hsa-miR-937 4.9785 3.8112 2.2459 0.4839 
hsa-miR-574-3p 7.9370 7.0906 1.7979 0.4839 
hsa-miR-1237 2.8475 2.0403 1.7498 0.4839 
hsa-miR-206 3.0294 1.7743 2.3868 0.4839 
hsa-miR-129-3p 1.7338 0.7030 2.0432 0.4839 
hsa-miR-885-5p 6.2987 5.1769 2.1761 0.4839 
hsa-miR-1227 4.6286 3.0407 3.0061 0.4839 
hsa-miR-631 4.3710 2.9772 2.6277 0.4839 
hsa-miR-487b 2.2864 0.5419 3.3507 0.8249 
hsa-miR-543 1.4302 0.1764 2.3847 0.8249 
hsa-miR-365 6.3121 5.3710 1.9200 0.8249 
hsa-let-7e 6.8754 5.9369 1.9166 0.8249 
hsa-miR-151-3p 3.8653 2.8896 1.9666 0.8249 
hsa-miR-106b 6.7077 5.7538 1.9371 0.8249 
hsa-miR-301a 4.3942 3.3753 2.0263 0.8249 
hsa-miR-382 2.2284 0.8661 2.5709 0.8249 
hsa-miR-100 4.7280 3.4939 2.3523 0.8249 
hsa-miR-452 2.3510 1.1210 2.3456 0.8249 
hsa-miR-371-5p 2.2495 1.1375 2.1614 0.8249 
hsa-miR-765 4.3601 3.6031 1.6900 0.8249 
hsa-miR-362-3p 2.5044 1.5707 1.9102 0.8249 
hsa-miR-183 1.8260 0.5658 2.3953 0.8249 
hsa-miR-423-5p 3.8130 3.0448 1.7032 0.8249 
hsa-miR-328 6.1455 5.1186 2.0377 0.8249 
hsa-miR-595 4.4023 3.3593 2.0605 0.8249 
hsa-miR-542-3p 2.8903 1.2498 3.1177 0.8249 
hsa-miR-550 1.9877 1.2773 1.6363 0.8249 
hsa-miR-634 2.1901 1.4710 1.6462 0.8249 
hsa-miR-188-5p 4.1637 3.5119 1.5711 1.3079 
hsa-miR-450a 2.6452 1.0431 3.0358 1.3079 
hsa-miR-181c 2.7493 1.7464 2.0040 1.3079 
hsa-miR-126 8.1419 7.2671 1.8338 1.3079 
hsa-miR-484 3.8659 3.2581 1.5239 1.3079 
hsa-miR-95 2.2341 1.4163 1.7627 1.3079 
hsa-miR-19a 7.0755 6.2318 1.7946 1.3079 
hsa-miR-181a 6.7619 5.9594 1.7441 1.3079 
hsa-miR-376a 4.3021 3.2870 2.0211 1.3079 
hsa-miR-425 4.4042 3.5379 1.8230 1.3079 
hsa-miR-455-5p 1.9466 0.6533 2.4510 1.3079 
hsa-miR-324-3p 5.1796 4.5422 1.5555 1.3079 
hsa-miR-622 2.5718 1.6357 1.9133 1.3079 
hsa-miR-613 1.6581 0.9437 1.6408 1.3079 
hsa-miR-575 3.9488 3.2521 1.6208 1.3079 
hsa-miR-222 3.2655 2.5148 1.6827 1.3079 
hsa-miR-32 2.5684 1.6119 1.9406 1.3079 
hsa-miR-197 7.9098 7.0983 1.7551 1.3079 
hsa-miR-766 6.9197 6.0107 1.8777 1.3079 
hsa-miR-876-5p 1.3810 0.6503 1.6595 1.9848 
hsa-miR-505 2.8023 2.0524 1.6817 1.9848 
hsa-let-7c 7.3815 6.6304 1.6831 1.9848 
hsa-miR-98 4.5308 3.7445 1.7247 1.9848 
hsa-miR-27a 9.1504 8.3246 1.7725 1.9848 
hsa-miR-324-5p 4.1229 3.2487 1.8330 1.9848 
hsa-miR-874 3.6715 3.0471 1.5416 1.9848 
hsa-miR-133a 1.3162 0.6960 1.5371 1.9848 
Systematic nameSignal in cancer stromal tissue (log2)Signal in normal stromal tissue (log2)Fold changeQ (%)
hsa-miR-214 7.3612 5.1633 4.5882 0.0000 
hsa-miR-21 13.7414 11.7651 3.9348 0.0000 
hsa-miR-455-3p 5.0351 3.4322 3.0376 0.0000 
hsa-miR-663 4.4278 2.7578 3.1820 0.0000 
hsa-miR-127-3p 3.7355 1.9303 3.4947 0.0000 
hsa-miR-92a 7.2865 5.7584 2.8842 0.0000 
hsa-miR-381 3.9035 2.5478 2.5593 0.0000 
hsa-miR-93 5.5042 4.0246 2.7886 0.0000 
hsa-miR-224 4.5767 2.2308 5.0840 0.0000 
hsa-miR-432 2.6401 1.3709 2.4102 0.0000 
hsa-miR-221 5.1187 3.6628 2.7433 0.0000 
hsa-miR-125b 7.7959 5.7433 4.1486 0.0000 
hsa-miR-17 6.3813 4.9449 2.7064 0.0000 
hsa-miR-337-5p 2.8150 1.4391 2.5952 0.0000 
hsa-miR-502-3p 1.8047 0.5097 2.4538 0.0000 
hsa-miR-300 3.6767 2.3557 2.4985 0.0000 
hsa-miR-128 2.5359 1.3263 2.3127 0.0000 
hsa-miR-532-5p 2.7469 1.5402 2.3080 0.0000 
hsa-miR-609 3.8840 2.8980 1.9806 0.0000 
hsa-miR-130b 4.1915 2.7904 2.6409 0.0000 
hsa-miR-18a 3.7635 1.3688 5.2588 0.0000 
hsa-miR-181d 3.1301 2.0477 2.1175 0.0000 
hsa-miR-362-5p 3.2330 2.0853 2.2156 0.0000 
hsa-miR-424 7.7230 5.9653 3.3816 0.0000 
hsa-miR-152 3.1990 1.6066 3.0156 0.0000 
hsa-miR-654-3p 4.3411 2.6129 3.3130 0.0000 
hsa-miR-149 3.8869 2.7086 2.2632 0.0000 
hsa-miR-7 4.7678 2.8344 3.8196 0.0000 
hsa-miR-485-3p 5.3006 4.2385 2.0880 0.0000 
hsa-miR-99b 3.9191 2.6203 2.4603 0.0000 
hsa-miR-933 2.7300 1.7161 2.0193 0.0000 
hsa-miR-615-3p 3.8335 2.6442 2.2804 0.0000 
hsa-miR-640 2.9456 1.4328 2.8535 0.0000 
hsa-miR-483-3p 6.2669 5.0075 2.3940 0.0000 
hsa-miR-605 3.8121 2.5917 2.3301 0.0000 
hsa-miR-135b 3.8255 0.7653 8.3408 0.0000 
hsa-miR-296-5p 1.9389 1.1457 1.7329 0.0000 
hsa-miR-647 3.8730 2.1903 3.2102 0.0000 
hsa-miR-1236 3.4103 1.8925 2.8635 0.0000 
hsa-miR-34b 4.2683 2.3956 3.6620 0.0000 
hsa-miR-592 2.5548 0.8012 3.3721 0.4839 
hsa-miR-25 5.9887 4.7911 2.2936 0.4839 
hsa-let-7i 8.6952 7.6400 2.0781 0.4839 
hsa-miR-20b 5.5041 4.2857 2.3269 0.4839 
hsa-miR-130a 7.3585 6.0856 2.4165 0.4839 
hsa-miR-199a-5p 7.4083 5.9666 2.7163 0.4839 
hsa-miR-1238 4.2775 3.5734 1.6291 0.4839 
hsa-miR-331-3p 6.6993 5.5074 2.2846 0.4839 
hsa-miR-24 9.5984 8.5492 2.0693 0.4839 
hsa-miR-20a 7.5321 6.3601 2.2532 0.4839 
hsa-miR-23a 10.0837 9.0655 2.0254 0.4839 
hsa-miR-199b-5p 6.7800 5.3520 2.6907 0.4839 
hsa-miR-181b 4.6692 3.7467 1.8955 0.4839 
hsa-miR-377 4.4965 3.2165 2.4283 0.4839 
hsa-miR-18b 2.5247 0.9391 3.0013 0.4839 
hsa-miR-539 2.0809 1.0446 2.0510 0.4839 
hsa-miR-22 9.7884 8.6262 2.2379 0.4839 
hsa-miR-223 8.8473 7.7969 2.0712 0.4839 
hsa-miR-491-3p 2.5471 0.9916 2.9393 0.4839 
hsa-miR-125a-5p 5.6168 4.5834 2.0469 0.4839 
hsa-miR-299-5p 3.1356 1.7790 2.5607 0.4839 
hsa-miR-483-5p 4.9531 3.9757 1.9689 0.4839 
hsa-miR-379 2.4551 0.4574 3.9938 0.4839 
hsa-miR-1234 5.0701 4.3136 1.6894 0.4839 
hsa-miR-99a 3.9888 2.3726 3.0657 0.4839 
hsa-miR-1225-3p 3.9937 3.3510 1.5613 0.4839 
hsa-miR-495 2.3389 0.7363 3.0369 0.4839 
hsa-miR-574-5p 7.1083 6.2371 1.8292 0.4839 
hsa-miR-125a-3p 3.2257 2.3094 1.8873 0.4839 
hsa-miR-326 1.9496 0.8913 2.0825 0.4839 
hsa-miR-1224-3p 4.8757 3.6868 2.2798 0.4839 
hsa-miR-1229 3.7613 2.5318 2.3448 0.4839 
hsa-miR-937 4.9785 3.8112 2.2459 0.4839 
hsa-miR-574-3p 7.9370 7.0906 1.7979 0.4839 
hsa-miR-1237 2.8475 2.0403 1.7498 0.4839 
hsa-miR-206 3.0294 1.7743 2.3868 0.4839 
hsa-miR-129-3p 1.7338 0.7030 2.0432 0.4839 
hsa-miR-885-5p 6.2987 5.1769 2.1761 0.4839 
hsa-miR-1227 4.6286 3.0407 3.0061 0.4839 
hsa-miR-631 4.3710 2.9772 2.6277 0.4839 
hsa-miR-487b 2.2864 0.5419 3.3507 0.8249 
hsa-miR-543 1.4302 0.1764 2.3847 0.8249 
hsa-miR-365 6.3121 5.3710 1.9200 0.8249 
hsa-let-7e 6.8754 5.9369 1.9166 0.8249 
hsa-miR-151-3p 3.8653 2.8896 1.9666 0.8249 
hsa-miR-106b 6.7077 5.7538 1.9371 0.8249 
hsa-miR-301a 4.3942 3.3753 2.0263 0.8249 
hsa-miR-382 2.2284 0.8661 2.5709 0.8249 
hsa-miR-100 4.7280 3.4939 2.3523 0.8249 
hsa-miR-452 2.3510 1.1210 2.3456 0.8249 
hsa-miR-371-5p 2.2495 1.1375 2.1614 0.8249 
hsa-miR-765 4.3601 3.6031 1.6900 0.8249 
hsa-miR-362-3p 2.5044 1.5707 1.9102 0.8249 
hsa-miR-183 1.8260 0.5658 2.3953 0.8249 
hsa-miR-423-5p 3.8130 3.0448 1.7032 0.8249 
hsa-miR-328 6.1455 5.1186 2.0377 0.8249 
hsa-miR-595 4.4023 3.3593 2.0605 0.8249 
hsa-miR-542-3p 2.8903 1.2498 3.1177 0.8249 
hsa-miR-550 1.9877 1.2773 1.6363 0.8249 
hsa-miR-634 2.1901 1.4710 1.6462 0.8249 
hsa-miR-188-5p 4.1637 3.5119 1.5711 1.3079 
hsa-miR-450a 2.6452 1.0431 3.0358 1.3079 
hsa-miR-181c 2.7493 1.7464 2.0040 1.3079 
hsa-miR-126 8.1419 7.2671 1.8338 1.3079 
hsa-miR-484 3.8659 3.2581 1.5239 1.3079 
hsa-miR-95 2.2341 1.4163 1.7627 1.3079 
hsa-miR-19a 7.0755 6.2318 1.7946 1.3079 
hsa-miR-181a 6.7619 5.9594 1.7441 1.3079 
hsa-miR-376a 4.3021 3.2870 2.0211 1.3079 
hsa-miR-425 4.4042 3.5379 1.8230 1.3079 
hsa-miR-455-5p 1.9466 0.6533 2.4510 1.3079 
hsa-miR-324-3p 5.1796 4.5422 1.5555 1.3079 
hsa-miR-622 2.5718 1.6357 1.9133 1.3079 
hsa-miR-613 1.6581 0.9437 1.6408 1.3079 
hsa-miR-575 3.9488 3.2521 1.6208 1.3079 
hsa-miR-222 3.2655 2.5148 1.6827 1.3079 
hsa-miR-32 2.5684 1.6119 1.9406 1.3079 
hsa-miR-197 7.9098 7.0983 1.7551 1.3079 
hsa-miR-766 6.9197 6.0107 1.8777 1.3079 
hsa-miR-876-5p 1.3810 0.6503 1.6595 1.9848 
hsa-miR-505 2.8023 2.0524 1.6817 1.9848 
hsa-let-7c 7.3815 6.6304 1.6831 1.9848 
hsa-miR-98 4.5308 3.7445 1.7247 1.9848 
hsa-miR-27a 9.1504 8.3246 1.7725 1.9848 
hsa-miR-324-5p 4.1229 3.2487 1.8330 1.9848 
hsa-miR-874 3.6715 3.0471 1.5416 1.9848 
hsa-miR-133a 1.3162 0.6960 1.5371 1.9848 

NOTE: miRNAs which are components of miR-17-92a cluster or miR-25-106b cluster are marked (gray).

The correlation coefficient ratio was significantly higher within miRNA clusters (Supplementary Table S1). These clusters are transcribed from another chromosome. The miR-17-92a cluster was located on chromosome 13 in the host gene C13ORF25, and its homolog miR-106b-25 cluster was located in the intron of the host gene MCM7 on chromosome 7 (Fig. 1, top part). Some components shared the same seed sequence (Fig. 1, bottom part), suggesting they regulated the same targets simultaneously.

Figure 1.

Top, schematic diagram showing the genomic structures of the 2 miRNA clusters located on chromosomes 13 and 7. The miR-17-92a cluster is located in the intron of host gene C13ORF25 and miR-25-106b is in MCM7. Bottom, sequences of 2 miRNA cluster components. The same marks indicate seed sequence (2–8 mer from the 5′side) homology between the individual miRNAs.

Figure 1.

Top, schematic diagram showing the genomic structures of the 2 miRNA clusters located on chromosomes 13 and 7. The miR-17-92a cluster is located in the intron of host gene C13ORF25 and miR-25-106b is in MCM7. Bottom, sequences of 2 miRNA cluster components. The same marks indicate seed sequence (2–8 mer from the 5′side) homology between the individual miRNAs.

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In 4 of the 13 samples of cancer tissue and the 4 normal tissues, both epithelial and stromal samples were available. Thus, we carried out combined analyses in epithelial and stromal tissues in these samples. The expression status of epithelial tissues was highly correlated with that of stromal tissues, and the expression of miRNA in the epithelium was always higher than that in the stroma in all components of the miR-17-92a cluster and the miR-106b-25 cluster (Fig. 2).

Figure 2.

Correlation between the expression status of epithelial tissue and stromal tissue of miR-17-92a and miR-25-106b clusters. In all components of the clusters, expression status of the epithelial tissues and the stromal tissues was highly correlated and expression was higher in epithelium than in stroma.

Figure 2.

Correlation between the expression status of epithelial tissue and stromal tissue of miR-17-92a and miR-25-106b clusters. In all components of the clusters, expression status of the epithelial tissues and the stromal tissues was highly correlated and expression was higher in epithelium than in stroma.

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Significantly downregulated genes which are putative targets of the miR-17-92a and miR-106b-25 clusters are involved in a variety of cellular functions

We also carried out gene expression array analyses using the same samples used in the miRNA microarray. We identified significantly downregulated genes (fold change < 0.5 and Q < 0.05), which were putative targets of the miR-17-92a and miR-106b-25 clusters. The number of genes that matched these criteria was 1,939. Gene set enrichment analysis using Gene Codis version 2.0 (16, 17) and KEGG revealed that the following molecules were enriched: KEGG 04060: cytokine–cytokine receptor interaction [chemokines, hematopoietins, platelet-derived growth factor (PDGF) family, TNF family, and TGFβ family], KEGG 04340: hedgehog signaling pathway, KEGG 05200: pathways in cancer, and KEGG 04514: cell adhesion molecules (CAM; Table 2, Supplementary Table S2A). These indicated the possibility that the miR-17-92a and miR-25-106b clusters were involved in those important pathways in colorectal cancer stroma.

Table 2.

Genes downregulated in cancer stroma, which are putative targets of miR-17-92a and/or miR-25-106b clusters

Gene symbolGenbank accessionDescriptionFold ChangeaQ (%)amiR-17/20a/106b/93 targetbmiR-18a targetbmiR-19a/19b targetbmiR-92a/25 targetb
KEGG 04061: Cytokine–cytokine receptor interaction (chemokines, hematopoietins, PDGF family, TNF family, and TGFβ family) 
IL10RA NM_001558 Homo sapiens interleukin 10 receptor, α (IL10RA) 0.2492 0.0000 ○ ○ ○  
TNFRSF17 NM_001192 Homo sapiens TNF receptor superfamily, member 17 (TNFRSF17) 0.1313 0.0000 ○ ○   
LIFR NM_002310 Homo sapiens leukemia inhibitory factor receptor alpha (LIFR) 0.1563 0.0000 ○ ○ ○  
CXCL12 NM_199168 Homo sapiens chemokine (C-X-C motif) ligand 12 (stromal cell–derived factor 1; CXCL12), transcript variant 1 0.1218 0.0000   ○  
BMP2 NM_001200 Homo sapiens bone morphogenetic protein 2 (BMP2) 0.1734 0.0748 ○ ○   
CNTFR NM_147164 Homo sapiens ciliary neurotrophic factor receptor (CNTFR), transcript variant 1 0.2142 0.0748   ○  
IL6R NM_000565 Homo sapiens interleukin 6 receptor (IL6R), transcript variant 1 0.2694 0.0748 ○ ○   
PDGFRA BC015186 Homo sapiens platelet-derived growth factor receptor, α polypeptide, mRNA (cDNA clone IMAGE: 4043984), complete cds. 0.2247 0.1256 ○   ○ 
TNFRSF13B NM_012452 Homo sapiens TNF receptor superfamily, member 13B (TNFRSF13B) 0.2033 0.1256    ○ 
XCL1 NM_002995 Homo sapiens chemokine (C motif) ligand 1 (XCL1) 0.1676 0.1256 ○ ○ ○ ○ 
CTF1 NM_001330 Homo sapiens cardiotrophin 1 (CTF1) 0.3178 0.2025  ○   
XCL2 NM_003175 Homo sapiens chemokine (C motif) ligand 2 (XCL2) 0.2028 0.2025 ○ ○ ○  
CCR9 NM_006641 Homo sapiens chemokine (C-C motif) receptor 9 (CCR9), transcript variant B 0.2514 0.2025 ○ ○ ○ ○ 
TGFBR2 NM_003242 Homo sapiens TGF, β receptor II (70/80 kDa) (TGFBR2), transcript variant 2 0.3819 0.3097 ○  ○  
CCL16 NM_004590 Homo sapiens chemokine (C-C motif) ligand 16 (CCL16) 0.3502 0.4833    ○ 
IFNA2 NM_000605 Homo sapiens IFN, α 2 (IFNA2) 0.4167 0.7799  ○ ○  
GHR NM_000163 Homo sapiens growth hormone receptor (GHR) 0.3538 0.7799   ○ ○ 
IFNW1 NM_002177 Homo sapiens IFN, omega 1 (IFNW1) 0.3926 0.7799   ○  
IL15 NM_172174 Homo sapiens interleukin 15 (IL15), transcript variant 1 0.4331 0.7799    ○ 
LEP NM_000230 Homo sapiens leptin (obesity homolog, mouse; LEP) 0.3591 0.7799 ○    
BMPR1B NM_001203 Homo sapiens bone morphogenetic protein receptor, type IB (BMPR1B) 0.4251 1.2419 ○ ○   
FASLG NM_000639 Homo sapiens Fas ligand (TNF superfamily, member 6; FASLG) 0.4284 1.2419 ○ ○ ○ ○ 
CCL13 NM_005408 Homo sapiens chemokine (C-C motif) ligand 13 (CCL13) 0.2404 1.2419    ○ 
CCL5 NM_002985 Homo sapiens chemokine (C-C motif) ligand 5 (CCL5) 0.3839 1.2419 ○ ○ ○  
IL9R NM_176786 Homo sapiens interleukin 9 receptor (IL9R), transcript variant 2 0.4804 1.2419  ○   
IL18R1 NM_003855 Homo sapiens interleukin 18 receptor 1 (IL18R1) 0.4667 1.2419   ○  
IL29 NM_172140 Homo sapiens interleukin 29 (IFN, lambda 1; IL29) 0.4156 2.2195  ○   
TNFRSF19 NM_018647 Homo sapiens TNF receptor superfamily, member 19 (TNFRSF19), transcript variant 1 0.4843 2.2195   ○  
TSLP NM_033035 Homo sapiens thymic stromal lymphopoietin (TSLP), transcript variant 1 0.3841 2.2195   ○  
IL25 NM_022789 Homo sapiens interleukin 25 (IL25), transcript variant 1 0.4775 2.2195 ○ ○   
KEGG 04340: Hedgehog signaling pathway 
WNT10A NM_025216 Homo sapiens wingless type MMTV integration site family, member 10A (WNT10A) 0.1264 0.0000   ○  
BMP2 NM_001200 Homo sapiens bone morphogenetic protein 2 (BMP2) 0.1734 0.0748 ○ ○   
BMP6 NM_001718 Homo sapiens bone morphogenetic protein 6 (BMP6) 0.2097 0.2025   ○  
BMP5 NM_021073 Homo sapiens bone morphogenetic protein 5 (BMP5) 0.1747 0.3097    ○ 
WNT9A NM_003395 Homo sapiens wingless-type MMTV integration site family, member 9A (WNT9A) 0.3830 0.7799   ○  
WNT1 NM_005430 Homo sapiens wingless-type MMTV integration site family, member 1 (WNT1) 0.4226 0.7799   ○  
CSNK1A1 AF447582 Homo sapiens HLCDGP1 mRNA, complete cds. 0.4952 1.2419   ○  
WNT7B NM_058238 Homo sapiens wingless-type MMTV integration site family, member 7B (WNT7B) 0.4704 1.2419 ○ ○ ○  
BMP8A NM_181809 Homo sapiens bone morphogenetic protein 8a (BMP8A) 0.3596 2.2195    ○ 
HHIP AK074711 Homo sapiens cDNA FLJ90230 fis, clone NT2RM2000410 0.4494 3.4908    ○ 
KEGG 05200: Pathways in cancer 
CASP8 NM_033356 Homo sapiens caspase 8, apoptosis-related cysteine peptidase (CASP8), transcript variant C 0.1313 0.0000 ○  ○  
COL4A4 NM_000092 Homo sapiens collagen, type IV, alpha 4 (COL4A4) 0.0790 0.0000 ○ ○   
WNT10A NM_025216 Homo sapiens wingless-type MMTV integration site family, member 10A (WNT10A) 0.1264 0.0000   ○  
BCL2 NM_000633 Homo sapiens B-cell CLL/lymphoma 2 (BCL2), nuclear gene encoding mitochondrial protein, transcript variant α 0.2490 0.0353 ○   ○ 
RASSF5 NM_182664 Homo sapiens Ras association (RalGDS/AF-6) domain family 5 (RASSF5), transcript variant 2 0.2031 0.0353   ○  
BMP2 NM_001200 Homo sapiens bone morphogenetic protein 2 (BMP2) 0.1734 0.0748 ○ ○   
VHL AF088066 Homo sapiens full-length insert cDNA clone ZD86C03. 0.3146 0.0748 ○ ○   
PDGFRA BC015186 Homo sapiens platelet-derived growth factor receptor, α polypeptide, mRNA (cDNA clone IMAGE:4043984), complete cds 0.2247 0.1256 ○   ○ 
IGF1 NM_000618 Homo sapiens insulin-like growth factor 1 (somatomedin C; IGF1) 0.2991 0.2025  ○ ○  
ETS1 NM_005238 Homo sapiens v-ets erythroblastosis virus E26 oncogene homolog 1 (avian; ETS1) 0.3762 0.3097   ○ ○ 
TGFBR2 NM_003242 Homo sapiens TGF, β receptor II (70/80 kDa) (TGFBR2), transcript variant 2 0.3819 0.3097 ○  ○  
NKX3-1 NM_006167 Homo sapiens NK3 transcription factor related, locus 1 (Drosophila; NKX3-1) 0.4231 0.4833 ○    
CBL NM_005188 Homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence (CBL) 0.4498 0.4833  ○ ○ ○ 
STK4 BC005231 Homo sapiens serine/threonine kinase 4, mRNA (cDNA clone IMAGE:3950315), complete cds. 0.4024 0.4833 ○ ○ ○  
SMAD2 NM_005901 Homo sapiens SMAD family member 2 (SMAD2), transcript variant 1 0.3846 0.4833  ○ ○ ○ 
FGFR2 NM_022970 Homo sapiens fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson–Weiss syndrome; FGFR2), transcript variant 2, mRNA 0.3038 0.4833   ○ ○ 
WNT9A NM_003395 Homo sapiens wingless-type MMTV integration site family, member 9A (WNT9A) 0.3830 0.7799   ○  
WNT1 NM_005430 Homo sapiens wingless-type MMTV integration site family, member 1 (WNT1) 0.4226 0.7799   ○  
COL4A6 NM_033641 Homo sapiens collagen, type IV, α 6 (COL4A6), transcript variant B 0.1932 0.7799    ○ 
PIK3CD NM_005026 Homo sapiens phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) 0.4580 0.7799    ○ 
DAPK2 NM_014326 Homo sapiens death-associated protein kinase 2 (DAPK2) 0.3810 0.7799 ○ ○  ○ 
FGF6 NM_020996 Homo sapiens fibroblast growth factor 6 (FGF6) 0.3913 0.7799   ○  
PLD1 NM_002662 Homo sapiens phospholipase D1, phosphatidylcholine specific (PLD1) 0.4685 0.7799 ○ ○ ○  
FASLG NM_000639 Homo sapiens Fas ligand (TNF superfamily, member 6; FASLG) 0.4284 1.2419 ○ ○ ○ ○ 
RUNX1 X90978 Homo sapiens mRNA for an acute myeloid leukaemia protein (1,793 bp) 0.4519 1.2419 ○ ○   
SMAD3 U68019 Homo sapiens mad protein homolog (hMAD-3) mRNA, complete cds 0.4943 1.2419  ○   
LAMA3 NM_000227 Homo sapiens laminin, alpha 3 (LAMA3), transcript variant 2 0.4329 1.2419 ○    
DCC NM_005215 Homo sapiens deleted in colorectal carcinoma (DCC) 0.4342 1.2419   ○  
WNT7B NM_058238 Homo sapiens wingless-type MMTV integration site family, member 7B (WNT7B) 0.4704 1.2419 ○ ○ ○  
FGF5 NM_004464 Homo sapiens fibroblast growth factor 5 (FGF5), transcript variant 1 0.4823 2.2195 ○   ○ 
HHIP AK074711 Homo sapiens cDNA FLJ90230 fis, clone NT2RM2000410. 0.4494 3.4908    ○ 
KEGG 04514: Cell adhesion molecules (CAM) 
CNTN2 NM_005076 Homo sapiens contactin 2 (axonal; CNTN2) 0.1659 0.0748 ○ ○ ○  
ALCAM NM_001627 Homo sapiens activated leukocyte cell adhesion molecule (ALCAM) 0.2975 0.1256  ○   
CD22 NM_001771 Homo sapiens CD22 molecule (CD22) 0.1359 0.1256   ○  
SPN NM_001030288 Homo sapiens sialophorin (leukosialin, CD43; SPN), transcript variant 1 0.3110 0.2025 ○ ○  ○ 
CLDN18 NM_016369 Homo sapiens claudin 18 (CLDN18), transcript variant 1 0.3400 0.2025 ○ ○   
CNTNAP2 NM_014141 Homo sapiens contactin-associated protein-like 2 (CNTNAP2) 0.3082 0.3097 ○   ○ 
CLDN19 NM_148960 Homo sapiens claudin 19 (CLDN19) 0.3840 0.4833 ○ ○ ○ ○ 
PTPRC NM_002838 Homo sapiens protein tyrosine phosphatase, receptor type, C (PTPRC), transcript variant 1 0.3629 0.7799    ○ 
NRXN1 NM_004801 Homo sapiens neurexin 1 (NRXN1), transcript variant alpha 0.3674 0.7799  ○   
CD8A NM_001768 Homo sapiens CD8a molecule (CD8A), transcript variant 1 0.4033 1.2419  ○   
CLDN16 NM_006580 Homo sapiens claudin 16 (CLDN16) 0.4543 1.2419  ○   
CNTNAP1 NM_003632 Homo sapiens contactin-associated protein 1 (CNTNAP1) 0.4725 1.2419  ○   
NCAM1 NM_000615 Homo sapiens neural cell adhesion molecule 1 (NCAM1), transcript variant 1 0.4247 1.2419   ○  
CD28 NM_006139 Homo sapiens CD28 molecule (CD28) 0.4997 2.2195 ○ ○ ○  
L1CAM NM_000425 Homo sapiens L1 cell adhesion molecule (L1CAM), transcript variant 1 0.4938 3.4908 ○ ○ ○  
WNT9A NM_003395 Homo sapiens wingless-type MMTV integration site family, member 9A (WNT9A) 0.3830 0.7799   ○  
WNT1 NM_005430 Homo sapiens wingless-type MMTV integration site family, member 1 (WNT1) 0.4226 0.7799   ○  
COL4A6 NM_033641 Homo sapiens collagen, type IV, alpha 6 (COL4A6), transcript variant B 0.1932 0.7799    ○ 
PIK3CD NM_005026 Homo sapiens phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) 0.4580 0.7799    ○ 
DAPK2 NM_014326 Homo sapiens death-associated protein kinase 2 (DAPK2) 0.3810 0.7799 ○ ○  ○ 
FGF6 NM_020996 Homo sapiens fibroblast growth factor 6 (FGF6) 0.3913 0.7799   ○  
PLD1 NM_002662 Homo sapiens phospholipase D1, phosphatidylcholine-specific (PLD1) 0.4685 0.7799 ○ ○ ○  
FASLG NM_000639 Homo sapiens Fas ligand (TNF superfamily, member 6; FASLG) 0.4284 1.2419 ○ ○ ○ ○ 
RUNX1 X90978 Homo sapiens mRNA for an acute myeloid leukaemia protein (1,793 bp). 0.4519 1.2419 ○ ○   
SMAD3 U68019 Homo sapiens mad protein homolog (hMAD-3) 0.4943 1.2419  ○   
LAMA3 NM_000227 Homo sapiens laminin, alpha 3 (LAMA3), transcript variant 2 0.4329 1.2419 ○    
DCC NM_005215 Homo sapiens deleted in colorectal carcinoma (DCC) 0.4342 1.2419   ○  
WNT7B NM_058238 Homo sapiens wingless-type MMTV integration site family, member 7B (WNT7B) 0.4704 1.2419 ○ ○ ○  
FGF5 NM_004464 Homo sapiens fibroblast growth factor 5 (FGF5), transcript variant 1 0.4823 2.2195 ○   ○ 
HHIP AK074711 Homo sapiens cDNA FLJ90230 fis, clone NT2RM2000410. 0.4494 3.4908    ○ 
Gene symbolGenbank accessionDescriptionFold ChangeaQ (%)amiR-17/20a/106b/93 targetbmiR-18a targetbmiR-19a/19b targetbmiR-92a/25 targetb
KEGG 04061: Cytokine–cytokine receptor interaction (chemokines, hematopoietins, PDGF family, TNF family, and TGFβ family) 
IL10RA NM_001558 Homo sapiens interleukin 10 receptor, α (IL10RA) 0.2492 0.0000 ○ ○ ○  
TNFRSF17 NM_001192 Homo sapiens TNF receptor superfamily, member 17 (TNFRSF17) 0.1313 0.0000 ○ ○   
LIFR NM_002310 Homo sapiens leukemia inhibitory factor receptor alpha (LIFR) 0.1563 0.0000 ○ ○ ○  
CXCL12 NM_199168 Homo sapiens chemokine (C-X-C motif) ligand 12 (stromal cell–derived factor 1; CXCL12), transcript variant 1 0.1218 0.0000   ○  
BMP2 NM_001200 Homo sapiens bone morphogenetic protein 2 (BMP2) 0.1734 0.0748 ○ ○   
CNTFR NM_147164 Homo sapiens ciliary neurotrophic factor receptor (CNTFR), transcript variant 1 0.2142 0.0748   ○  
IL6R NM_000565 Homo sapiens interleukin 6 receptor (IL6R), transcript variant 1 0.2694 0.0748 ○ ○   
PDGFRA BC015186 Homo sapiens platelet-derived growth factor receptor, α polypeptide, mRNA (cDNA clone IMAGE: 4043984), complete cds. 0.2247 0.1256 ○   ○ 
TNFRSF13B NM_012452 Homo sapiens TNF receptor superfamily, member 13B (TNFRSF13B) 0.2033 0.1256    ○ 
XCL1 NM_002995 Homo sapiens chemokine (C motif) ligand 1 (XCL1) 0.1676 0.1256 ○ ○ ○ ○ 
CTF1 NM_001330 Homo sapiens cardiotrophin 1 (CTF1) 0.3178 0.2025  ○   
XCL2 NM_003175 Homo sapiens chemokine (C motif) ligand 2 (XCL2) 0.2028 0.2025 ○ ○ ○  
CCR9 NM_006641 Homo sapiens chemokine (C-C motif) receptor 9 (CCR9), transcript variant B 0.2514 0.2025 ○ ○ ○ ○ 
TGFBR2 NM_003242 Homo sapiens TGF, β receptor II (70/80 kDa) (TGFBR2), transcript variant 2 0.3819 0.3097 ○  ○  
CCL16 NM_004590 Homo sapiens chemokine (C-C motif) ligand 16 (CCL16) 0.3502 0.4833    ○ 
IFNA2 NM_000605 Homo sapiens IFN, α 2 (IFNA2) 0.4167 0.7799  ○ ○  
GHR NM_000163 Homo sapiens growth hormone receptor (GHR) 0.3538 0.7799   ○ ○ 
IFNW1 NM_002177 Homo sapiens IFN, omega 1 (IFNW1) 0.3926 0.7799   ○  
IL15 NM_172174 Homo sapiens interleukin 15 (IL15), transcript variant 1 0.4331 0.7799    ○ 
LEP NM_000230 Homo sapiens leptin (obesity homolog, mouse; LEP) 0.3591 0.7799 ○    
BMPR1B NM_001203 Homo sapiens bone morphogenetic protein receptor, type IB (BMPR1B) 0.4251 1.2419 ○ ○   
FASLG NM_000639 Homo sapiens Fas ligand (TNF superfamily, member 6; FASLG) 0.4284 1.2419 ○ ○ ○ ○ 
CCL13 NM_005408 Homo sapiens chemokine (C-C motif) ligand 13 (CCL13) 0.2404 1.2419    ○ 
CCL5 NM_002985 Homo sapiens chemokine (C-C motif) ligand 5 (CCL5) 0.3839 1.2419 ○ ○ ○  
IL9R NM_176786 Homo sapiens interleukin 9 receptor (IL9R), transcript variant 2 0.4804 1.2419  ○   
IL18R1 NM_003855 Homo sapiens interleukin 18 receptor 1 (IL18R1) 0.4667 1.2419   ○  
IL29 NM_172140 Homo sapiens interleukin 29 (IFN, lambda 1; IL29) 0.4156 2.2195  ○   
TNFRSF19 NM_018647 Homo sapiens TNF receptor superfamily, member 19 (TNFRSF19), transcript variant 1 0.4843 2.2195   ○  
TSLP NM_033035 Homo sapiens thymic stromal lymphopoietin (TSLP), transcript variant 1 0.3841 2.2195   ○  
IL25 NM_022789 Homo sapiens interleukin 25 (IL25), transcript variant 1 0.4775 2.2195 ○ ○   
KEGG 04340: Hedgehog signaling pathway 
WNT10A NM_025216 Homo sapiens wingless type MMTV integration site family, member 10A (WNT10A) 0.1264 0.0000   ○  
BMP2 NM_001200 Homo sapiens bone morphogenetic protein 2 (BMP2) 0.1734 0.0748 ○ ○   
BMP6 NM_001718 Homo sapiens bone morphogenetic protein 6 (BMP6) 0.2097 0.2025   ○  
BMP5 NM_021073 Homo sapiens bone morphogenetic protein 5 (BMP5) 0.1747 0.3097    ○ 
WNT9A NM_003395 Homo sapiens wingless-type MMTV integration site family, member 9A (WNT9A) 0.3830 0.7799   ○  
WNT1 NM_005430 Homo sapiens wingless-type MMTV integration site family, member 1 (WNT1) 0.4226 0.7799   ○  
CSNK1A1 AF447582 Homo sapiens HLCDGP1 mRNA, complete cds. 0.4952 1.2419   ○  
WNT7B NM_058238 Homo sapiens wingless-type MMTV integration site family, member 7B (WNT7B) 0.4704 1.2419 ○ ○ ○  
BMP8A NM_181809 Homo sapiens bone morphogenetic protein 8a (BMP8A) 0.3596 2.2195    ○ 
HHIP AK074711 Homo sapiens cDNA FLJ90230 fis, clone NT2RM2000410 0.4494 3.4908    ○ 
KEGG 05200: Pathways in cancer 
CASP8 NM_033356 Homo sapiens caspase 8, apoptosis-related cysteine peptidase (CASP8), transcript variant C 0.1313 0.0000 ○  ○  
COL4A4 NM_000092 Homo sapiens collagen, type IV, alpha 4 (COL4A4) 0.0790 0.0000 ○ ○   
WNT10A NM_025216 Homo sapiens wingless-type MMTV integration site family, member 10A (WNT10A) 0.1264 0.0000   ○  
BCL2 NM_000633 Homo sapiens B-cell CLL/lymphoma 2 (BCL2), nuclear gene encoding mitochondrial protein, transcript variant α 0.2490 0.0353 ○   ○ 
RASSF5 NM_182664 Homo sapiens Ras association (RalGDS/AF-6) domain family 5 (RASSF5), transcript variant 2 0.2031 0.0353   ○  
BMP2 NM_001200 Homo sapiens bone morphogenetic protein 2 (BMP2) 0.1734 0.0748 ○ ○   
VHL AF088066 Homo sapiens full-length insert cDNA clone ZD86C03. 0.3146 0.0748 ○ ○   
PDGFRA BC015186 Homo sapiens platelet-derived growth factor receptor, α polypeptide, mRNA (cDNA clone IMAGE:4043984), complete cds 0.2247 0.1256 ○   ○ 
IGF1 NM_000618 Homo sapiens insulin-like growth factor 1 (somatomedin C; IGF1) 0.2991 0.2025  ○ ○  
ETS1 NM_005238 Homo sapiens v-ets erythroblastosis virus E26 oncogene homolog 1 (avian; ETS1) 0.3762 0.3097   ○ ○ 
TGFBR2 NM_003242 Homo sapiens TGF, β receptor II (70/80 kDa) (TGFBR2), transcript variant 2 0.3819 0.3097 ○  ○  
NKX3-1 NM_006167 Homo sapiens NK3 transcription factor related, locus 1 (Drosophila; NKX3-1) 0.4231 0.4833 ○    
CBL NM_005188 Homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence (CBL) 0.4498 0.4833  ○ ○ ○ 
STK4 BC005231 Homo sapiens serine/threonine kinase 4, mRNA (cDNA clone IMAGE:3950315), complete cds. 0.4024 0.4833 ○ ○ ○  
SMAD2 NM_005901 Homo sapiens SMAD family member 2 (SMAD2), transcript variant 1 0.3846 0.4833  ○ ○ ○ 
FGFR2 NM_022970 Homo sapiens fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson–Weiss syndrome; FGFR2), transcript variant 2, mRNA 0.3038 0.4833   ○ ○ 
WNT9A NM_003395 Homo sapiens wingless-type MMTV integration site family, member 9A (WNT9A) 0.3830 0.7799   ○  
WNT1 NM_005430 Homo sapiens wingless-type MMTV integration site family, member 1 (WNT1) 0.4226 0.7799   ○  
COL4A6 NM_033641 Homo sapiens collagen, type IV, α 6 (COL4A6), transcript variant B 0.1932 0.7799    ○ 
PIK3CD NM_005026 Homo sapiens phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) 0.4580 0.7799    ○ 
DAPK2 NM_014326 Homo sapiens death-associated protein kinase 2 (DAPK2) 0.3810 0.7799 ○ ○  ○ 
FGF6 NM_020996 Homo sapiens fibroblast growth factor 6 (FGF6) 0.3913 0.7799   ○  
PLD1 NM_002662 Homo sapiens phospholipase D1, phosphatidylcholine specific (PLD1) 0.4685 0.7799 ○ ○ ○  
FASLG NM_000639 Homo sapiens Fas ligand (TNF superfamily, member 6; FASLG) 0.4284 1.2419 ○ ○ ○ ○ 
RUNX1 X90978 Homo sapiens mRNA for an acute myeloid leukaemia protein (1,793 bp) 0.4519 1.2419 ○ ○   
SMAD3 U68019 Homo sapiens mad protein homolog (hMAD-3) mRNA, complete cds 0.4943 1.2419  ○   
LAMA3 NM_000227 Homo sapiens laminin, alpha 3 (LAMA3), transcript variant 2 0.4329 1.2419 ○    
DCC NM_005215 Homo sapiens deleted in colorectal carcinoma (DCC) 0.4342 1.2419   ○  
WNT7B NM_058238 Homo sapiens wingless-type MMTV integration site family, member 7B (WNT7B) 0.4704 1.2419 ○ ○ ○  
FGF5 NM_004464 Homo sapiens fibroblast growth factor 5 (FGF5), transcript variant 1 0.4823 2.2195 ○   ○ 
HHIP AK074711 Homo sapiens cDNA FLJ90230 fis, clone NT2RM2000410. 0.4494 3.4908    ○ 
KEGG 04514: Cell adhesion molecules (CAM) 
CNTN2 NM_005076 Homo sapiens contactin 2 (axonal; CNTN2) 0.1659 0.0748 ○ ○ ○  
ALCAM NM_001627 Homo sapiens activated leukocyte cell adhesion molecule (ALCAM) 0.2975 0.1256  ○   
CD22 NM_001771 Homo sapiens CD22 molecule (CD22) 0.1359 0.1256   ○  
SPN NM_001030288 Homo sapiens sialophorin (leukosialin, CD43; SPN), transcript variant 1 0.3110 0.2025 ○ ○  ○ 
CLDN18 NM_016369 Homo sapiens claudin 18 (CLDN18), transcript variant 1 0.3400 0.2025 ○ ○   
CNTNAP2 NM_014141 Homo sapiens contactin-associated protein-like 2 (CNTNAP2) 0.3082 0.3097 ○   ○ 
CLDN19 NM_148960 Homo sapiens claudin 19 (CLDN19) 0.3840 0.4833 ○ ○ ○ ○ 
PTPRC NM_002838 Homo sapiens protein tyrosine phosphatase, receptor type, C (PTPRC), transcript variant 1 0.3629 0.7799    ○ 
NRXN1 NM_004801 Homo sapiens neurexin 1 (NRXN1), transcript variant alpha 0.3674 0.7799  ○   
CD8A NM_001768 Homo sapiens CD8a molecule (CD8A), transcript variant 1 0.4033 1.2419  ○   
CLDN16 NM_006580 Homo sapiens claudin 16 (CLDN16) 0.4543 1.2419  ○   
CNTNAP1 NM_003632 Homo sapiens contactin-associated protein 1 (CNTNAP1) 0.4725 1.2419  ○   
NCAM1 NM_000615 Homo sapiens neural cell adhesion molecule 1 (NCAM1), transcript variant 1 0.4247 1.2419   ○  
CD28 NM_006139 Homo sapiens CD28 molecule (CD28) 0.4997 2.2195 ○ ○ ○  
L1CAM NM_000425 Homo sapiens L1 cell adhesion molecule (L1CAM), transcript variant 1 0.4938 3.4908 ○ ○ ○  
WNT9A NM_003395 Homo sapiens wingless-type MMTV integration site family, member 9A (WNT9A) 0.3830 0.7799   ○  
WNT1 NM_005430 Homo sapiens wingless-type MMTV integration site family, member 1 (WNT1) 0.4226 0.7799   ○  
COL4A6 NM_033641 Homo sapiens collagen, type IV, alpha 6 (COL4A6), transcript variant B 0.1932 0.7799    ○ 
PIK3CD NM_005026 Homo sapiens phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) 0.4580 0.7799    ○ 
DAPK2 NM_014326 Homo sapiens death-associated protein kinase 2 (DAPK2) 0.3810 0.7799 ○ ○  ○ 
FGF6 NM_020996 Homo sapiens fibroblast growth factor 6 (FGF6) 0.3913 0.7799   ○  
PLD1 NM_002662 Homo sapiens phospholipase D1, phosphatidylcholine-specific (PLD1) 0.4685 0.7799 ○ ○ ○  
FASLG NM_000639 Homo sapiens Fas ligand (TNF superfamily, member 6; FASLG) 0.4284 1.2419 ○ ○ ○ ○ 
RUNX1 X90978 Homo sapiens mRNA for an acute myeloid leukaemia protein (1,793 bp). 0.4519 1.2419 ○ ○   
SMAD3 U68019 Homo sapiens mad protein homolog (hMAD-3) 0.4943 1.2419  ○   
LAMA3 NM_000227 Homo sapiens laminin, alpha 3 (LAMA3), transcript variant 2 0.4329 1.2419 ○    
DCC NM_005215 Homo sapiens deleted in colorectal carcinoma (DCC) 0.4342 1.2419   ○  
WNT7B NM_058238 Homo sapiens wingless-type MMTV integration site family, member 7B (WNT7B) 0.4704 1.2419 ○ ○ ○  
FGF5 NM_004464 Homo sapiens fibroblast growth factor 5 (FGF5), transcript variant 1 0.4823 2.2195 ○   ○ 
HHIP AK074711 Homo sapiens cDNA FLJ90230 fis, clone NT2RM2000410. 0.4494 3.4908    ○ 

NOTE: KEGG (ref. 18).

aFold change < 0.50, Q < 0.05

bBased on the computational analysis by TargetScan (ref. 19, 20). Genes which are targets of each miRNA are marked with circle.

Identification of a putative miRNA gene pathway focusing on inverse correlations between miRNAs and genes

We used different approaches to identify putative targets of the miR-17-92a and miR-106b-25 clusters. Combining the microarray data of miRNAs and genes in stromal samples, we identified highly inversely correlated miRNA gene pairs, which were putative pathways predicted by TargetScan (refs. 19, 20; correlation coefficient >0.65 and P < 0.05; Table 3). These included a number of crucial regulators of cellular function, such as the apoptosis-related miR-17 death–associated protein kinase 2 (DAPK) pairing, the miR-18a–caspase 7 pairing, and the miR-17 cancer-related transcription factor 7 (TCF7) pairing.

Table 3.

Highly inverse-correlated miRNA and putative target gene pairs

miRNA namemRNA nameGenbank accessionDescriptioncorrelation coefficientaPaPathway (KEGG)b
hsa-miR-17 ACPP NM_001099 Acid phosphatase, prostate –0.8549 0.0002 00361: gamma-Hexachlorocyclohexane degradation, 00740: Riboflavin metabolism 
hsa-miR-18a HTR1D NM_000864 5-Hydroxytryptamine (serotonin) receptor 1D –0.8467 0.0003 04080: Neuroactive ligand–receptor interaction 
hsa-miR-17 DAPK2 NM_014326 Death-associated protein kinase 2 –0.8073 0.0009 05200: Pathways in cancer, 05219: Bladder cancer 
hsa-miR-17 LY75 NM_002349 Lymphocyte antigen 75 –0.8072 0.0009  
hsa-miR-17 TRIM7 NM_203297 Tripartite motif-containing 7 –0.7794 0.0017  
hsa-miR-19b CASP7 NM_033338 Caspase 7, apoptosis-related cysteine peptidase –0.7728 0.0020 04210: Apoptosis, 05010: Alzheimer's disease 
hsa-miR-17 SHROOM1 NM_133456 shroom family member 1 –0.7553 0.0028  
hsa-miR-19a PDE11A NM_016953 Phosphodiesterase 11A –0.7406 0.0038 00230: Purine metabolism 
hsa-miR-20a SYT13 NM_020826 Synaptotagmin XIII –0.7385 0.0039  
hsa-miR-18a PDE11A NM_016953 Phosphodiesterase 11A –0.7377 0.0040 00230: Purine metabolism 
hsa-miR-18a SLC18A1 NM_003053 Solute carrier family 18 (vesicular monoamine), member 1 –0.7342 0.0043 05012: Parkinson's disease 
hsa-miR-19a CLIC5 NM_016929 Chloride intracellular channel 5 –0.7231 0.0052  
hsa-miR-19a ARFIP1 NM_001025595 ADP-ribosylation factor interacting protein 1 –0.7229 0.0052  
hsa-miR-19a KCNV1 NM_014379 Potassium channel, subfamily V, member 1 –0.7190 0.0056  
hsa-miR-18a MYO7B BC035615 Myosin VIIB –0.7151 0.0060  
hsa-miR-17 PKDREJ NM_006071 Polycystic kidney disease (polycystin) and REJ homolog (sperm receptor for egg jelly homolog, sea urchin) –0.7108 0.0065  
hsa-miR-19a STX18 NM_016930 syntaxin 18 –0.7068 0.0069 04130: SNARE interactions in vesicular transport 
hsa-miR-17 TRIM68 NM_018073 Tripartite motif-containing 68 –0.7058 0.0070  
hsa-miR-18a SLC35D2 NM_007001 Solute carrier family 35, member D2 –0.7051 0.0071  
hsa-miR-17 TCF7 NM_003202 Transcription factor 7(T-cell specific, HMG box) –0.7023 0.0074 04310: Wnt signaling pathway, 04520: Adherens junction, 04916: Melanogenesis, 05200: Pathways in cancer, 05210: Colorectal cancer, 05213: Endometrial cancer, 05215: Prostate cancer, 05216: Thyroid cancer, 05217: Basal cell carcinoma, 05221: Acute myeloid leukemia, 05412: Arrhythmogenic right ventricular cardiomyopathy (ARVC) 
hsa-miR-18a SLC5A9 NM_001011547 Solute carrier family 5 (sodium/glucose cotransporter), member 9 –0.7009 0.0076  
hsa-miR-19a ABLIM1 NM_001003408 Actin binding LIM protein 1 –0.6966 0.0082 04360: Axon guidance 
hsa-miR-18a CASP7 NM_033338 Caspase 7, apoptosis-related cysteine peptidase –0.6960 0.0082 04210: Apoptosis, 05010: Alzheimer's disease 
hsa-miR-20a H2AFJ NM_177925 H2A histone family, member J –0.6939 0.0085 05322: Systemic lupus erythematosus 
hsa-miR-17 SLC39A8 NM_022154 Solute carrier family 39 (zinc transporter), member 8 –0.6910 0.0089  
hsa-miR-17 CLIC5 NM_016929 Chloride intracellular channel 5 –0.6887 0.0092  
hsa-miR-20a KIAA0319 NM_014809 KIAA0319 –0.6783 0.0108  
hsa-miR-19a CEACAM8 NM_001816 Carcinoembryonic antigen-related cell adhesion molecule 8 –0.6751 0.0114  
hsa-miR-19a TRIM68 NM_018073 Tripartite motif-containing 68 –0.6714 0.0120  
hsa-miR-19a SFT2D3 NM_032740 SFT2 domain containing 3 –0.6684 0.0125  
hsa-miR-17 GGT6 NM_153338 γ-glutamyltransferase 6 –0.6676 0.0127 00430: Taurine and hypotaurine metabolism, 00450: Selenoamino acid metabolism, 00460: Cyanoamino acid metabolism, 00480: Glutathione metabolism, 00590: Arachidonic acid metabolism, 01100: Metabolic pathways 
hsa-miR-20a XDH NM_000379 Xanthine dehydrogenase –0.6673 0.0127 00230: Purine metabolism, 00232: Caffeine metabolism, 00983: Drug metabolism–other enzymes, 01100: Metabolic pathways 
hsa-miR-20a PPARA NM_005036 Peroxisome proliferator-activated receptor α –0.6654 0.0131 03320: PPAR signaling pathway, 04920: Adipocytokine signaling pathway 
hsa-miR-17 C2orf15 NM_144706 Chromosome 2 open reading frame 15 –0.6653 0.0131  
hsa-miR-17 GK5 BX648681 Glycerol kinase 5 (putative) –0.6634 0.0134  
hsa-miR-17 SIDT1 NM_017699 SID1 transmembrane family, member 1 –0.6619 0.0137  
hsa-miR-25 ATP10B AB018258 ATPase, class V, type 10B –0.6605 0.0140  
hsa-miR-17 TMC5 NM_024780 Transmembrane channel-like 5 –0.6590 0.0143  
hsa-miR-18a ALDH1A2 NM_170697 Aldehyde dehydrogenase 1 family, member A2 –0.6586 0.0144 00830: Retinol metabolism, 01100: Metabolic pathways 
hsa-miR-17 INADL NM_176877 InaD-like (Drosophila–0.6516 0.0158 04530: Tight junction 
miRNA namemRNA nameGenbank accessionDescriptioncorrelation coefficientaPaPathway (KEGG)b
hsa-miR-17 ACPP NM_001099 Acid phosphatase, prostate –0.8549 0.0002 00361: gamma-Hexachlorocyclohexane degradation, 00740: Riboflavin metabolism 
hsa-miR-18a HTR1D NM_000864 5-Hydroxytryptamine (serotonin) receptor 1D –0.8467 0.0003 04080: Neuroactive ligand–receptor interaction 
hsa-miR-17 DAPK2 NM_014326 Death-associated protein kinase 2 –0.8073 0.0009 05200: Pathways in cancer, 05219: Bladder cancer 
hsa-miR-17 LY75 NM_002349 Lymphocyte antigen 75 –0.8072 0.0009  
hsa-miR-17 TRIM7 NM_203297 Tripartite motif-containing 7 –0.7794 0.0017  
hsa-miR-19b CASP7 NM_033338 Caspase 7, apoptosis-related cysteine peptidase –0.7728 0.0020 04210: Apoptosis, 05010: Alzheimer's disease 
hsa-miR-17 SHROOM1 NM_133456 shroom family member 1 –0.7553 0.0028  
hsa-miR-19a PDE11A NM_016953 Phosphodiesterase 11A –0.7406 0.0038 00230: Purine metabolism 
hsa-miR-20a SYT13 NM_020826 Synaptotagmin XIII –0.7385 0.0039  
hsa-miR-18a PDE11A NM_016953 Phosphodiesterase 11A –0.7377 0.0040 00230: Purine metabolism 
hsa-miR-18a SLC18A1 NM_003053 Solute carrier family 18 (vesicular monoamine), member 1 –0.7342 0.0043 05012: Parkinson's disease 
hsa-miR-19a CLIC5 NM_016929 Chloride intracellular channel 5 –0.7231 0.0052  
hsa-miR-19a ARFIP1 NM_001025595 ADP-ribosylation factor interacting protein 1 –0.7229 0.0052  
hsa-miR-19a KCNV1 NM_014379 Potassium channel, subfamily V, member 1 –0.7190 0.0056  
hsa-miR-18a MYO7B BC035615 Myosin VIIB –0.7151 0.0060  
hsa-miR-17 PKDREJ NM_006071 Polycystic kidney disease (polycystin) and REJ homolog (sperm receptor for egg jelly homolog, sea urchin) –0.7108 0.0065  
hsa-miR-19a STX18 NM_016930 syntaxin 18 –0.7068 0.0069 04130: SNARE interactions in vesicular transport 
hsa-miR-17 TRIM68 NM_018073 Tripartite motif-containing 68 –0.7058 0.0070  
hsa-miR-18a SLC35D2 NM_007001 Solute carrier family 35, member D2 –0.7051 0.0071  
hsa-miR-17 TCF7 NM_003202 Transcription factor 7(T-cell specific, HMG box) –0.7023 0.0074 04310: Wnt signaling pathway, 04520: Adherens junction, 04916: Melanogenesis, 05200: Pathways in cancer, 05210: Colorectal cancer, 05213: Endometrial cancer, 05215: Prostate cancer, 05216: Thyroid cancer, 05217: Basal cell carcinoma, 05221: Acute myeloid leukemia, 05412: Arrhythmogenic right ventricular cardiomyopathy (ARVC) 
hsa-miR-18a SLC5A9 NM_001011547 Solute carrier family 5 (sodium/glucose cotransporter), member 9 –0.7009 0.0076  
hsa-miR-19a ABLIM1 NM_001003408 Actin binding LIM protein 1 –0.6966 0.0082 04360: Axon guidance 
hsa-miR-18a CASP7 NM_033338 Caspase 7, apoptosis-related cysteine peptidase –0.6960 0.0082 04210: Apoptosis, 05010: Alzheimer's disease 
hsa-miR-20a H2AFJ NM_177925 H2A histone family, member J –0.6939 0.0085 05322: Systemic lupus erythematosus 
hsa-miR-17 SLC39A8 NM_022154 Solute carrier family 39 (zinc transporter), member 8 –0.6910 0.0089  
hsa-miR-17 CLIC5 NM_016929 Chloride intracellular channel 5 –0.6887 0.0092  
hsa-miR-20a KIAA0319 NM_014809 KIAA0319 –0.6783 0.0108  
hsa-miR-19a CEACAM8 NM_001816 Carcinoembryonic antigen-related cell adhesion molecule 8 –0.6751 0.0114  
hsa-miR-19a TRIM68 NM_018073 Tripartite motif-containing 68 –0.6714 0.0120  
hsa-miR-19a SFT2D3 NM_032740 SFT2 domain containing 3 –0.6684 0.0125  
hsa-miR-17 GGT6 NM_153338 γ-glutamyltransferase 6 –0.6676 0.0127 00430: Taurine and hypotaurine metabolism, 00450: Selenoamino acid metabolism, 00460: Cyanoamino acid metabolism, 00480: Glutathione metabolism, 00590: Arachidonic acid metabolism, 01100: Metabolic pathways 
hsa-miR-20a XDH NM_000379 Xanthine dehydrogenase –0.6673 0.0127 00230: Purine metabolism, 00232: Caffeine metabolism, 00983: Drug metabolism–other enzymes, 01100: Metabolic pathways 
hsa-miR-20a PPARA NM_005036 Peroxisome proliferator-activated receptor α –0.6654 0.0131 03320: PPAR signaling pathway, 04920: Adipocytokine signaling pathway 
hsa-miR-17 C2orf15 NM_144706 Chromosome 2 open reading frame 15 –0.6653 0.0131  
hsa-miR-17 GK5 BX648681 Glycerol kinase 5 (putative) –0.6634 0.0134  
hsa-miR-17 SIDT1 NM_017699 SID1 transmembrane family, member 1 –0.6619 0.0137  
hsa-miR-25 ATP10B AB018258 ATPase, class V, type 10B –0.6605 0.0140  
hsa-miR-17 TMC5 NM_024780 Transmembrane channel-like 5 –0.6590 0.0143  
hsa-miR-18a ALDH1A2 NM_170697 Aldehyde dehydrogenase 1 family, member A2 –0.6586 0.0144 00830: Retinol metabolism, 01100: Metabolic pathways 
hsa-miR-17 INADL NM_176877 InaD-like (Drosophila–0.6516 0.0158 04530: Tight junction 

NOTE: Based on the computational analysis by TargetScan (ref. 19, 20).

aCorrelation coefficient <–0.65, P < 0.05 Ranked by correlation coefficient.

bKEGG, kyoto encyclopedia of genes and genomes (ref. 18).

miR-25 and miR-92a expression status in colorectal cancer stromal tissue

The miRNA array analysis revealed that 2 oncogenic miRNA clusters were upregulated in colorectal cancer stroma. Therefore, we confirmed the expression status of 2 representative miRNAs from the 2 clusters, miR-25, and miR-92a in clinical samples. MiR-25 and miR-92a are selected because of relative high expression in cancer stroma and because they have the same seed sequence and share the same targets and could work coordinately. In samples of normal epithelial tissue (n = 4), cancer epithelial tissue (n = 10), normal stromal tissue (n = 4), and cancer stromal tissue (n = 26), which included 13 cancer samples and 4 normal samples used for microarray analysis, we carried out qRT-PCR to investigate the expression of miR-25 and miR-92a. The data confirmed the upregulation of these miRNAs in cancer stroma compared with normal stroma in accordance with upregulation in cancer epithelium (Fig. 3).

Figure 3.

Expression of miR-25 (left) and miR-92a (right) analyzed by quantitative RT-PCR in epithelium and stroma of cancer and normal tissue. Dots, expression of each sample; horizontal line, median; box, 25th through 75th percentile; error bars, range. Normal epithelium: n = 4, cancer epithelium: n = 10, normal stroma: n = 4, cancer stroma: n = 26.

Figure 3.

Expression of miR-25 (left) and miR-92a (right) analyzed by quantitative RT-PCR in epithelium and stroma of cancer and normal tissue. Dots, expression of each sample; horizontal line, median; box, 25th through 75th percentile; error bars, range. Normal epithelium: n = 4, cancer epithelium: n = 10, normal stroma: n = 4, cancer stroma: n = 26.

Close modal

miR-25 and miR-92a expression in colorectal cancer stroma was associated with clinicopathologic factors

For the 26 colorectal cancer stromal samples for which we used RT-PCR analysis, clinicopathologic data were available in 24 cases. Clinicopathologic analysis revealed that the high miR-25 expression group (values > the 0.25 quartile; 0.54, normalized to RNU6B) had more advanced venous invasion compared with the low expression group (values < the 0.25 quartile; P = 0.046, Table 4). In the high miR-92a expression group (values > the 0.75 quartile; 1.16, normalized to RNU6B), there was greater lymphatic invasion (P = 0.005), venous invasion (P = 0.016), and liver metastasis (P = 0.018) compared with the low miR-92a expression group (values < the 0.75 quartile; Table 5). However, no significant differences were observed regarding age, gender, histology, lymphatic invasion, venous invasion, lymph node metastasis, peritoneal dissemination, or distant metastasis.

Table 4.

miR-25 expression in cancer stroma and clinocopathologic factors

High expressionLow expression
Factors(n = 18)(n = 6)P
Age (mean ± SD) 62.8 ± 2.95 66.5 ± 5.11 0.54 
Sex    
 Male 0.13 
 Female  
Histologic grade    
 Well/moderately 11 0.81 
 Poorly/others  
Size    
 50 mm≥ (small) 10 0.63 
 51 mm≤ (large)  
Depth of tumor invasiona 
m, sm, mpa 
ss, se, si 12  
Lymph node metastasis 
 Absent 13 0.092 
 Present  
Lymphatic invasion 
 Absent 0.15 
 Present 12  
Venous invasion    
 Absent 0.046b 
 Present 14  
Liver metastasis    
 Absent 15 
 Present  
Dukes stage    
 AB 11 0.24 
 CD  
High expressionLow expression
Factors(n = 18)(n = 6)P
Age (mean ± SD) 62.8 ± 2.95 66.5 ± 5.11 0.54 
Sex    
 Male 0.13 
 Female  
Histologic grade    
 Well/moderately 11 0.81 
 Poorly/others  
Size    
 50 mm≥ (small) 10 0.63 
 51 mm≤ (large)  
Depth of tumor invasiona 
m, sm, mpa 
ss, se, si 12  
Lymph node metastasis 
 Absent 13 0.092 
 Present  
Lymphatic invasion 
 Absent 0.15 
 Present 12  
Venous invasion    
 Absent 0.046b 
 Present 14  
Liver metastasis    
 Absent 15 
 Present  
Dukes stage    
 AB 11 0.24 
 CD  

aTumor invasion of mucosa (m), submucosa (sm), muscularis propria (mp), subserosa (ss), penetration of serosa (se), and invasion of adjacent strucures (si)

bP < 0.05.

Table 5.

miR-92a expression in cancer stroma and clinocopathologic factors

High expressionLow expression
Factors(n = 6)(n = 18)P
Age (mean ± SD) 72.0 ± 4.74 61.0 ± 2.74 0.057 
Sex    
 Male 10 0.63 
 Female  
Histologic grade    
 Well/moderately 11 0.81 
 Poorly/others  
Size    
 50 mm> (small) 11 0.63 
 51 mm< (large)  
Depth of tumor invasiona 
m, sm, mpa 0.3 
ss, se, si 11  
Lymph node metastasis 
 Absent 12 0.47 
 Present  
Lymphatic invasion 
 Absent 10 0.0050b 
 Present  
Venous invasion    
 Absent 0.016b 
 Present 10  
Liver metastasis    
 Absent 17 0.018b 
 Present  
Dukes stage    
 AB 11 0.24 
 CD  
High expressionLow expression
Factors(n = 6)(n = 18)P
Age (mean ± SD) 72.0 ± 4.74 61.0 ± 2.74 0.057 
Sex    
 Male 10 0.63 
 Female  
Histologic grade    
 Well/moderately 11 0.81 
 Poorly/others  
Size    
 50 mm> (small) 11 0.63 
 51 mm< (large)  
Depth of tumor invasiona 
m, sm, mpa 0.3 
ss, se, si 11  
Lymph node metastasis 
 Absent 12 0.47 
 Present  
Lymphatic invasion 
 Absent 10 0.0050b 
 Present  
Venous invasion    
 Absent 0.016b 
 Present 10  
Liver metastasis    
 Absent 17 0.018b 
 Present  
Dukes stage    
 AB 11 0.24 
 CD  

aTumor invasion of mucosa (m), submucosa (sm), muscularis propria (mp), subserosa (ss), penetration of serosa (se), and invasion of adjacent strucures (si).

bP < 0.05.

Downregulated miRNAs in colorectal cancer stromal tissue compared with normal stromal tissue

We also investigated downregulated miRNAs in cancer stroma compared with normal stroma. As a result, previously reported tumor-suppressive miRNAs, such as the miR-192-miR-194 cluster, miR-215, miR-29c, miR-26b, and let-7g were all downregulated in cancer stromal tissues compared with normal stromal tissues (Supplementary Table S3) (22, 23). Downregulation of tumor-suppressive miRNAs in cancer stroma as well as upregulation of oncogenic miRNAs in cancer stroma indicated that alterations of miRNA expression in stromal tissues are similar to that in epithelial tissues.

Comparison of miRNA expression status in cancer stroma and cancer epithelium

Significantly upregulated or downregulated miRNAs in cancer stroma compared with cancer epithelium are listed in Supplementary Table S4A and S4B.

In this study, we analyzed expression levels of miRNAs expressed in cancer stroma and revealed that many oncogenic miRNAs including miR-21 (24), miR-221 (25), and almost all components of the miR-17-92a cluster and its homolog, and the miR-106b-25 cluster were upregulated in cancer stroma compared with normal stroma (Table 1). Previous reports showed that the miR-17-92a cluster is upregulated in lung cancer, colorectal cancer, lymphoma, multiple myeloma, and medulloblastoma, whereas the miR-106b-25 cluster is upregulated in gastric, colon, and prostate cancer, neuroblastoma, and multiple myeloma (9, 26, 27). Importantly, the expression of these miRNAs in stromal tissues had not been explored. We focused on these well-characterized miRNAs which are upregulated in cancer stroma as well as epithelial tissues.

The reason for the upregulation of such oncogenic miRNAs in stromal tissue is still unclear. However, previous studies have revealed genetic alterations such as LOH of cancer stromal cells (28) or epigenetic modification of cancer-associated fibroblasts (CAF; ref. 29). Hence, genetic or epigenetic changes of cancer stromal cells could be one explanation for this, although it remains controversial. Another possibility is intercellular transfer or penetration of miRNAs. It has been shown that secreted miRNAs from donor cells are transferred to and function in recipient cells. Some reports have shown that miRNAs are transferred in exosomes, vesicles of endocytic origin (30). If this occurred between cancer cells and stromal cells, profiles of miRNAs could become similar in both. We revealed that oncogenic miRNAs such as miR-135b, miR-221, and the miR-17-92a cluster were expressed at relatively higher levels in cancer epithelium than in stroma (Supplementary Table S4B). These findings suggest that upregulation of such oncogenic miRNAs in cancer stroma follows the upregulation of oncogenic miRNA in epithelium during carcinogenesis and cancer progression. However, further studies are required to confirm the interaction between cancer and its stroma.

Recently, Mestdagh and colleagues reported that the miR-17-92 miRNA cluster regulated key components of the TGFβ pathway such as SMAD2, SMAD4, and TGFBR2 in neuroblastoma (31). With regard to epithelial–mesenchymal interactions, Bhowmick and colleagues showed that ablation of TGFBR2 in stromal tissue can lead to carcinogenesis and cancer progression in vivo (3). In our data, TGFBR2, SMAD2, and SMAD3, which are putative targets of the miR-17-92a and miR-106b-25 cluster, were shown to be significantly repressed in cancer stroma (Table 2 and Table 3). These results suggest that, at least in part, inhibition of the TGFβ pathway in stromal tissue is associated with tumorigenesis and cancer progression. However, the putative miRNA target gene pathways we showed in this study have not been experimentally established, therefore, further exploration is needed to confirm that they function in stromal tissue.

Clinicopathologic analysis showed that miRNAs expression in stromal tissue is associated with the malignant potential of cancer (Table 4 and Table 5). High expression of miR-25 was associated with venous invasion, and miR-92a with lymphatic and venous invasion and liver metastasis. These data indicate that miRNA expression in stroma as well as in the epithelium can influence tumor aggressiveness in colorectal cancer. Lymphatic or venous invasion occurs dominantly in stromal tissue; therefore these results are entirely reasonable. Although validation in a larger number of samples is required in future studies, our data indicate the potential clinical significance of miRNAs in cancer stroma.

Stromal tissues are constituted by various kinds of cells, including immune cells, endothelial cells, and fibroblasts. Previous reports suggested CAF or tumor-associated macrophage could enhance tumor progression and metastasis (4, 32), and Vermeulen and colleagues showed that myofibroblasts play a role in the maintenance of cancer stem cell properties (33). In this study, we analyzed unsorted samples of stromal tissue, therefore the miRNA profiling for each cell type was not available. Sorting according to surface antigen, such as CD31 for endothelial cells or CD14 for macrophage, will be required for cell type–specific investigations in future studies.

Our findings suggest the possibility that oncogenic miRNAs including the miR-17-92a and miR-25-106b clusters in colorectal cancer stromal tissues are functionally associated with cancer progression. Because our results are based on microarray data derived from a small sample size, further validation is required.

No potential conflicts of interest were disclosed.

Conception and design: N. Nishida, M. Nagahara, K. Mimori, H. Ishii, M. Mori

Development of methodology: N. Nishida, M. Nagahara, F. Tanaka, H. Ishii, M. Mori

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Nishida, H. Ishii, M. Mori

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Nishida, T. Sato, H. Ishii, M. Mori

Writing, review, and/or revision of the manuscript: N. Nishida, K. Mimori, T. Sudo, F. Tanaka, H. Ishii, M. Mori

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N. Nishida, T. Sudo, H. Ishii, M. Mori

Study supervision: N. Nishida, K. Mimori, F. Tanaka, K. Shibata, K. Sugihara, Y. Doki, M. Mori

The authors thank T. Shimooka, K. Ogata, M. Kasagi, and T. Kawano for their excellent technical assistance.

This work was supported in part by the following grants and foundations: CREST, Japan Science and Technology Agency (JST); Japan Society for the Promotion of Science (JSPS) grant-in-aid for Scientific Research: 21679006, 20390360, 20590313, 20591547, 21591644, 21592014, 20790960, 21791297, 21229015, 20659209, and 20012039; NEDO (New Energy and Industrial Technology Development Organization) Technological Development for Chromosome Analysis; The Ministry of Education, Culture, Sports, Science and Technology of Japan for Scientific Research on Priority Areas, Cancer Translational Research Project, Japan; and LS094, Bureau of Science, Technology and Innovation Policy, Cabinet Office, Government Of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data