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. 2009 Oct 20:9:374.
doi: 10.1186/1471-2407-9-374.

Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer

Greg M Arndt  1 , Lesley Dossey, Lara M Cullen, Angela Lai, Riki Druker, Michael Eisbacher, Chunyan Zhang, Nham Tran, Hongtao Fan, Kathy Retzlaff, Anton Bittner, Mitch Raponi
Affiliations

Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer

Greg M Arndt et al. BMC Cancer. .

Abstract

Background: MicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis.

Results: The expression analysis of clinical CRC samples identified 37 miRNAs that were differentially expressed between CRC and normal tissue. Furthermore, several of these miRNAs were associated with CRC tumor progression including loss of miR-133a and gain of miR-224. We identified 11 common miRNAs that were differentially expressed between normal colon and CRC in both the cell line models and clinical samples. In vitro functional studies indicated that miR-143 and miR-145 appear to function in opposing manners to either inhibit or augment cell proliferation in a metastatic CRC model. The pathways targeted by miR-143 and miR-145 showed no significant overlap. Furthermore, gene expression analysis of metastatic versus non-metastatic isogenic cell lines indicated that miR-145 targets involved in cell cycle and neuregulin pathways were significantly down-regulated in the metastatic context.

Conclusion: MiRNAs showing altered expression at different stages of CRC could be targets for CRC therapies and be further developed as potential diagnostic and prognostic analytes. The identified biological processes and signalling pathways collectively targeted by co-expressed miRNAs in CRC provide a basis for understanding the functional role of miRNAs in cancer.

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Figures

Figure 1
Figure 1
Two-way hierarchical clustering of CRC and normal colorectal tissue using 37 differentially expressed miRNAs. The hypergeometric mean of the log2 signal intensity was calculated across all samples using a euclidean distance metric with complete linkage. Red and blue indicates miRNAs expressed at a high or low level relative to other samples in the dataset, respectively. The miR-143-145 and miR-17-92 clusters are indicated by vertical blue and red bars, respectively. Samples are grouped into two main clusters: Cluster I primarily represents normal colorectal tissue, and clusters II represents CRC samples. Replicate samples are indicated by the suffix "2".
Figure 2
Figure 2
Correlation of miRNA expression comparing the mirVana Bioarray and ABI Taqman platforms. Linear regression was performed on miRNAs that were measured on both platforms. Four different CRC clinical samples were examined. Taqman and Ambion miRNA expression is measured in Ct and log2 units, respectively. The Pearson correlation was calculated for each comparison.
Figure 3
Figure 3
miRNA expression in fresh frozen versus formalin-fixed paraffin embedded CRC samples. Taqman miRNA expression assays were performed on 169 miRNAs from 4 matched fresh frozen and FFPE samples and Ct measurements were compared by linear regression. The Pearson correlation was calculated for each comparison.
Figure 4
Figure 4
Expression of 14 miRNAs in 8 CRC cell lines and normal colon total RNA. Northern blots were performed using U6 snRNA as a normalization control.
Figure 5
Figure 5
Over-expression of miR-143 in the SW620 cell line affects cell morphology and proliferation. (A) MiR-143 genomic DNA was cloned under control of the U6 promoter (in pSilencer 2.1) and transfected into SW620 cells. Seven stable SW620 clones were identified that expressed miR-143 (A4, B3, B5, B6, C1, C5, and D2). U6 snRNA was used as a loading control. (B) The seven SW620/miR-143 clones were examined for cell morphology compared to vector control. (C) Western analysis of the SW620/miR-143 clones and control cells shows the steady-state levels of E-cadherin. E-cadherin ratios were calculated using β-actin as a normalization control. (D) Seven SW620/miR-143 clones were assayed for proliferation/metabolic activity compared with vector control when grown in the absence (open bars) or presence of serum (solid bars). (E) The same clones were examined for anchorage-independent cell growth in the rapid soft agar assay in the presence of serum. Two independent experiments were performed. ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
Over-expression of miR-145 in SW620 cell line affects cell morphology and proliferation. (A) The genomic region surrounding the miR-145 gene was PCR-amplified and cloned into pSilencer 4.1 under control of the CMV promoter. Mature miR-145 was detected by Northern analysis in a pooled population of SW620 cells following transfection. U6 snRNA was used as a loading control. (B) A major distinguishing feature of the cell population over-expressing miR-145 was the change in cell morphology from the round single cells of SW620 to elongated cells with extended processes typical of fibroblast-like cells. (C) The miR-145-expressing SW620 cell population showed a two-fold increase in anchorage-independent growth when grown in the presence of serum and a greater than 50% increase in cell proliferation/metabolic activity when grown in the presence (solid bars) or absence (open bars) of serum. *** p < 0.001. (D) Western analysis of the SW620/miR-145 cells and control cells shows the steady-state levels of E-cadherin were 50% lower in SW620 cells expressing the mature miR-145. FBS = fetal bovine serum, SF = serum-free.
Figure 7
Figure 7
Antisense-mediated reversion of miR-145-induced proliferation. (A) Total RNA from treated samples showed that miR-145 was depleted in cells receiving the miR-145-specific 2'Ome antisense RNA but not in the mock treated, or miR-145 sense treated controls. (B) As expected, SW620/miR-145 expressing pools showed increased proliferation compared to vector controls in the presence (solid bars) or absence (open bars) of serum. When treated with miR-145 antisense RNA, a reduction in proliferation was seen in both SW620/vector and SW620/miR-145 pools. Three independent experiments were performed. * p < 0.05; ** p < 0.01, *** p < 0.001.
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