Potential role of miR-9 and miR-223 in recurrent ovarian cancer

Abstract

Background: MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression by binding to target mRNAs. miRNAs have not been comprehensively studied in recurrent ovarian cancer, yet an incurable disease.

Results: Using real-time RT-PCR, we obtained distinct miRNA expression profiles between primary and recurrent serous papillary ovarian adenocarcinomas (n = 6) in a subset of samples previously used in a transcriptome approach. Expression levels of top dysregulated miRNA genes, miR-223 and miR-9, were examined using TaqMan PCR in independent cohorts of fresh frozen (n = 18) and FFPE serous ovarian tumours (n = 22). Concordance was observed on TaqMan analysis for miR-223 and miR-9 between the training cohort and the independent test cohorts. Target prediction analysis for the above miRNA "recurrent metastatic signature" identified genes previously validated in our transcriptome study. Common biological pathways well characterised in ovarian cancer were shared by miR-9 and miR-223 lists of predicted target genes. We provide strong evidence that miR-9 acts as a putative tumour suppressor gene in recurrent ovarian cancer. Components of the miRNA processing machinery, such as Dicer and Drosha are not responsible for miRNA deregulation in recurrent ovarian cancer, as deluded by TaqMan and immunohistochemistry.

Conclusion: We propose a miRNA model for the molecular pathogenesis of recurrent ovarian cancer. Some of the differentially deregulated miRNAs identified correlate with our previous transcriptome findings. Based on integrated transcriptome and miRNA analysis, miR-9 and miR-223 can be of potential importance as biomarkers in recurrent ovarian cancer.

Figure 1

Flow chart of our study design. miRNA profiling was performed using TaqMan PCR in a homogenous set of 3 primary advanced and 3 recurrent serous papillary ovarian adenocarcinomas from different patients. Selected miRNA targets (n = 2) were examined in two independent sets of primary and recurrent fresh frozen (n = 18) and FFPE serous papillary adenocarcinomas (n = 22). We also attempted to determine whether components of the miRNA processing machinery (Dicer and Drosha) are responsible for miRNA dysregulation in recurrent ovarian cancer.

Figure 2

Differentially expressed miRNAs in recurrent versus primary ovarian cancers. Relative fold changes are displayed on the y axis and the miRNAs are on the x axis. The most significant altered expression was observed for mir-223 (up) and mir-9 (down).

Figure 3

Unsupervised hierarchical cluster heatmap based on differential miRNA expression patterns identified in the initial cohort. Vertical bars represent the samples and the horizontal bars represent the miRNA genes. Green bars reflect downregulated genes and red bars upregulated genes. Interestingly, P3 clusters with the recurrent samples on the left of the heatmap. P3 relapsed within 6 months post completion of treatment and should be considered "chemoresistant" P, primary tumours; R, recurrent tumors.

Figure 4

Independent TaqMan^® PCR validation of top dysregulated miRNAs. Concordance was observed on TaqMan analysis for miR223 and miR9 between the training and expanded independent cohort of serous ovarian carcinomas (comprised of snap frozen tissues).

Figure 5

TaqMan^® PCR validation in training cohort and FFPE cohort. The relative quantitation from TaqMan^® in recurrent vs primary tumours are plotted for both cohorts.

Figure 6

Figure illustrating common biological pathways identified when the lists of predicted targets for miR-9 and miR-223 were exported in the Panther classification system and examined against the H. sapiens reference list to determine the percentage of genes compared to what expected. Common pathways between the top dysregulated genes were significant at a p < 0.05 as highlighted by the orange bars.

Figure 7

Expression of eIF6 and Dicer in normal ovarian surface epithelium and ovarian serous adenocarcinoma (×40): No staining ('0') for eIF6 (A) and Dicer (B) in normal ovarian surface epithelium. Strong ('3') staining for eIF6 (C) and Dicer (D) in ovarian serous adenocarcinomas.

Figure 8

Global staining pattern for eIF6 (e) and Dicer (f) in a TMA of primary serous papillary ovarian adenocarcinomas. Global staining pattern for eiF6 and Dicer in the TMA of recurrent ovarian tumours is not shown. Analysis was based on relative expression levels of eIF6 and Dicer in primary vs recurrent tumours.

Figure 9

A miRNA model for the molecular pathogenesis of recurrent ovarian cancer. Based on integrated transcriptome and miRNA analysis, we identified a "recurrent metastatic signature" comprised top dysregulated miRNAs, miR-9 (down) and miR-223 (up) that correlate with our previous transcriptome findings. miR-9 appears to be a tumour suppressor gene and when downregulated, it can lead to drug resistance. miR-223 can act either as oncogene or tumour suppressor gene. Genes highlighted in red were previously identified in our transcriptome study. Genes in blue have been well characterised in ovarian cancer. FGF signalling pathway appears to be commonly shared between our identified miRNAs.