pone-0002436-g005: Validation of illumina arrays data for let-7f.Transcripts identified by illumina arrays to be altered following let-7f overexpression are validated by RT-PCR. Fold changes for genes KIF1A, ASS, FDPS, NTS (in UCI-101 cells), and TFF1, EEF1A2, ESM1, VIM (in BG-1 cells) are shown and confirm the changes identified by illumina arrays.

Mentions: To further study the potential targets of key ovarian miRNAs, we overexpressed miR-34c, miR-98, miR-424, and let-7f in 2 ovarian cancer cell lines (BG-1 and UCI-101). miR-424 and let-7f were downregulated in both tissues and cell lines (Figure 3A), while miR-34c and miR-98 were found downregulated in ovarian cancer tissues and in cell lines, respectively. Figure 4 shows the successful over-expression of the miRNAs 72 hours following transfection. We then used Illumina microarrays to investigate mRNA level changes following the overexpression of these candidates. Tables 3 and 4 list the transcripts that were most significantly altered following over-expression of the miRNAs in BG-1 and UCI-101 cell lines, respectively. Interestingly, these tables include genes such as GPX3, MCM7, MSN that have previously been implicated in ovarian cancer. Among the significantly altered genes (absolute Z-ratio>1.5), there are 10 known cancer genes (MSN, PIM1, CBL, COL1A1, COX6C, EVI1, EXT1, HLXB9, PTPN11, TCEA1), 4 tumor suppressor genes (HIF1A, CAV1, GADD45A, PTTG1IP), 26 cell cycle genes (ACAT2, CDK5, FDPS, ID1, LLGL1, MAD2L1, ANLN, ATAD2, C12orf48, CD9, ECT2, GADD45A, GSTO1, HSPD1, IPO7, KNTC2, KRT18, MRPS17, NUDT1, PFN1, PRKCA, SFRP1, SKP2, SNRPA1, VAMP8, WEE1), and 6 genes involved in chromatin remodeling (ASF1A, GCN5L2, ATAD2, CBX2, CBX4, NCOA3). Very little overlap was found between the predicted (Pictar and Target Scan) and experimental targets. Interestingly, the experimental targets varied according to the cell line used, suggesting a significant influence of the molecular background on miRNA target selection. In order to validate the Illumina data, RT-PCR was performed on 8 genes found to be targets of let-7f (KIF1A, ASS, FDPS, NTS in UCI-101 cells, and TFF1, EEF1A2, ESM1, VIM, in BG-1 cells) (Figure 5). We found that, while the absolute values were different, the trends were the same. KIF1A and ASS were found elevated in UCI-101, while FDPS and NTS were downregulated in these cells. TFF1 and EEF1A2 were confirmed to be elevated in BG-1, while ESM1 and VIM were downregulated.

Dahiya N, Sherman-Baust CA, Wang TL, Davidson B, Shih IeM, Zhang Y, Wood W, Becker KG, Morin PJ - PLoS ONE (2008)

Bottom Line: Public databases were used to reveal potential targets for the highly differentially expressed miRNAs.In order to experimentally identify transcripts whose stability may be affected by the differentially expressed miRNAs, we transfected precursor miRNAs into human cancer cell lines and used oligonucleotide microarrays to examine changes in the mRNA levels.These miRNAs and their targets may have important roles in the initiation and development of ovarian cancer.

Affiliation: Laboratory of Cellular and Molecular Biology, Hopkins Medical Institutions, Baltimore, Maryland, United States of America.

Abstract: MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of cancers, and these genes are thought to function as both tumor suppressors and oncogenes.Using microRNA microarrays, we identify several miRNAs aberrantly expressed in human ovarian cancer tissues and cell lines. miR-221 stands out as a highly elevated miRNA in ovarian cancer, while miR-21 and several members of the let-7 family are found downregulated. Public databases were used to reveal potential targets for the highly differentially expressed miRNAs. In order to experimentally identify transcripts whose stability may be affected by the differentially expressed miRNAs, we transfected precursor miRNAs into human cancer cell lines and used oligonucleotide microarrays to examine changes in the mRNA levels. Interestingly, there was little overlap between the predicted and the experimental targets or pathways, or between experimental targets/pathways obtained using different cell lines, highlighting the complexity of miRNA target selection.Our results identify several differentially expressed miRNAs in ovarian cancer and identify potential target transcripts that may be regulated by these miRNAs. These miRNAs and their targets may have important roles in the initiation and development of ovarian cancer.