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EphB6 receptor modulates micro RNA profile of breast carcinoma cells.

Bhushan L, Kandpal RP - PLoS ONE (2011)

Bottom Line: Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6.These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion.The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Medical Sciences, Western University of Health Sciences, Pomona, California, United States of America.

ABSTRACT
Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.

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Significantly altered miRNAs in EphB6 transfected MDA-MB-231 cells.A. Volcano plot indicating miRNAs with dLMR values greater than 0.5 or less than −0.5 at a p-value of less than 0.001. The symbols in red color represent the miRNAs meeting the cut-off values. B. Variations in the values of delta LogMeadianRatio (dLMR) for selected miRNAs are indicated by standard deviations among three replicates.
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pone-0022484-g002: Significantly altered miRNAs in EphB6 transfected MDA-MB-231 cells.A. Volcano plot indicating miRNAs with dLMR values greater than 0.5 or less than −0.5 at a p-value of less than 0.001. The symbols in red color represent the miRNAs meeting the cut-off values. B. Variations in the values of delta LogMeadianRatio (dLMR) for selected miRNAs are indicated by standard deviations among three replicates.

Mentions: The array data were filtered by applying absolute Δ LogMedianRatios (dLMR), and the results are shown as a Volcano plot (Figure 2A). These analyses revealed significant alterations in 8 miRNAs comprising of miR-26a, miRPlus-E1172, miR-100, miR-23a, miR-16, miR-29a, miR-24 and miRPlus-E1258 that were altered with a p-value of less than 0.001 and dLMR either greater than 0.5 or less than −0.5. Among these miRNAs, seven were up-regulated and miRPlus-1258 was significantly down-regulated in EphB6 transfected cells. The differences in the above miRNAs were further confirmed by plotting the dLMR with standard deviations. The standard deviations for triplicate measurements of vector-transfected and EphB6-transfected samples were well within the acceptable range (Figure 2B). A coordinated expression pattern was observed for members of two polycistronic miRNA clusters containing miR-23a, miR-23b, miR-24 and miR-27. The levels of miR-23a, miR-23b and miR-24 were comparable in all vector-transfected MDA-MB-231 clones. However, their levels in EphB6-transfected clones were significantly higher than the vector-transfected clones. Similar changes were observed in the levels of miR-27 between vector-transfected and EphB6-transfected clones (Figure 3).


EphB6 receptor modulates micro RNA profile of breast carcinoma cells.

Bhushan L, Kandpal RP - PLoS ONE (2011)

Significantly altered miRNAs in EphB6 transfected MDA-MB-231 cells.A. Volcano plot indicating miRNAs with dLMR values greater than 0.5 or less than −0.5 at a p-value of less than 0.001. The symbols in red color represent the miRNAs meeting the cut-off values. B. Variations in the values of delta LogMeadianRatio (dLMR) for selected miRNAs are indicated by standard deviations among three replicates.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139643&req=5

pone-0022484-g002: Significantly altered miRNAs in EphB6 transfected MDA-MB-231 cells.A. Volcano plot indicating miRNAs with dLMR values greater than 0.5 or less than −0.5 at a p-value of less than 0.001. The symbols in red color represent the miRNAs meeting the cut-off values. B. Variations in the values of delta LogMeadianRatio (dLMR) for selected miRNAs are indicated by standard deviations among three replicates.
Mentions: The array data were filtered by applying absolute Δ LogMedianRatios (dLMR), and the results are shown as a Volcano plot (Figure 2A). These analyses revealed significant alterations in 8 miRNAs comprising of miR-26a, miRPlus-E1172, miR-100, miR-23a, miR-16, miR-29a, miR-24 and miRPlus-E1258 that were altered with a p-value of less than 0.001 and dLMR either greater than 0.5 or less than −0.5. Among these miRNAs, seven were up-regulated and miRPlus-1258 was significantly down-regulated in EphB6 transfected cells. The differences in the above miRNAs were further confirmed by plotting the dLMR with standard deviations. The standard deviations for triplicate measurements of vector-transfected and EphB6-transfected samples were well within the acceptable range (Figure 2B). A coordinated expression pattern was observed for members of two polycistronic miRNA clusters containing miR-23a, miR-23b, miR-24 and miR-27. The levels of miR-23a, miR-23b and miR-24 were comparable in all vector-transfected MDA-MB-231 clones. However, their levels in EphB6-transfected clones were significantly higher than the vector-transfected clones. Similar changes were observed in the levels of miR-27 between vector-transfected and EphB6-transfected clones (Figure 3).

Bottom Line: Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6.These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion.The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Medical Sciences, Western University of Health Sciences, Pomona, California, United States of America.

ABSTRACT
Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.

Show MeSH
Related in: MedlinePlus