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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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Related in: MedlinePlus

Amplification of mRNAs targeted by various miRNAs.Each panel shows a representative gel picture for the transcript levels in empty vector-transfected (left lane) and EphB6-transfected (right lane) MDA-MB-231 cells. The bar diagram on the right shows quantification of three gels for the levels of transcripts. The Y-axis represents arbitrary units of intensity. Empty vector-transfected and EphB6-transfected MDA-MB-231 cells are designated as −Eph and +Eph. The transcript designations are indicated in each panel.
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pone-0022484-g005: Amplification of mRNAs targeted by various miRNAs.Each panel shows a representative gel picture for the transcript levels in empty vector-transfected (left lane) and EphB6-transfected (right lane) MDA-MB-231 cells. The bar diagram on the right shows quantification of three gels for the levels of transcripts. The Y-axis represents arbitrary units of intensity. Empty vector-transfected and EphB6-transfected MDA-MB-231 cells are designated as −Eph and +Eph. The transcript designations are indicated in each panel.

Mentions: We selected SMARCC1, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB3, BMPR2 and BMPR1A for validation by PCR. As shown in Figures 5 and 6, the levels of these transcripts were significantly down-regulated in MDA-MB-231 cells transfected with EphB6. Noteworthy among these targets were BMPR2 and eIF4EB2. The mRNAs for BMPR2 and eIF4EB2 were barely detectable in EphB6 transfected cells. The levels of SMARCC1, eIF2C4, FKABP5, FKBP1A, TRIB1, TRIB3 and BMPR1A in the transfected cells were between 20% and 30% of the native cells. These results validate the rationale for the selection approach, and clearly demonstrate the low abundance of a target mRNA for which the relevant miRNA is up-regulated in the cell. These results clearly demonstrate an inverse relationship between the levels of miRNAs and their mRNA targets.


EphB6 receptor modulates micro RNA profile of breast carcinoma cells.

Bhushan L, Kandpal RP - PLoS ONE (2011)

Amplification of mRNAs targeted by various miRNAs.Each panel shows a representative gel picture for the transcript levels in empty vector-transfected (left lane) and EphB6-transfected (right lane) MDA-MB-231 cells. The bar diagram on the right shows quantification of three gels for the levels of transcripts. The Y-axis represents arbitrary units of intensity. Empty vector-transfected and EphB6-transfected MDA-MB-231 cells are designated as −Eph and +Eph. The transcript designations are indicated in each panel.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022484-g005: Amplification of mRNAs targeted by various miRNAs.Each panel shows a representative gel picture for the transcript levels in empty vector-transfected (left lane) and EphB6-transfected (right lane) MDA-MB-231 cells. The bar diagram on the right shows quantification of three gels for the levels of transcripts. The Y-axis represents arbitrary units of intensity. Empty vector-transfected and EphB6-transfected MDA-MB-231 cells are designated as −Eph and +Eph. The transcript designations are indicated in each panel.
Mentions: We selected SMARCC1, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB3, BMPR2 and BMPR1A for validation by PCR. As shown in Figures 5 and 6, the levels of these transcripts were significantly down-regulated in MDA-MB-231 cells transfected with EphB6. Noteworthy among these targets were BMPR2 and eIF4EB2. The mRNAs for BMPR2 and eIF4EB2 were barely detectable in EphB6 transfected cells. The levels of SMARCC1, eIF2C4, FKABP5, FKBP1A, TRIB1, TRIB3 and BMPR1A in the transfected cells were between 20% and 30% of the native cells. These results validate the rationale for the selection approach, and clearly demonstrate the low abundance of a target mRNA for which the relevant miRNA is up-regulated in the cell. These results clearly demonstrate an inverse relationship between the levels of miRNAs and their mRNA targets.

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