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Identification and characterization of new miRNAs cloned from normal mouse mammary gland.

Sdassi N, Silveri L, Laubier J, Tilly G, Costa J, Layani S, Vilotte JL, Le Provost F - BMC Genomics (2009)

Bottom Line: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes.Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them.If none of them is mammary gland specific, a few of them are not ubiquitously expressed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR 339, Laboratoire de Génétique Biochimique et Cytogénétique, Jouy-en-Josas, France. Nezha.Sdassi@jouy.inra.fr

ABSTRACT

Background: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Until now their implication in the mammary gland biology was suggested by few studies mainly focusing on pathological situations allowing the characterization of miRNAs as markers of breast cancer tumour classes. If in the normal mammary gland, the expression of known miRNAs has been studied in human and mice but the full repertoire of miRNAs expressed in this tissue is not yet available.

Results: To extend the repertoire of mouse mammary gland expressed miRNAs, we have constructed several libraries of small miRNAs allowing the cloning of 455 sequences. After bioinformatics' analysis, 3 known miRNA (present in miRbase) and 33 new miRNAs were identified. Expression of 24 out of the 33 has been confirmed by RT-PCR. Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them. No correlation could be established between their expression pattern and evolutionary conservation. Six of them appear to be mouse specific. In several cases, multiple potential precursors of miRNA were present in the genome and we have developed a strategy to determine which of them was able to mature the miRNA.

Conclusion: The cloning approach has allowed improving the repertoire of miRNAs in the mammary gland, an evolutionary recent organ. This tissue is a good candidate to find tissue-specific miRNAs and to detect miRNA specific to mammals. We provide evidence for 24 new miRNA. If none of them is mammary gland specific, a few of them are not ubiquitously expressed. For the first time 6 mouse specific miRNA have been identified.

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Related in: MedlinePlus

Functional validation of precursors MG008-X and MG053-01. Detection of miRNAs MG008 and MG053 after transfection of COS-7 cell with expression vectors vMG008 and vMG053 by Northern blot analysis. RNAs were extracted from cells transfected with expression vectors (T) and with the control empty vector (C), separated onto a 15% denaturing PAGE and transferred onto a nylon membrane Blots were hybridized with miRNA antisense oligonucleotides and a U6 probe used as an internal loading control.
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Figure 4: Functional validation of precursors MG008-X and MG053-01. Detection of miRNAs MG008 and MG053 after transfection of COS-7 cell with expression vectors vMG008 and vMG053 by Northern blot analysis. RNAs were extracted from cells transfected with expression vectors (T) and with the control empty vector (C), separated onto a 15% denaturing PAGE and transferred onto a nylon membrane Blots were hybridized with miRNA antisense oligonucleotides and a U6 probe used as an internal loading control.

Mentions: The bioinformatics' analysis allowed the detection of potential precursors, but these results could not determine if these hairpin structures will be matured by RNase III, Drosha in the nucleus and Dicer in the cytoplasm. The use of expression vectors in cell culture, allowing the synthesis of hairpin structures that are matured into miRNAs, has already been demonstrated [44,45]. To test the functional validity of the precursors obtained by bioinformatics' analysis, we expressed them in transfected cells and checked for the presence of the mature miRNAs. The constructs carrying the precursors (MG008-X and MG053-01) were transiently transfected in COS-7 cells and the expression of the mature miRNAs (MG008 and MG053) was studied by Northern blot analysis (Figure 4). As a control the precursor of a known miRNA (let-7b) was used (data not shown). This approach could be used to validate precursor(s) of miRNA.


Identification and characterization of new miRNAs cloned from normal mouse mammary gland.

Sdassi N, Silveri L, Laubier J, Tilly G, Costa J, Layani S, Vilotte JL, Le Provost F - BMC Genomics (2009)

Functional validation of precursors MG008-X and MG053-01. Detection of miRNAs MG008 and MG053 after transfection of COS-7 cell with expression vectors vMG008 and vMG053 by Northern blot analysis. RNAs were extracted from cells transfected with expression vectors (T) and with the control empty vector (C), separated onto a 15% denaturing PAGE and transferred onto a nylon membrane Blots were hybridized with miRNA antisense oligonucleotides and a U6 probe used as an internal loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683868&req=5

Figure 4: Functional validation of precursors MG008-X and MG053-01. Detection of miRNAs MG008 and MG053 after transfection of COS-7 cell with expression vectors vMG008 and vMG053 by Northern blot analysis. RNAs were extracted from cells transfected with expression vectors (T) and with the control empty vector (C), separated onto a 15% denaturing PAGE and transferred onto a nylon membrane Blots were hybridized with miRNA antisense oligonucleotides and a U6 probe used as an internal loading control.
Mentions: The bioinformatics' analysis allowed the detection of potential precursors, but these results could not determine if these hairpin structures will be matured by RNase III, Drosha in the nucleus and Dicer in the cytoplasm. The use of expression vectors in cell culture, allowing the synthesis of hairpin structures that are matured into miRNAs, has already been demonstrated [44,45]. To test the functional validity of the precursors obtained by bioinformatics' analysis, we expressed them in transfected cells and checked for the presence of the mature miRNAs. The constructs carrying the precursors (MG008-X and MG053-01) were transiently transfected in COS-7 cells and the expression of the mature miRNAs (MG008 and MG053) was studied by Northern blot analysis (Figure 4). As a control the precursor of a known miRNA (let-7b) was used (data not shown). This approach could be used to validate precursor(s) of miRNA.

Bottom Line: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes.Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them.If none of them is mammary gland specific, a few of them are not ubiquitously expressed.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR 339, Laboratoire de Génétique Biochimique et Cytogénétique, Jouy-en-Josas, France. Nezha.Sdassi@jouy.inra.fr

ABSTRACT

Background: MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Until now their implication in the mammary gland biology was suggested by few studies mainly focusing on pathological situations allowing the characterization of miRNAs as markers of breast cancer tumour classes. If in the normal mammary gland, the expression of known miRNAs has been studied in human and mice but the full repertoire of miRNAs expressed in this tissue is not yet available.

Results: To extend the repertoire of mouse mammary gland expressed miRNAs, we have constructed several libraries of small miRNAs allowing the cloning of 455 sequences. After bioinformatics' analysis, 3 known miRNA (present in miRbase) and 33 new miRNAs were identified. Expression of 24 out of the 33 has been confirmed by RT-PCR. Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them. No correlation could be established between their expression pattern and evolutionary conservation. Six of them appear to be mouse specific. In several cases, multiple potential precursors of miRNA were present in the genome and we have developed a strategy to determine which of them was able to mature the miRNA.

Conclusion: The cloning approach has allowed improving the repertoire of miRNAs in the mammary gland, an evolutionary recent organ. This tissue is a good candidate to find tissue-specific miRNAs and to detect miRNA specific to mammals. We provide evidence for 24 new miRNA. If none of them is mammary gland specific, a few of them are not ubiquitously expressed. For the first time 6 mouse specific miRNA have been identified.

Show MeSH
Related in: MedlinePlus