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Identification of differentially expressed sense and antisense transcript pairs in breast epithelial tissues.

Grigoriadis A, Oliver GR, Tanney A, Kendrick H, Smalley MJ, Jat P, Neville AM - BMC Genomics (2009)

Bottom Line: Expression of 431 NATs were confirmed by either of the other two technologies.Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated.Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ludwig Institute for Cancer Research, 605 Third Avenue, New York, NY 10158, USA. anita.grigoriadis@kcl.ac.uk

ABSTRACT

Background: More than 20% of human transcripts have naturally occurring antisense products (or natural antisense transcripts--NATs), some of which may play a key role in a range of human diseases. To date, several databases of in silico defined human sense-antisense (SAS) pairs have appeared, however no study has focused on differential expression of SAS pairs in breast tissue. We therefore investigated the expression levels of sense and antisense transcripts in normal and malignant human breast epithelia using the Affymetrix HG-U133 Plus 2.0 and Almac Diagnostics Breast Cancer DSA microarray technologies as well as massively parallel signature sequencing (MPSS) data.

Results: The expression of more than 2500 antisense transcripts were detected in normal breast duct luminal cells and in primary breast tumors substantially enriched for their epithelial cell content by DSA microarray. Expression of 431 NATs were confirmed by either of the other two technologies. A corresponding sense transcript could be identified on DSA for 257 antisense transcripts. Of these SAS pairs, 163 have not been previously reported. A positive correlation of differential expression between normal and malignant breast samples was observed for most SAS pairs. Orientation specific RT-QPCR of selected SAS pairs validated their expression in several breast cancer cell lines and solid breast tumours.

Conclusion: Disease-focused and antisense enriched microarray platforms (such as Breast Cancer DSA) confirm the assumption that antisense transcription in the human breast is more prevalent than previously anticipated. Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated. Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs.

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Quantitative strand-specific RT-PCR analysis of SAS pair expression in breast cancer cell lines and solid primary breast tumours. cDNAs of 15 breast cancer cell lines and 10 primary breast tumours (BC) were analyzed using the ΔΔCT relative quantification real-time qPCR. Red bars represent sense transcripts, green bars the corresponding antisense transcripts. Analysis of qPCR data was performed using the immortalised luminal cell line (226L) as comparator for all the breast cell lines and solid primary breast tumours (indicates as BC). ACTB was used as endogenous control throughout all analysis. ΔΔCT relative quantification data are expressed as mean fold changes across samples together with 95% confidence intervals.
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Figure 4: Quantitative strand-specific RT-PCR analysis of SAS pair expression in breast cancer cell lines and solid primary breast tumours. cDNAs of 15 breast cancer cell lines and 10 primary breast tumours (BC) were analyzed using the ΔΔCT relative quantification real-time qPCR. Red bars represent sense transcripts, green bars the corresponding antisense transcripts. Analysis of qPCR data was performed using the immortalised luminal cell line (226L) as comparator for all the breast cell lines and solid primary breast tumours (indicates as BC). ACTB was used as endogenous control throughout all analysis. ΔΔCT relative quantification data are expressed as mean fold changes across samples together with 95% confidence intervals.

Mentions: Since none of the identified SAS pairs exhibited an opposite fold change between the normal and the malignant breast epithelium, we wanted to explore further whether these SAS pairs were exclusively showing positive differential expression patterns. Three of the 163 novel SAS pairs were randomly selected and strand-specific RT-PCR used to measure their expression. The 3 pairs, corresponding to discoidin, CUB and LCCL domain containing 2 (DCBLD2, NM_080927.3), matrix metallopeptidase 24 (MMP24, NM_006690.3) and keratin 81 (KRT81, NM_002281.3) had their expression interrogated in 16 breast cancer cell lines and ten solid primary breast tumours (Figure 4). To distinguish expression coming from the sense or the antisense strand, the first strand cDNA synthesis of the RT-PCR was set up either with the sense primer (generating cDNA from sense strand mRNA transcripts) or the antisense primer (generating cDNA from RNA from the antisense strand). FAM labeled fluorescent probes for each gene were used to determine the relative expression levels of the sense and antisense by qPCR. KRT81 showed similar expression patterns in all tested samples with regards to sense and antisense transcription and overall expression levels. In contrast, DCBLD2 and MMP24 showed significantly higher expression ratios in the solid tumours in comparison with the breast cancer cell lines. Furthermore the sense and antisense transcripts of DCBLD2 had different expression levels in BT474 and T47D, two hormone-receptor positive luminal-specific breast cancer cell lines. When the expression of the antisense and sense MMP24 transcript was interrogated, opposite expression levels were observed in five breast cancer cell lines (namely MDA-MB-231 and HMT3552 of basal-subtype; and BT474, MDA-MB-453, SKBR5 and SKBR7 of luminal-subtype) as well as in five solid breast tumours.


Identification of differentially expressed sense and antisense transcript pairs in breast epithelial tissues.

Grigoriadis A, Oliver GR, Tanney A, Kendrick H, Smalley MJ, Jat P, Neville AM - BMC Genomics (2009)

Quantitative strand-specific RT-PCR analysis of SAS pair expression in breast cancer cell lines and solid primary breast tumours. cDNAs of 15 breast cancer cell lines and 10 primary breast tumours (BC) were analyzed using the ΔΔCT relative quantification real-time qPCR. Red bars represent sense transcripts, green bars the corresponding antisense transcripts. Analysis of qPCR data was performed using the immortalised luminal cell line (226L) as comparator for all the breast cell lines and solid primary breast tumours (indicates as BC). ACTB was used as endogenous control throughout all analysis. ΔΔCT relative quantification data are expressed as mean fold changes across samples together with 95% confidence intervals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2721853&req=5

Figure 4: Quantitative strand-specific RT-PCR analysis of SAS pair expression in breast cancer cell lines and solid primary breast tumours. cDNAs of 15 breast cancer cell lines and 10 primary breast tumours (BC) were analyzed using the ΔΔCT relative quantification real-time qPCR. Red bars represent sense transcripts, green bars the corresponding antisense transcripts. Analysis of qPCR data was performed using the immortalised luminal cell line (226L) as comparator for all the breast cell lines and solid primary breast tumours (indicates as BC). ACTB was used as endogenous control throughout all analysis. ΔΔCT relative quantification data are expressed as mean fold changes across samples together with 95% confidence intervals.
Mentions: Since none of the identified SAS pairs exhibited an opposite fold change between the normal and the malignant breast epithelium, we wanted to explore further whether these SAS pairs were exclusively showing positive differential expression patterns. Three of the 163 novel SAS pairs were randomly selected and strand-specific RT-PCR used to measure their expression. The 3 pairs, corresponding to discoidin, CUB and LCCL domain containing 2 (DCBLD2, NM_080927.3), matrix metallopeptidase 24 (MMP24, NM_006690.3) and keratin 81 (KRT81, NM_002281.3) had their expression interrogated in 16 breast cancer cell lines and ten solid primary breast tumours (Figure 4). To distinguish expression coming from the sense or the antisense strand, the first strand cDNA synthesis of the RT-PCR was set up either with the sense primer (generating cDNA from sense strand mRNA transcripts) or the antisense primer (generating cDNA from RNA from the antisense strand). FAM labeled fluorescent probes for each gene were used to determine the relative expression levels of the sense and antisense by qPCR. KRT81 showed similar expression patterns in all tested samples with regards to sense and antisense transcription and overall expression levels. In contrast, DCBLD2 and MMP24 showed significantly higher expression ratios in the solid tumours in comparison with the breast cancer cell lines. Furthermore the sense and antisense transcripts of DCBLD2 had different expression levels in BT474 and T47D, two hormone-receptor positive luminal-specific breast cancer cell lines. When the expression of the antisense and sense MMP24 transcript was interrogated, opposite expression levels were observed in five breast cancer cell lines (namely MDA-MB-231 and HMT3552 of basal-subtype; and BT474, MDA-MB-453, SKBR5 and SKBR7 of luminal-subtype) as well as in five solid breast tumours.

Bottom Line: Expression of 431 NATs were confirmed by either of the other two technologies.Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated.Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ludwig Institute for Cancer Research, 605 Third Avenue, New York, NY 10158, USA. anita.grigoriadis@kcl.ac.uk

ABSTRACT

Background: More than 20% of human transcripts have naturally occurring antisense products (or natural antisense transcripts--NATs), some of which may play a key role in a range of human diseases. To date, several databases of in silico defined human sense-antisense (SAS) pairs have appeared, however no study has focused on differential expression of SAS pairs in breast tissue. We therefore investigated the expression levels of sense and antisense transcripts in normal and malignant human breast epithelia using the Affymetrix HG-U133 Plus 2.0 and Almac Diagnostics Breast Cancer DSA microarray technologies as well as massively parallel signature sequencing (MPSS) data.

Results: The expression of more than 2500 antisense transcripts were detected in normal breast duct luminal cells and in primary breast tumors substantially enriched for their epithelial cell content by DSA microarray. Expression of 431 NATs were confirmed by either of the other two technologies. A corresponding sense transcript could be identified on DSA for 257 antisense transcripts. Of these SAS pairs, 163 have not been previously reported. A positive correlation of differential expression between normal and malignant breast samples was observed for most SAS pairs. Orientation specific RT-QPCR of selected SAS pairs validated their expression in several breast cancer cell lines and solid breast tumours.

Conclusion: Disease-focused and antisense enriched microarray platforms (such as Breast Cancer DSA) confirm the assumption that antisense transcription in the human breast is more prevalent than previously anticipated. Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated. Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs.

Show MeSH
Related in: MedlinePlus