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. 2011 May;17(5):878-91.
doi: 10.1261/rna.2528811. Epub 2011 Apr 1.

SNORD-host RNA Zfas1 is a regulator of mammary development and a potential marker for breast cancer

Marjan E Askarian-Amiri  1 , Joanna Crawford, Juliet D French, Chanel E Smart, Martin A Smith, Michael B Clark, Kelin Ru, Tim R Mercer, Ella R Thompson, Sunil R Lakhani, Ana C Vargas, Ian G Campbell, Melissa A Brown, Marcel E Dinger, John S Mattick
Affiliations

SNORD-host RNA Zfas1 is a regulator of mammary development and a potential marker for breast cancer

Marjan E Askarian-Amiri et al. RNA. 2011 May.

Abstract

Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript (Zfas1) antisense to the 5' end of the protein-coding gene Znfx1. In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12, Snord12b, and Snord12c. In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life >16 h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1, their predicted secondary structures have similar features. Like Zfas1, ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.

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Figures

FIGURE 1.
FIGURE 1.
Relationship and expression of Znfx1 and its associated ncRNA Zfas1. (A) Genomic context of Znfx1 and its associated ncRNA. The enlarged Zfas1 indicates the location of three snoRNAs derived from this gene. The high degree of conservation of these regions across mammalian species is indicated. (B) Relative expression of Znfx1 (left) and Zfas1 (right) in mammary epithelial cells during different developmental stages of mammary gland development to Tubulin delta 1 (Tubd1). Expression levels of three biological replicates for each stage were measured in triplicate by qPCR. (C) Relative expression profile of Znfx1 and Zfas1 to Tubd1 in different tissues by qPCR. Technical replicates were performed in triplicate for each sample. (D) Decay curve of Znfx1 and Zfas1 in N2A cells. Transcription was blocked by treatment with actinomycin D, and expression levels of three biological replicates were detected by qPCR. Error bars in B, C, and D are the standard error of the mean (SEM). (E) Northern blot analysis of Znfx1 and Zfas1 on RNA derived from total, cytoplasmic, and nuclear fractions of HC11 cells. Arrows indicate size of transcript for each gene.
FIGURE 2.
FIGURE 2.
ISH mammary gland sections from pregnant mice. Panels illustrate mammary gland sections hybridized with no probe (top; negative control), Zfas1 antisense probe (middle), and Zfas1 sense probe (bottom; negative control). Images in dotted boxed areas increase in magnification from left to right. The arrows show ductal and alveolar structure and the expression of Zfas1 within these structures. Scale bars in each panel are indicated. The genomic context of the ISH probe is shown in Supplemental Fig. S3.
FIGURE 3.
FIGURE 3.
Effect of Zfas1 knockdown by RNA interference. (A) The expression level, normalized to Tubd1, of Znfx1 and Zfas1 genes in HC11 cells transfected with Zfas1 siRNA relative to the respective expression of each gene in HC11 cells transfected with scrambled siRNA measured by qPCR 1–5 d after siRNA transfection. Technical replicates were performed in triplicate for each time point, with error bars indicating SEM. (B) Proliferation rates based on level of BrdU incorporation measured 48 h after cells were transfected with Zfas1 versus scrambled siRNA. Six technical replicates were performed with error bars indicating SEM. (C) MTT assay measuring the metabolic rate of HC11 cells transfected with Zfas1 versus scrambled siRNA. Six technical replicates were performed with error bars indicating SEM. (D) Effect of Zfas1 knockdown compared to the scrambled siRNA control on dome formation in differentiated HC11 cells measured on day 8. (E) Quantitative PCR, relative to Tubd1, of β-casein (Csn2) levels in differentiated (day 8) cells relative to undifferentiated (day 2) in HC11 cells transfected with Zfas1 or scrambled siRNA. The results in D and E represent data from three experiments, each with three technical replicates, with error bars indicating the SEM of the three experiments.
FIGURE 4.
FIGURE 4.
Expression of snoRNAs that are intronic to Zfas1. (A) Relative expression (from left to right) of Snord12, Snord12b, and Snord12c to Snord68 during different mammary gland developmental stages. Expression levels of three biological replicates for each stage were measured in triplicate by qPCR. (B) Expression levels (from left to right) of Snord12, Snord12b, and Snord12c during HC11 cell differentiation relative to Snord68. Expression levels of two biological replicates for each stage were measured in triplicate by qPCR. Error bars in both A and B are SEM of the biological replicates. (C) Decay curve of Snord12, Snord12b, and Snord12c in N2A cells. Transcription was blocked by treatment with actinomycin D, and expression levels were detected in triplicate by qPCR. Errors are the standard error of the mean (SEM). (D) Northern blot analysis of Snord12, Snord12b, and Snord12c (see Fig. 1A and Supplemental Fig. S3 for genomic positions) expression in total, cytoplasmic, and nuclear RNA derived from undifferentiated HC11 cells. (E) Expression levels of Snord12, Snord12b, and Snord12c, normalized to Snord68, in undifferentiated (day 2) HC11 cells transfected with Zfas1 siRNA relative to expression levels of each normalized gene in HC11 cells transfected with scrambled siRNA. Technical replicates were performed in triplicate, with error bars indicating SEM.
FIGURE 5.
FIGURE 5.
Expression analysis of human ZFAS1. (A) Genomic context of human ZNFX1 and ZFAS1. The 5′ ends of ZNFX1 and ZFAS1 are oriented head-to-head on opposite strands. The zoomed-in regions show five different ZFAS1 isoforms that are represented by ESTs. The positions of the intronically-derived snoRNAs—SNORD12, SNORD12B, and SNORD12C—are also shown with the degree of conservation across mammalian species indicated. (B) Comparative expression levels (tpm) of ZNFX1 and ZFAS1 based on RNA deep sequencing of human breast tissue and mammary epithelium. (C) Relative abundance of alternate isoforms of ZFAS1 in various human tissues and cell lines based on exon-exon junction spanning deep sequence tags. The number on top of each bar represents the number of informative tags. (D) Relative expression level of ZNFX1 and ZFAS1 to GAPDH in the five paired normal and invasive ductal carcinoma (IDC) samples detected by qPCR with technical replicates performed in triplicate. Error bars indicate SEM of the biological replicates.
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