The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis

Abstract

Background:To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer.Methods:Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis.Results:Small-nucleolar RNA expression was as variable as miRNA expression (miR-21, miR-210, miR-10b). Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer.Conclusion:Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression.

Regulation of apoptosis by long non-coding RNA GAS5 in breast cancer cells: implications for chemotherapy

“…In this regard, a wide range of mTOR inhibitors may prove useful in patients with receptor-positive disease, whereas treatment options may be limited to dual PI3K/mTOR inhibitors in TNBC patients. Findings here in breast cancer cell lines may be relevant to the therapy of a wide range of other cancers, including head and neck squamous cell carcinoma [10] …”

“…The identification of novel therapeutic targets for multiple breast cancer subtypes is therefore important, and lncRNAs may offer new opportunities in this regard, given their emerging roles as regulators of fundamental biological processes [1][2][3][4][5]. Indeed, GAS5 is already of particular interest in relation to breast cancer, since its expression is down-regulated in tumour tissue [9], and patient prognosis is significantly related to GAS5 transcript levels [10]. Current findings in preclinical models demonstrate here, for the first time, that breast cancer cell responses to several apoptotic stimuli are quantitatively related to cellular GAS5 levels, suggesting a potential functional basis for the latter clinically important relationship.…”

“…Although functional studies have previously reported that GAS5 transcripts exert apoptosispromoting effects in breast cancer cells and in a range of other cell types [9, 15, 18- Thus, even small reductions in endogenous GAS5 expression may adversely impact upon the responses of breast cancer cells to certain death-inducing stimuli, including conventional chemotherapeutic agents, and this resistance to therapy may explain why breast cancer patient survival is related to tumour GAS5 levels [10].…”

“…If impaired responses to chemotherapeutic drugs underlie the clinical relationship between patient survival and breast cancer GAS5 levels [10], then parameters of breast cancer cell survival should show quantitative relationships with cellular GAS5 levels. Regression analysis was therefore performed on data from the GAS5 silencing experiments; cellular GAS5 levels were expressed relative to those in negative control siRNA-treated cultures to facilitate comparisons between the two cell lines.…”

“…The lncRNA and small nucleolar RNA (snoRNA) host gene, growth arrest-specific 5 (GAS5), is of particular interest in breast cancer, since its expression is down-regulated in tumour tissue [9], and patient prognosis is related to GAS5 transcript levels [10]. GAS5 is a 5′-terminal oligopyrimidine tract (5′-TOP) gene, and comprises 12 exons which are alternatively spliced to yield two possible mature lncRNAs [11].…”