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Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation.

Christov CP, Trivier E, Krude T - Br. J. Cancer (2008)

Bottom Line: In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22).A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi).Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

ABSTRACT
Noncoding Y RNAs have recently been identified as essential factors for chromosomal DNA replication in human cell nuclei. Here, we investigate the expression of human Y RNAs in tumours and test their requirement for cell proliferation. Relative expression levels of all four human Y RNAs (hY1, hY3, hY4 and hY5 RNA) were determined by quantitative RT-PCR in extracts from human solid tumours, corresponding nonmalignant normal tissues and derived cultured cells. On average, all four hY RNAs are significantly overexpressed in solid tumours between 4- and 13-fold, compared to the corresponding normal tissues. In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22). A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi). Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation. We conclude that noncoding hY RNAs have potential both as new cancer biomarkers and as molecular targets for anti-proliferative intervention.

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RNA interference against hY RNAs. (A) Quantification of RNA levels after RNAi. Three small interfering RNAs (designated as siRNAs a, b and c) directed against hY1, hY3, hY4 and hY5 RNA were transfected into proliferating HeLa cells. RNA was isolated at 48 h after transfection and the amounts of each targeted hY RNA relative to a calibrator RNA were determined by qRT–PCR. 5.8S rRNA was used as calibrator for hY1, and HPRT mRNA for the other hY RNAs. The expression of each target hY RNA after the experimental RNAi is shown as the percentage of the relative expression levels observed after a control RNAi against nontarget firefly luciferase mRNA. Mean values are shown for n independent experiments as indicated. (B) Quantification of replicating S phase cells after RNAi. At 47 h after transfection of asynchronously proliferating HeLa cells with the indicated siRNAs, replicating cells in the population were labelled for 1 h with BrdU. At 48 h, percentages of S phase cells incorporating BrdU into their chromosomal DNA were determined by immunofluorescence microscopy. Mean values and s.d. are shown for n independent experiments.
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fig4: RNA interference against hY RNAs. (A) Quantification of RNA levels after RNAi. Three small interfering RNAs (designated as siRNAs a, b and c) directed against hY1, hY3, hY4 and hY5 RNA were transfected into proliferating HeLa cells. RNA was isolated at 48 h after transfection and the amounts of each targeted hY RNA relative to a calibrator RNA were determined by qRT–PCR. 5.8S rRNA was used as calibrator for hY1, and HPRT mRNA for the other hY RNAs. The expression of each target hY RNA after the experimental RNAi is shown as the percentage of the relative expression levels observed after a control RNAi against nontarget firefly luciferase mRNA. Mean values are shown for n independent experiments as indicated. (B) Quantification of replicating S phase cells after RNAi. At 47 h after transfection of asynchronously proliferating HeLa cells with the indicated siRNAs, replicating cells in the population were labelled for 1 h with BrdU. At 48 h, percentages of S phase cells incorporating BrdU into their chromosomal DNA were determined by immunofluorescence microscopy. Mean values and s.d. are shown for n independent experiments.

Mentions: In the next set of experiments, we investigated whether degradation of hY RNAs in proliferating human cells leads to an inhibition of cell proliferation. All four hY RNAs were expressed in several cell lines investigated (Supplementary Figure S1), in agreement with earlier reports (Hendrick et al, 1981; Pruijn et al, 1993). Our previous experiments have already established that RNA interference (RNAi) against hY1 RNAs is feasible in human cells, and we reported that degradation of hY1 RNA by two separate siRNAs results in a reduced proportion of replicating cells (Christov et al, 2006). We have therefore extended this analysis and conducted a systematic survey of RNAi on all four hY RNAs and analysed the consequences on cell proliferation (Figure 4) and cell viability (Figure 5).


Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation.

Christov CP, Trivier E, Krude T - Br. J. Cancer (2008)

RNA interference against hY RNAs. (A) Quantification of RNA levels after RNAi. Three small interfering RNAs (designated as siRNAs a, b and c) directed against hY1, hY3, hY4 and hY5 RNA were transfected into proliferating HeLa cells. RNA was isolated at 48 h after transfection and the amounts of each targeted hY RNA relative to a calibrator RNA were determined by qRT–PCR. 5.8S rRNA was used as calibrator for hY1, and HPRT mRNA for the other hY RNAs. The expression of each target hY RNA after the experimental RNAi is shown as the percentage of the relative expression levels observed after a control RNAi against nontarget firefly luciferase mRNA. Mean values are shown for n independent experiments as indicated. (B) Quantification of replicating S phase cells after RNAi. At 47 h after transfection of asynchronously proliferating HeLa cells with the indicated siRNAs, replicating cells in the population were labelled for 1 h with BrdU. At 48 h, percentages of S phase cells incorporating BrdU into their chromosomal DNA were determined by immunofluorescence microscopy. Mean values and s.d. are shown for n independent experiments.
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getmorefigures.php?uid=PMC2266855&req=5

fig4: RNA interference against hY RNAs. (A) Quantification of RNA levels after RNAi. Three small interfering RNAs (designated as siRNAs a, b and c) directed against hY1, hY3, hY4 and hY5 RNA were transfected into proliferating HeLa cells. RNA was isolated at 48 h after transfection and the amounts of each targeted hY RNA relative to a calibrator RNA were determined by qRT–PCR. 5.8S rRNA was used as calibrator for hY1, and HPRT mRNA for the other hY RNAs. The expression of each target hY RNA after the experimental RNAi is shown as the percentage of the relative expression levels observed after a control RNAi against nontarget firefly luciferase mRNA. Mean values are shown for n independent experiments as indicated. (B) Quantification of replicating S phase cells after RNAi. At 47 h after transfection of asynchronously proliferating HeLa cells with the indicated siRNAs, replicating cells in the population were labelled for 1 h with BrdU. At 48 h, percentages of S phase cells incorporating BrdU into their chromosomal DNA were determined by immunofluorescence microscopy. Mean values and s.d. are shown for n independent experiments.
Mentions: In the next set of experiments, we investigated whether degradation of hY RNAs in proliferating human cells leads to an inhibition of cell proliferation. All four hY RNAs were expressed in several cell lines investigated (Supplementary Figure S1), in agreement with earlier reports (Hendrick et al, 1981; Pruijn et al, 1993). Our previous experiments have already established that RNA interference (RNAi) against hY1 RNAs is feasible in human cells, and we reported that degradation of hY1 RNA by two separate siRNAs results in a reduced proportion of replicating cells (Christov et al, 2006). We have therefore extended this analysis and conducted a systematic survey of RNAi on all four hY RNAs and analysed the consequences on cell proliferation (Figure 4) and cell viability (Figure 5).

Bottom Line: In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22).A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi).Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

ABSTRACT
Noncoding Y RNAs have recently been identified as essential factors for chromosomal DNA replication in human cell nuclei. Here, we investigate the expression of human Y RNAs in tumours and test their requirement for cell proliferation. Relative expression levels of all four human Y RNAs (hY1, hY3, hY4 and hY5 RNA) were determined by quantitative RT-PCR in extracts from human solid tumours, corresponding nonmalignant normal tissues and derived cultured cells. On average, all four hY RNAs are significantly overexpressed in solid tumours between 4- and 13-fold, compared to the corresponding normal tissues. In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22). A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi). Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation. We conclude that noncoding hY RNAs have potential both as new cancer biomarkers and as molecular targets for anti-proliferative intervention.

Show MeSH
Related in: MedlinePlus