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LncRNA loc285194 is a p53-regulated tumor suppressor.

Liu Q, Huang J, Zhou N, Zhang Z, Zhang A, Lu Z, Wu F, Mo YY - Nucleic Acids Res. (2013)

Bottom Line: We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth.Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays.Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, USA.

ABSTRACT
Protein-coding genes account for only a small part of the human genome, whereas the vast majority of transcripts make up the non-coding RNAs including long non-coding RNAs (lncRNAs). Accumulating evidence indicates that lncRNAs could play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. LncRNA loc285194 was previously shown to be within a tumor suppressor unit in osteosarcoma and to suppress tumor cell growth. However, it is unknown regarding the regulation of loc285194. Moreover, the underlying mechanism by which loc285194 functions as a potential tumor suppressor is elusive. In this study, we show that loc285194 is a p53 transcription target; ectopic expression of loc285194 inhibits tumor cell growth both in vitro and in vivo. Through deletion analysis, we identify an active region responsible for tumor cell growth inhibition within exon 4, which harbors two miR-211 binding sites. Importantly, this loc285194-mediated growth inhibition is in part due to specific suppression of miR-211. We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth. Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays. Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.

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Identification of loc285194 as a p53-induced lncRNA. (A) Detection of p53 induction in response to doxo by western blot. (B) Effect of doxo on loc285154 expression in HCT-116-WT and HCT-116  cells. The cells were treated with doxo at 1 µg/ml for 24 h before extraction of total RNA for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (C) Induction of loc285194 in HCT-116, MCF-7, HCT-8 and A549 cells in response to doxo. The cells were treated the same way as in (B). Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (D) Induction of loc285194 in doxo-treated cells as detected by ISH. (E) Induction of loc285194 by ectopically expressed p53. HCT-116 WT cells were first transfected with vector or wild-type p53 or mutant p53 (R175H) overnight; their expression was confirmed by western blot (top panel). After changing medium, the cells were allowed to further grow for 24 h before extraction of total RNA (bottom panel). Error bars represent SEM, n = 3. *P < 0.05.
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gkt182-F1: Identification of loc285194 as a p53-induced lncRNA. (A) Detection of p53 induction in response to doxo by western blot. (B) Effect of doxo on loc285154 expression in HCT-116-WT and HCT-116 cells. The cells were treated with doxo at 1 µg/ml for 24 h before extraction of total RNA for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (C) Induction of loc285194 in HCT-116, MCF-7, HCT-8 and A549 cells in response to doxo. The cells were treated the same way as in (B). Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (D) Induction of loc285194 in doxo-treated cells as detected by ISH. (E) Induction of loc285194 by ectopically expressed p53. HCT-116 WT cells were first transfected with vector or wild-type p53 or mutant p53 (R175H) overnight; their expression was confirmed by western blot (top panel). After changing medium, the cells were allowed to further grow for 24 h before extraction of total RNA (bottom panel). Error bars represent SEM, n = 3. *P < 0.05.

Mentions: As p53 is the well-known tumor suppressor that regulates a variety of cellular functions and disease processes, we simply asked whether any of these lncRNAs is induced by p53. We first treated HCT-116 cells expressing wild-type p53 (HCT-116 WT) with various concentrations of doxorubicin (doxo), a known DNA damaging agent, for 24 h and detected p53 induction in a dose-dependent manner, as expected (Figure 1A). Therefore, we then chose doxo at 1 µg/ml for 24 h for profiling experiments and identified 7 lncRNAs with over a 10-fold of induction by p53 from the preliminary profiling (Supplementary Figure S2). We were particularly interested in loc285194 because a previous report suggests its potential tumor suppressive role in osteosarcoma (18). To further confirm the specific effect of p53 on loc285194 expression, we treated HCT-116 p53 (HCT-116 ) cells at the same concentration of doxo. Although doxo also induced loc285294 in HCT-116 cells, the level of induction was much lower than that in HCT-116-WT cells (Figure 1B). Next, we examined loc285294 expression using a different set of primers derived from a different region of loc285194 (Figure 1C), further confirming this p53-mediated induction. In addition, such induction was also detected in other cell lines expressing wild-type p53, i.e. MCF-7, HCT-8 and A549 (Figure 1C) with ∼8-, 5- and 7-fold, respectively. Moreover, we performed in situ hybridization to detect the level of loc285194 in the cells after doxo treatment. We observed a substantial increase of loc285194 in the doxo-treated cells compared with no doxo control (Figure 1D). Of note, we found that a majority signal came from the cytoplasm. As doxo may also induce other cellular responses independent of p53, leading to gene expression, we ectopically expressed p53 to determine whether this loc285194 induction is specific to p53. As shown in Figure 1E, we detected over a 3-fold increase in the loc285194 level, similar to the induction of a known p53-regulated gene, miR-145 (22) (Figure 1E, right). Of considerable interest, p53 with a point mutation (R175H) at the DNA-binding domain, a frequent mutant in cancer (28), had no effect on loc285194 expression, suggesting that loc285194 is specifically induced by wild-type p53. Therefore, loc285194 was selected for further characterization of the p53-mediated induction and its role in tumorigenesis.Figure 1.


LncRNA loc285194 is a p53-regulated tumor suppressor.

Liu Q, Huang J, Zhou N, Zhang Z, Zhang A, Lu Z, Wu F, Mo YY - Nucleic Acids Res. (2013)

Identification of loc285194 as a p53-induced lncRNA. (A) Detection of p53 induction in response to doxo by western blot. (B) Effect of doxo on loc285154 expression in HCT-116-WT and HCT-116  cells. The cells were treated with doxo at 1 µg/ml for 24 h before extraction of total RNA for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (C) Induction of loc285194 in HCT-116, MCF-7, HCT-8 and A549 cells in response to doxo. The cells were treated the same way as in (B). Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (D) Induction of loc285194 in doxo-treated cells as detected by ISH. (E) Induction of loc285194 by ectopically expressed p53. HCT-116 WT cells were first transfected with vector or wild-type p53 or mutant p53 (R175H) overnight; their expression was confirmed by western blot (top panel). After changing medium, the cells were allowed to further grow for 24 h before extraction of total RNA (bottom panel). Error bars represent SEM, n = 3. *P < 0.05.
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Related In: Results  -  Collection

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gkt182-F1: Identification of loc285194 as a p53-induced lncRNA. (A) Detection of p53 induction in response to doxo by western blot. (B) Effect of doxo on loc285154 expression in HCT-116-WT and HCT-116 cells. The cells were treated with doxo at 1 µg/ml for 24 h before extraction of total RNA for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (C) Induction of loc285194 in HCT-116, MCF-7, HCT-8 and A549 cells in response to doxo. The cells were treated the same way as in (B). Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (D) Induction of loc285194 in doxo-treated cells as detected by ISH. (E) Induction of loc285194 by ectopically expressed p53. HCT-116 WT cells were first transfected with vector or wild-type p53 or mutant p53 (R175H) overnight; their expression was confirmed by western blot (top panel). After changing medium, the cells were allowed to further grow for 24 h before extraction of total RNA (bottom panel). Error bars represent SEM, n = 3. *P < 0.05.
Mentions: As p53 is the well-known tumor suppressor that regulates a variety of cellular functions and disease processes, we simply asked whether any of these lncRNAs is induced by p53. We first treated HCT-116 cells expressing wild-type p53 (HCT-116 WT) with various concentrations of doxorubicin (doxo), a known DNA damaging agent, for 24 h and detected p53 induction in a dose-dependent manner, as expected (Figure 1A). Therefore, we then chose doxo at 1 µg/ml for 24 h for profiling experiments and identified 7 lncRNAs with over a 10-fold of induction by p53 from the preliminary profiling (Supplementary Figure S2). We were particularly interested in loc285194 because a previous report suggests its potential tumor suppressive role in osteosarcoma (18). To further confirm the specific effect of p53 on loc285194 expression, we treated HCT-116 p53 (HCT-116 ) cells at the same concentration of doxo. Although doxo also induced loc285294 in HCT-116 cells, the level of induction was much lower than that in HCT-116-WT cells (Figure 1B). Next, we examined loc285294 expression using a different set of primers derived from a different region of loc285194 (Figure 1C), further confirming this p53-mediated induction. In addition, such induction was also detected in other cell lines expressing wild-type p53, i.e. MCF-7, HCT-8 and A549 (Figure 1C) with ∼8-, 5- and 7-fold, respectively. Moreover, we performed in situ hybridization to detect the level of loc285194 in the cells after doxo treatment. We observed a substantial increase of loc285194 in the doxo-treated cells compared with no doxo control (Figure 1D). Of note, we found that a majority signal came from the cytoplasm. As doxo may also induce other cellular responses independent of p53, leading to gene expression, we ectopically expressed p53 to determine whether this loc285194 induction is specific to p53. As shown in Figure 1E, we detected over a 3-fold increase in the loc285194 level, similar to the induction of a known p53-regulated gene, miR-145 (22) (Figure 1E, right). Of considerable interest, p53 with a point mutation (R175H) at the DNA-binding domain, a frequent mutant in cancer (28), had no effect on loc285194 expression, suggesting that loc285194 is specifically induced by wild-type p53. Therefore, loc285194 was selected for further characterization of the p53-mediated induction and its role in tumorigenesis.Figure 1.

Bottom Line: We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth.Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays.Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, USA.

ABSTRACT
Protein-coding genes account for only a small part of the human genome, whereas the vast majority of transcripts make up the non-coding RNAs including long non-coding RNAs (lncRNAs). Accumulating evidence indicates that lncRNAs could play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. LncRNA loc285194 was previously shown to be within a tumor suppressor unit in osteosarcoma and to suppress tumor cell growth. However, it is unknown regarding the regulation of loc285194. Moreover, the underlying mechanism by which loc285194 functions as a potential tumor suppressor is elusive. In this study, we show that loc285194 is a p53 transcription target; ectopic expression of loc285194 inhibits tumor cell growth both in vitro and in vivo. Through deletion analysis, we identify an active region responsible for tumor cell growth inhibition within exon 4, which harbors two miR-211 binding sites. Importantly, this loc285194-mediated growth inhibition is in part due to specific suppression of miR-211. We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth. Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays. Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.

Show MeSH
Related in: MedlinePlus