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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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p53 induces loc285194 by direct interacting with loc285194 promoter. (A) Schematic description of loc285194 promoter with a potential p53 response element (p53RE). Also shown are sequences for mutant p53RE and p53RE consensus. Relative position of primers used for ChIP assay (E) was indicated by arrows. (B) Induction of loc285194 promoter activity by wild-type p53, but not mutant p53. HCT-116 WT cells were transfected with loc285194 luciferase reporter (loc-p-FL) along with vector, wild-type p53 or mutant p53 (R175H). The cells were harvested for luciferase assays 24 h after transfection. The miR-145 promoter reporter served as a positive control. Error bars represent SEM, n = 3. **P < 0.01. (C) Deletion analysis of the promoter activity to determine the role of the potential p53RE in p53 regulation of loc285194. Loc-p-FL is a full-length promoter; Loc-p-d1 and Loc-p-d2 carry a deletion involving p53RE; Loc-p-m is a mutant at p53RE, as indicated in Figure 2A. (D) Relative luciferase activity for corresponding constructs in Figure 2C. Luciferase assay was conducted as in Figure 2B. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (E) Confirmation of p53 binding to p53RE in loc285194 promoter as detected by ChIP assay. Control primers Loc285194-ChIP-Ctrl-5.1 and Loc285194-ChIP-Ctrl-5.1 were derived from outside of p53RE. Immunoglobulin G and SUMO antibody were used as negative controls; p21 served as a positive control.
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gkt182-F2: p53 induces loc285194 by direct interacting with loc285194 promoter. (A) Schematic description of loc285194 promoter with a potential p53 response element (p53RE). Also shown are sequences for mutant p53RE and p53RE consensus. Relative position of primers used for ChIP assay (E) was indicated by arrows. (B) Induction of loc285194 promoter activity by wild-type p53, but not mutant p53. HCT-116 WT cells were transfected with loc285194 luciferase reporter (loc-p-FL) along with vector, wild-type p53 or mutant p53 (R175H). The cells were harvested for luciferase assays 24 h after transfection. The miR-145 promoter reporter served as a positive control. Error bars represent SEM, n = 3. **P < 0.01. (C) Deletion analysis of the promoter activity to determine the role of the potential p53RE in p53 regulation of loc285194. Loc-p-FL is a full-length promoter; Loc-p-d1 and Loc-p-d2 carry a deletion involving p53RE; Loc-p-m is a mutant at p53RE, as indicated in Figure 2A. (D) Relative luciferase activity for corresponding constructs in Figure 2C. Luciferase assay was conducted as in Figure 2B. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (E) Confirmation of p53 binding to p53RE in loc285194 promoter as detected by ChIP assay. Control primers Loc285194-ChIP-Ctrl-5.1 and Loc285194-ChIP-Ctrl-5.1 were derived from outside of p53RE. Immunoglobulin G and SUMO antibody were used as negative controls; p21 served as a positive control.

Mentions: p53 is also a well-known transcription factor. To determine whether p53 transcriptionally regulates loc285194, we used the p53 scan program (29) to analyze the upstream region of loc285194 and identified a putative p53 response element (p53RE) at ∼1500 upstream of loc285194 (Figure 2A). Thus, we cloned this 2-kb fragment into pGL3 basic (Promega). Luciferase assays indicated that p53 induced the promoter activity by over a 2.5-fold (Figure 2B), which was comparable with the induction of the miR-145 promoter (22). Consistent with the induction of loc285194 at the RNA level (Figure 1E), we found that mutant p53 had no effect on the promoter activity (Figure 2B). To further determine the function of this p53RE, we made two deletions involving p53RE (Figure 2C) and demonstrated that these deletions caused a significant reduction of the activity compared with the full-length (2 kb) promoter construct (Figure 2D). For example, the luciferase activity for deletions involving p53RE was <40% of the full length promoter. Furthermore, site-directed mutagenesis involving the conserved C and G of p53RE (Figure 2A) also yielded <40% of luciferase activity (Figure 2D). Finally, we showed that p53 directly interacted with p53RE by ChIP assays (Figure 2E). Together, these results suggest that p53 interacts with the p53RE in the loc285194 promoter to induce its transcription.Figure 2.


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)

p53 induces loc285194 by direct interacting with loc285194 promoter. (A) Schematic description of loc285194 promoter with a potential p53 response element (p53RE). Also shown are sequences for mutant p53RE and p53RE consensus. Relative position of primers used for ChIP assay (E) was indicated by arrows. (B) Induction of loc285194 promoter activity by wild-type p53, but not mutant p53. HCT-116 WT cells were transfected with loc285194 luciferase reporter (loc-p-FL) along with vector, wild-type p53 or mutant p53 (R175H). The cells were harvested for luciferase assays 24 h after transfection. The miR-145 promoter reporter served as a positive control. Error bars represent SEM, n = 3. **P < 0.01. (C) Deletion analysis of the promoter activity to determine the role of the potential p53RE in p53 regulation of loc285194. Loc-p-FL is a full-length promoter; Loc-p-d1 and Loc-p-d2 carry a deletion involving p53RE; Loc-p-m is a mutant at p53RE, as indicated in Figure 2A. (D) Relative luciferase activity for corresponding constructs in Figure 2C. Luciferase assay was conducted as in Figure 2B. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (E) Confirmation of p53 binding to p53RE in loc285194 promoter as detected by ChIP assay. Control primers Loc285194-ChIP-Ctrl-5.1 and Loc285194-ChIP-Ctrl-5.1 were derived from outside of p53RE. Immunoglobulin G and SUMO antibody were used as negative controls; p21 served as a positive control.
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Related In: Results  -  Collection

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gkt182-F2: p53 induces loc285194 by direct interacting with loc285194 promoter. (A) Schematic description of loc285194 promoter with a potential p53 response element (p53RE). Also shown are sequences for mutant p53RE and p53RE consensus. Relative position of primers used for ChIP assay (E) was indicated by arrows. (B) Induction of loc285194 promoter activity by wild-type p53, but not mutant p53. HCT-116 WT cells were transfected with loc285194 luciferase reporter (loc-p-FL) along with vector, wild-type p53 or mutant p53 (R175H). The cells were harvested for luciferase assays 24 h after transfection. The miR-145 promoter reporter served as a positive control. Error bars represent SEM, n = 3. **P < 0.01. (C) Deletion analysis of the promoter activity to determine the role of the potential p53RE in p53 regulation of loc285194. Loc-p-FL is a full-length promoter; Loc-p-d1 and Loc-p-d2 carry a deletion involving p53RE; Loc-p-m is a mutant at p53RE, as indicated in Figure 2A. (D) Relative luciferase activity for corresponding constructs in Figure 2C. Luciferase assay was conducted as in Figure 2B. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (E) Confirmation of p53 binding to p53RE in loc285194 promoter as detected by ChIP assay. Control primers Loc285194-ChIP-Ctrl-5.1 and Loc285194-ChIP-Ctrl-5.1 were derived from outside of p53RE. Immunoglobulin G and SUMO antibody were used as negative controls; p21 served as a positive control.
Mentions: p53 is also a well-known transcription factor. To determine whether p53 transcriptionally regulates loc285194, we used the p53 scan program (29) to analyze the upstream region of loc285194 and identified a putative p53 response element (p53RE) at ∼1500 upstream of loc285194 (Figure 2A). Thus, we cloned this 2-kb fragment into pGL3 basic (Promega). Luciferase assays indicated that p53 induced the promoter activity by over a 2.5-fold (Figure 2B), which was comparable with the induction of the miR-145 promoter (22). Consistent with the induction of loc285194 at the RNA level (Figure 1E), we found that mutant p53 had no effect on the promoter activity (Figure 2B). To further determine the function of this p53RE, we made two deletions involving p53RE (Figure 2C) and demonstrated that these deletions caused a significant reduction of the activity compared with the full-length (2 kb) promoter construct (Figure 2D). For example, the luciferase activity for deletions involving p53RE was <40% of the full length promoter. Furthermore, site-directed mutagenesis involving the conserved C and G of p53RE (Figure 2A) also yielded <40% of luciferase activity (Figure 2D). Finally, we showed that p53 directly interacted with p53RE by ChIP assays (Figure 2E). Together, these results suggest that p53 interacts with the p53RE in the loc285194 promoter to induce its transcription.Figure 2.

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