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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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Reciprocal negative regulation of miR-211 and loc285194. (A) Loc285194 carries two miR-211 binding sites at exon 4. Alignment between them is shown underneath with miR-211 seed sequences underlined. (B) Suppression of miR-211 expression by loc285194. HCT-116 cells were first transfected with vector, wild-type loc285194 (Loc-WT) or mutant loc285194 (Loc-d) with a deletion of two miR-211 binding sites, and 24 h after transfection, total RNA was isolated for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01. (C) Effect of miR-211 on loc285194 expression. HCT-116 WT cells were transfected with vector or miR-211, and total RNA was isolated for qRT-PCR 24 h after transfection. Error bars represent SEM, n = 3. *P < 0.05. (D) Effect of loc285194 on mature miR-211, pri-miR-211 and pre-miR-211. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (E) Pulldown of Ago2 by biotin-labeled loc285194 or GAS5 RNA probe, as detected by western blot. The GAS5 lane was composed from the same gel with the same contrast. (F) Detection of miR-211 in the pellet precipitated by the loc285194 probe, but not in the pellet precipitated by the GAS5 probe. Error bars represent SEM, n = 3. **P < 0.01.
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gkt182-F5: Reciprocal negative regulation of miR-211 and loc285194. (A) Loc285194 carries two miR-211 binding sites at exon 4. Alignment between them is shown underneath with miR-211 seed sequences underlined. (B) Suppression of miR-211 expression by loc285194. HCT-116 cells were first transfected with vector, wild-type loc285194 (Loc-WT) or mutant loc285194 (Loc-d) with a deletion of two miR-211 binding sites, and 24 h after transfection, total RNA was isolated for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01. (C) Effect of miR-211 on loc285194 expression. HCT-116 WT cells were transfected with vector or miR-211, and total RNA was isolated for qRT-PCR 24 h after transfection. Error bars represent SEM, n = 3. *P < 0.05. (D) Effect of loc285194 on mature miR-211, pri-miR-211 and pre-miR-211. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (E) Pulldown of Ago2 by biotin-labeled loc285194 or GAS5 RNA probe, as detected by western blot. The GAS5 lane was composed from the same gel with the same contrast. (F) Detection of miR-211 in the pellet precipitated by the loc285194 probe, but not in the pellet precipitated by the GAS5 probe. Error bars represent SEM, n = 3. **P < 0.01.

Mentions: Therefore, we searched for potential interactions with microRNAs because emerging evidence suggests that non-coding RNAs may participate in ‘competitive endogenous RNAs’ regulatory network (19). For example, there is a negative correlation between miR-155 and the ncRNA UC34A (31). Moreover, a muscle-specific lncRNA, linc-MD1, may act as a ‘sponges’ for miR-133 (16). Using microRNA.org-target program (www.microRNA.org), we found that 32 microRNAs formed complementary base paring with loc285194, and their relative positions were listed in Supplementary Figure S5. To determine whether any of them is truly regulated by loc285194, we profiled expression of these 32 microRNAs in HCT-116 WT transfected with vector or loc285194. The initial profiling identified 3 microRNAs (miR-24, miR-376c and miR-211) were reduced to <25% of the control by loc285194 (Supplementary Figure S6). As there were two miR-211 binding sites in exon 4 of loc285194 (Figure 5A), we deleted these two sites (Loc-d), and this construct was no longer able to suppress miR-211 (Figure 5B). To determine whether miR-211 is able to negatively regulate loc285194, we also cloned miR-211 and then introduced it into HCT-116 WT cells. As shown in Figure 5C, ectopic expression of miR-211 reduced the loc285194 level by almost 60%.Figure 5.


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)

Reciprocal negative regulation of miR-211 and loc285194. (A) Loc285194 carries two miR-211 binding sites at exon 4. Alignment between them is shown underneath with miR-211 seed sequences underlined. (B) Suppression of miR-211 expression by loc285194. HCT-116 cells were first transfected with vector, wild-type loc285194 (Loc-WT) or mutant loc285194 (Loc-d) with a deletion of two miR-211 binding sites, and 24 h after transfection, total RNA was isolated for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01. (C) Effect of miR-211 on loc285194 expression. HCT-116 WT cells were transfected with vector or miR-211, and total RNA was isolated for qRT-PCR 24 h after transfection. Error bars represent SEM, n = 3. *P < 0.05. (D) Effect of loc285194 on mature miR-211, pri-miR-211 and pre-miR-211. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (E) Pulldown of Ago2 by biotin-labeled loc285194 or GAS5 RNA probe, as detected by western blot. The GAS5 lane was composed from the same gel with the same contrast. (F) Detection of miR-211 in the pellet precipitated by the loc285194 probe, but not in the pellet precipitated by the GAS5 probe. Error bars represent SEM, n = 3. **P < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643595&req=5

gkt182-F5: Reciprocal negative regulation of miR-211 and loc285194. (A) Loc285194 carries two miR-211 binding sites at exon 4. Alignment between them is shown underneath with miR-211 seed sequences underlined. (B) Suppression of miR-211 expression by loc285194. HCT-116 cells were first transfected with vector, wild-type loc285194 (Loc-WT) or mutant loc285194 (Loc-d) with a deletion of two miR-211 binding sites, and 24 h after transfection, total RNA was isolated for qRT-PCR. Error bars represent SEM, n = 3. **P < 0.01. (C) Effect of miR-211 on loc285194 expression. HCT-116 WT cells were transfected with vector or miR-211, and total RNA was isolated for qRT-PCR 24 h after transfection. Error bars represent SEM, n = 3. *P < 0.05. (D) Effect of loc285194 on mature miR-211, pri-miR-211 and pre-miR-211. Error bars represent SEM, n = 3. **P < 0.01; n.s., not significant. (E) Pulldown of Ago2 by biotin-labeled loc285194 or GAS5 RNA probe, as detected by western blot. The GAS5 lane was composed from the same gel with the same contrast. (F) Detection of miR-211 in the pellet precipitated by the loc285194 probe, but not in the pellet precipitated by the GAS5 probe. Error bars represent SEM, n = 3. **P < 0.01.
Mentions: Therefore, we searched for potential interactions with microRNAs because emerging evidence suggests that non-coding RNAs may participate in ‘competitive endogenous RNAs’ regulatory network (19). For example, there is a negative correlation between miR-155 and the ncRNA UC34A (31). Moreover, a muscle-specific lncRNA, linc-MD1, may act as a ‘sponges’ for miR-133 (16). Using microRNA.org-target program (www.microRNA.org), we found that 32 microRNAs formed complementary base paring with loc285194, and their relative positions were listed in Supplementary Figure S5. To determine whether any of them is truly regulated by loc285194, we profiled expression of these 32 microRNAs in HCT-116 WT transfected with vector or loc285194. The initial profiling identified 3 microRNAs (miR-24, miR-376c and miR-211) were reduced to <25% of the control by loc285194 (Supplementary Figure S6). As there were two miR-211 binding sites in exon 4 of loc285194 (Figure 5A), we deleted these two sites (Loc-d), and this construct was no longer able to suppress miR-211 (Figure 5B). To determine whether miR-211 is able to negatively regulate loc285194, we also cloned miR-211 and then introduced it into HCT-116 WT cells. As shown in Figure 5C, ectopic expression of miR-211 reduced the loc285194 level by almost 60%.Figure 5.

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