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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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Loc285194 inhibits tumor cell growth in vitro. (A) Inhibition of cell growth by loc285194 in HCT-116 WT cells. The cells were first transfected with vector or loc285194 overnight and then split into 12-well plates. Cell number was counted from day 1 to day 5 by trypan blue method. Error bars represent SEM, n = 3. **P < 0.01. (B) Inhibition of cell growth by loc285194 in MCF-7 cells. The experiment was conducted the same way as in Figure 3A. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (C) Identification of the minimal region of loc285194 capable of inhibiting cell growth. Deletion constructs were made by PCR using primers (Supplementary Table S1) and then cloned into pCDH-MSCV-EF1-GFP-T2A-Puro. The length of each of 4 exons for loc285194 is indicated by number of nucleotides above E1∼4. E1∼4 is consisted of all 4 exons; E2∼4 is consisted of exon 2, 3 and 4; E1∼3 is consisted of exon 1, 2 and 3; E4 carries exon 4 only; and E4-d carries a deletion of the first 247 nucleotides of exon 4. (D) Effect of loc285194 and its deletion constructs corresponding to Figure 3C on cell growth. The experiment was conducted in HCT-116 WT cells with the same way as in Figure3 A. Error bars represent SEM, n = 3. *P < 0.05. (E) and (F) Suppression of loc285197 by RNAi increases cell growth in HCT-116 WT (E) and HCT-116  cells (F). Cells were first transfected with control siRNA or loc285192 siRNAs overnight and then split into 12-well plates. The cell number was counted from day 1 as 100% to day 4. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05.
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gkt182-F3: Loc285194 inhibits tumor cell growth in vitro. (A) Inhibition of cell growth by loc285194 in HCT-116 WT cells. The cells were first transfected with vector or loc285194 overnight and then split into 12-well plates. Cell number was counted from day 1 to day 5 by trypan blue method. Error bars represent SEM, n = 3. **P < 0.01. (B) Inhibition of cell growth by loc285194 in MCF-7 cells. The experiment was conducted the same way as in Figure 3A. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (C) Identification of the minimal region of loc285194 capable of inhibiting cell growth. Deletion constructs were made by PCR using primers (Supplementary Table S1) and then cloned into pCDH-MSCV-EF1-GFP-T2A-Puro. The length of each of 4 exons for loc285194 is indicated by number of nucleotides above E1∼4. E1∼4 is consisted of all 4 exons; E2∼4 is consisted of exon 2, 3 and 4; E1∼3 is consisted of exon 1, 2 and 3; E4 carries exon 4 only; and E4-d carries a deletion of the first 247 nucleotides of exon 4. (D) Effect of loc285194 and its deletion constructs corresponding to Figure 3C on cell growth. The experiment was conducted in HCT-116 WT cells with the same way as in Figure3 A. Error bars represent SEM, n = 3. *P < 0.05. (E) and (F) Suppression of loc285197 by RNAi increases cell growth in HCT-116 WT (E) and HCT-116 cells (F). Cells were first transfected with control siRNA or loc285192 siRNAs overnight and then split into 12-well plates. The cell number was counted from day 1 as 100% to day 4. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05.

Mentions: It is known that p53 can induce a large number of genes including tumor suppressors such as p21. To evaluate the biological consequence of p53 induction of loc285194 and the role of loc285194 in tumorigenesis, we ectopically expressed loc285194 in HCT-116 WT and MCF-7 cells (Supplementary Figure S3 and S4A) for cell proliferation assays. As expected, loc285194 significantly inhibited tumor cell growth (Figure 3A). This loc285194-mediated cell growth inhibition was also seen in MCF-7 cells (Figure 3B). Loc285194 consists of 4 exons with a total length of ∼2.1 kb. Thus, we made various deletion constructs to define which region of loc285194 is responsible for cell growth inhibition (Figure 3C). This deletion analysis identified the active region within exon 4 and the C-terminal 600 bp fragment of exon 4 (E4-d) was still able to suppress cell growth (Figure 3D). To further determine the role of loc285194 in cell growth inhibition, we suppressed loc285194 by RNAi (Supplementary Figure S4B), and both loc285194 siRNA-1 (18) and siRNA-2 (Supplementary Table S1) significantly suppressed the endogenous loc285194. At the same time, loc-siRNA increased cell growth in HCT-116-WT cells (Figure 3E) as well as in HCT-116 cells (Figure 3F), suggesting that loc285194 may target genes involved in cell growth and proliferation.Figure 3.


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)

Loc285194 inhibits tumor cell growth in vitro. (A) Inhibition of cell growth by loc285194 in HCT-116 WT cells. The cells were first transfected with vector or loc285194 overnight and then split into 12-well plates. Cell number was counted from day 1 to day 5 by trypan blue method. Error bars represent SEM, n = 3. **P < 0.01. (B) Inhibition of cell growth by loc285194 in MCF-7 cells. The experiment was conducted the same way as in Figure 3A. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (C) Identification of the minimal region of loc285194 capable of inhibiting cell growth. Deletion constructs were made by PCR using primers (Supplementary Table S1) and then cloned into pCDH-MSCV-EF1-GFP-T2A-Puro. The length of each of 4 exons for loc285194 is indicated by number of nucleotides above E1∼4. E1∼4 is consisted of all 4 exons; E2∼4 is consisted of exon 2, 3 and 4; E1∼3 is consisted of exon 1, 2 and 3; E4 carries exon 4 only; and E4-d carries a deletion of the first 247 nucleotides of exon 4. (D) Effect of loc285194 and its deletion constructs corresponding to Figure 3C on cell growth. The experiment was conducted in HCT-116 WT cells with the same way as in Figure3 A. Error bars represent SEM, n = 3. *P < 0.05. (E) and (F) Suppression of loc285197 by RNAi increases cell growth in HCT-116 WT (E) and HCT-116  cells (F). Cells were first transfected with control siRNA or loc285192 siRNAs overnight and then split into 12-well plates. The cell number was counted from day 1 as 100% to day 4. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05.
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gkt182-F3: Loc285194 inhibits tumor cell growth in vitro. (A) Inhibition of cell growth by loc285194 in HCT-116 WT cells. The cells were first transfected with vector or loc285194 overnight and then split into 12-well plates. Cell number was counted from day 1 to day 5 by trypan blue method. Error bars represent SEM, n = 3. **P < 0.01. (B) Inhibition of cell growth by loc285194 in MCF-7 cells. The experiment was conducted the same way as in Figure 3A. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05. (C) Identification of the minimal region of loc285194 capable of inhibiting cell growth. Deletion constructs were made by PCR using primers (Supplementary Table S1) and then cloned into pCDH-MSCV-EF1-GFP-T2A-Puro. The length of each of 4 exons for loc285194 is indicated by number of nucleotides above E1∼4. E1∼4 is consisted of all 4 exons; E2∼4 is consisted of exon 2, 3 and 4; E1∼3 is consisted of exon 1, 2 and 3; E4 carries exon 4 only; and E4-d carries a deletion of the first 247 nucleotides of exon 4. (D) Effect of loc285194 and its deletion constructs corresponding to Figure 3C on cell growth. The experiment was conducted in HCT-116 WT cells with the same way as in Figure3 A. Error bars represent SEM, n = 3. *P < 0.05. (E) and (F) Suppression of loc285197 by RNAi increases cell growth in HCT-116 WT (E) and HCT-116 cells (F). Cells were first transfected with control siRNA or loc285192 siRNAs overnight and then split into 12-well plates. The cell number was counted from day 1 as 100% to day 4. Error bars represent SEM, n = 3. **P < 0.01; *P < 0.05.
Mentions: It is known that p53 can induce a large number of genes including tumor suppressors such as p21. To evaluate the biological consequence of p53 induction of loc285194 and the role of loc285194 in tumorigenesis, we ectopically expressed loc285194 in HCT-116 WT and MCF-7 cells (Supplementary Figure S3 and S4A) for cell proliferation assays. As expected, loc285194 significantly inhibited tumor cell growth (Figure 3A). This loc285194-mediated cell growth inhibition was also seen in MCF-7 cells (Figure 3B). Loc285194 consists of 4 exons with a total length of ∼2.1 kb. Thus, we made various deletion constructs to define which region of loc285194 is responsible for cell growth inhibition (Figure 3C). This deletion analysis identified the active region within exon 4 and the C-terminal 600 bp fragment of exon 4 (E4-d) was still able to suppress cell growth (Figure 3D). To further determine the role of loc285194 in cell growth inhibition, we suppressed loc285194 by RNAi (Supplementary Figure S4B), and both loc285194 siRNA-1 (18) and siRNA-2 (Supplementary Table S1) significantly suppressed the endogenous loc285194. At the same time, loc-siRNA increased cell growth in HCT-116-WT cells (Figure 3E) as well as in HCT-116 cells (Figure 3F), suggesting that loc285194 may target genes involved in cell growth and proliferation.Figure 3.

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