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Knockdown of a HIF-2α promoter upstream long noncoding RNA impairs colorectal cancer stem cell properties in vitro through HIF-2α downregulation.

Yao J, Li J, Geng P, Li Y, Chen H, Zhu Y - Onco Targets Ther (2015)

Bottom Line: In this study, we found an lncRNA that is a promoter upstream transcript of hypoxia-inducible factor-2α (HIF-2α), and we named it "lncRNA-HIF2PUT".Herein, we showed that the expression of lncRNA-HIF2PUT was significantly correlated with HIF-2α in colorectal cancer (CRC) tissues.LncRNA-HIF2PUTsmall interfering RNA transfection resulted in decreased stemness genes expression, impaired colony formation, and spheroid formation ability, retarded migration, and invasion of the cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, People's Liberation Army No 161 Hospital, Wuhan, People's Republic of China.

ABSTRACT
Currently, various long noncoding RNAs (lncRNAs) have been identified as key regulators of multiple cancers. However, cancer stem cell (CSC)-related lncRNAs have rarely been reported. In this study, we found an lncRNA that is a promoter upstream transcript of hypoxia-inducible factor-2α (HIF-2α), and we named it "lncRNA-HIF2PUT". The function of HIF-2α is closely connected with "stem cell-like" properties, and the function of PROMPTs is often associated with the adjacent protein-coding transcripts. Herein, we showed that the expression of lncRNA-HIF2PUT was significantly correlated with HIF-2α in colorectal cancer (CRC) tissues. Knockdown of lncRNA-HIF2PUT blocked the HIF-2α expression and inhibited the CSC properties in CRC cell lines DLD-1 and HT29. LncRNA-HIF2PUTsmall interfering RNA transfection resulted in decreased stemness genes expression, impaired colony formation, and spheroid formation ability, retarded migration, and invasion of the cells. These data suggest that lncRNA-HIF2PUT may be a regulator of HIF-2α and a mediator of CSCs in CRC.

No MeSH data available.


Related in: MedlinePlus

Knockdown of lncRNA-HIF2PUT impairs CSC properties.Notes: (A) HIF2PUT lncRNA level and six stemness genes’ mRNA levels were measured by real-time PCR in DLD-1 and HT29 cells transfected with lncRNA-HIF2PUT siRNA and negative control siRNA. The expression of OCT4, SOX2, and CD44 was significantly downregulated. (B) Western blot showed that the expression of OCT4, SOX2, and CD44 protein was also significantly reduced in lncRNA-HIF2PUT siRNA groups compared with negative control groups. (C) Colony formation assay showed that CSC proliferation was retarded by lncRNA-HIF2PUT siRNA (P<0.05). (D) Spheroid formation assay showed that the self-renewal capacity in lncRNA-HIF2PUT siRNA groups was remarkably impaired. (E) The wound healing assay showed remarkable cell migration inhibition by lncRNA-HIF2PUT siRNA. (F) The matrigel invasion assay also showed significant cell invasion inhibition by lncRNA-HIF2PUT siRNA. *P<0.05.Abbreviations: lncRNA-HIF2PUT, long noncoding RNA that is a promoter upstream transcript (PROMPT) of hypoxia-inducible factor-2α; HIF-2α, hypoxia-inducible factor-2 alpha; PCR, polymerase chain reaction; siRNA, small interfering RNA; NC, noncancerous; mRNA, messenger RNA; CSC, cancer stem cell.
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f3-ott-8-3467: Knockdown of lncRNA-HIF2PUT impairs CSC properties.Notes: (A) HIF2PUT lncRNA level and six stemness genes’ mRNA levels were measured by real-time PCR in DLD-1 and HT29 cells transfected with lncRNA-HIF2PUT siRNA and negative control siRNA. The expression of OCT4, SOX2, and CD44 was significantly downregulated. (B) Western blot showed that the expression of OCT4, SOX2, and CD44 protein was also significantly reduced in lncRNA-HIF2PUT siRNA groups compared with negative control groups. (C) Colony formation assay showed that CSC proliferation was retarded by lncRNA-HIF2PUT siRNA (P<0.05). (D) Spheroid formation assay showed that the self-renewal capacity in lncRNA-HIF2PUT siRNA groups was remarkably impaired. (E) The wound healing assay showed remarkable cell migration inhibition by lncRNA-HIF2PUT siRNA. (F) The matrigel invasion assay also showed significant cell invasion inhibition by lncRNA-HIF2PUT siRNA. *P<0.05.Abbreviations: lncRNA-HIF2PUT, long noncoding RNA that is a promoter upstream transcript (PROMPT) of hypoxia-inducible factor-2α; HIF-2α, hypoxia-inducible factor-2 alpha; PCR, polymerase chain reaction; siRNA, small interfering RNA; NC, noncancerous; mRNA, messenger RNA; CSC, cancer stem cell.

Mentions: Next, we determined the effects of lncRNA-HIF2PUT knockdown on stem cell-like properties in DLD-1 and HT29 cells in vitro. After lncRNA-HIF2PUT siRNA treatment, the stemness genes OCT4, SOX2, and CD44 decreased significantly (Figure 3A, B), which was coincident with decreased colony formation rates in medium containing fetal bovine serum, and spheroid formation ability in serum-free medium (Figure 3C, D). To further identify the functional role of lncRNA-HIF2PUT RNA in cell migration and invasion, the scratch wound healing assay and matrigel invasion assay were performed in DLD-1 and HT29 cells in vitro. The wound healing assay showed remarkable cell migration inhibition in DLD-1 and HT29 siRNA groups compared with the cells in the noncancerous group (Figure 3E). The matrigel invasion assay also showed significant cell invasion inhibition in the lncRNA-HIF2PUT siRNA group compared with the noncancerous groups in the DLD-1 and HT29 cell lines (Figure 3F).


Knockdown of a HIF-2α promoter upstream long noncoding RNA impairs colorectal cancer stem cell properties in vitro through HIF-2α downregulation.

Yao J, Li J, Geng P, Li Y, Chen H, Zhu Y - Onco Targets Ther (2015)

Knockdown of lncRNA-HIF2PUT impairs CSC properties.Notes: (A) HIF2PUT lncRNA level and six stemness genes’ mRNA levels were measured by real-time PCR in DLD-1 and HT29 cells transfected with lncRNA-HIF2PUT siRNA and negative control siRNA. The expression of OCT4, SOX2, and CD44 was significantly downregulated. (B) Western blot showed that the expression of OCT4, SOX2, and CD44 protein was also significantly reduced in lncRNA-HIF2PUT siRNA groups compared with negative control groups. (C) Colony formation assay showed that CSC proliferation was retarded by lncRNA-HIF2PUT siRNA (P<0.05). (D) Spheroid formation assay showed that the self-renewal capacity in lncRNA-HIF2PUT siRNA groups was remarkably impaired. (E) The wound healing assay showed remarkable cell migration inhibition by lncRNA-HIF2PUT siRNA. (F) The matrigel invasion assay also showed significant cell invasion inhibition by lncRNA-HIF2PUT siRNA. *P<0.05.Abbreviations: lncRNA-HIF2PUT, long noncoding RNA that is a promoter upstream transcript (PROMPT) of hypoxia-inducible factor-2α; HIF-2α, hypoxia-inducible factor-2 alpha; PCR, polymerase chain reaction; siRNA, small interfering RNA; NC, noncancerous; mRNA, messenger RNA; CSC, cancer stem cell.
© Copyright Policy
Related In: Results  -  Collection

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f3-ott-8-3467: Knockdown of lncRNA-HIF2PUT impairs CSC properties.Notes: (A) HIF2PUT lncRNA level and six stemness genes’ mRNA levels were measured by real-time PCR in DLD-1 and HT29 cells transfected with lncRNA-HIF2PUT siRNA and negative control siRNA. The expression of OCT4, SOX2, and CD44 was significantly downregulated. (B) Western blot showed that the expression of OCT4, SOX2, and CD44 protein was also significantly reduced in lncRNA-HIF2PUT siRNA groups compared with negative control groups. (C) Colony formation assay showed that CSC proliferation was retarded by lncRNA-HIF2PUT siRNA (P<0.05). (D) Spheroid formation assay showed that the self-renewal capacity in lncRNA-HIF2PUT siRNA groups was remarkably impaired. (E) The wound healing assay showed remarkable cell migration inhibition by lncRNA-HIF2PUT siRNA. (F) The matrigel invasion assay also showed significant cell invasion inhibition by lncRNA-HIF2PUT siRNA. *P<0.05.Abbreviations: lncRNA-HIF2PUT, long noncoding RNA that is a promoter upstream transcript (PROMPT) of hypoxia-inducible factor-2α; HIF-2α, hypoxia-inducible factor-2 alpha; PCR, polymerase chain reaction; siRNA, small interfering RNA; NC, noncancerous; mRNA, messenger RNA; CSC, cancer stem cell.
Mentions: Next, we determined the effects of lncRNA-HIF2PUT knockdown on stem cell-like properties in DLD-1 and HT29 cells in vitro. After lncRNA-HIF2PUT siRNA treatment, the stemness genes OCT4, SOX2, and CD44 decreased significantly (Figure 3A, B), which was coincident with decreased colony formation rates in medium containing fetal bovine serum, and spheroid formation ability in serum-free medium (Figure 3C, D). To further identify the functional role of lncRNA-HIF2PUT RNA in cell migration and invasion, the scratch wound healing assay and matrigel invasion assay were performed in DLD-1 and HT29 cells in vitro. The wound healing assay showed remarkable cell migration inhibition in DLD-1 and HT29 siRNA groups compared with the cells in the noncancerous group (Figure 3E). The matrigel invasion assay also showed significant cell invasion inhibition in the lncRNA-HIF2PUT siRNA group compared with the noncancerous groups in the DLD-1 and HT29 cell lines (Figure 3F).

Bottom Line: In this study, we found an lncRNA that is a promoter upstream transcript of hypoxia-inducible factor-2α (HIF-2α), and we named it "lncRNA-HIF2PUT".Herein, we showed that the expression of lncRNA-HIF2PUT was significantly correlated with HIF-2α in colorectal cancer (CRC) tissues.LncRNA-HIF2PUTsmall interfering RNA transfection resulted in decreased stemness genes expression, impaired colony formation, and spheroid formation ability, retarded migration, and invasion of the cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, People's Liberation Army No 161 Hospital, Wuhan, People's Republic of China.

ABSTRACT
Currently, various long noncoding RNAs (lncRNAs) have been identified as key regulators of multiple cancers. However, cancer stem cell (CSC)-related lncRNAs have rarely been reported. In this study, we found an lncRNA that is a promoter upstream transcript of hypoxia-inducible factor-2α (HIF-2α), and we named it "lncRNA-HIF2PUT". The function of HIF-2α is closely connected with "stem cell-like" properties, and the function of PROMPTs is often associated with the adjacent protein-coding transcripts. Herein, we showed that the expression of lncRNA-HIF2PUT was significantly correlated with HIF-2α in colorectal cancer (CRC) tissues. Knockdown of lncRNA-HIF2PUT blocked the HIF-2α expression and inhibited the CSC properties in CRC cell lines DLD-1 and HT29. LncRNA-HIF2PUTsmall interfering RNA transfection resulted in decreased stemness genes expression, impaired colony formation, and spheroid formation ability, retarded migration, and invasion of the cells. These data suggest that lncRNA-HIF2PUT may be a regulator of HIF-2α and a mediator of CSCs in CRC.

No MeSH data available.


Related in: MedlinePlus