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

Figures & data

Table 1 Primers for real-time PCR analysis

Gene name	Forward	Reverse
β-Actin	5′-CCACTGGCATCGTGATGGA-3′	5′-CGCTCGGTGAGGATCTTCAT-3′
HIF2PUT	5′-CGGAGGTGTTCTATGAGCTGG-3′	5′-AGCTTGTGTGTTCGCAGGAA-3′
HIF-2α	5′-TGGGATCTAACAGGAACAGC-3′	5′-CTAAATAGCCAGACAAGGGT-3′
OCT4	5′-TATGCAAAGCAGAAACCCTCGTGC-3′	5′-TTCGGGCACTGCAGGAACAAATTC-3′
SOX2	5′-GCCGAGTGGAAACTTTTGTCG-3′	5′-GGCAGCGTGTACTTATCCTTCT-3′
NANOG	5′-TCCAGCAGATGCAAGAACTCTCCA-3′	5′-CACACCATTGCTATTCTTCGGCCA-3′
KLF4	5′-CCCACATGAAGCGACTTCCC-3′	5′-CAGGTCCAGGAGATCGTTGAA-3′
CD44	5′-CTGCCGCTTTGCAGGTGTA-3′	5′-CATTGTGGGCAAGGTGCTATT-3′
CD133	5′-AGTCGGAAACTGGCAGATAGC-3′	5′-GGTAGTGTTGTACTGGGCCAAT-3′

Abbreviations: PCR, polymerase chain reaction; HIF-2α, hypoxia-inducible factor-2 alpha.

Figure 1 IncRNA-HIF2PUT and HIF-2α expression levels were analyzed by real-time PCR in CRC tissue samples and in eight cell lines.

Notes: (A) The expression levels of lncRNA-HIF2PUT and (B) HIF-2α were calculated as the ratio of targets in cancerous tissue/targets in adjacent normal tissue (R [C/N]). (C) The expression of lncRNA-HIF2PUT was significantly correlated with that of HIF-2α (P<0.0001). (D) The expression of lncRNA-HIF2PUT was coincident with that of HIF-2α in the eight cell lines (P<0.0001). (E and F) The expression of both lncRNA-HIF2PUT and HIF-2α were significantly higher in each of the seven CRC cell lines than in the normal colon epithelial cell line FHC. The expression of lncRNA-HIF2PUT and HIF-2α was highest in DLD-1 and HT29 cells. *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; CRC, colorectal cancer; PCR, polymerase chain reaction.

Figure 2 Knockdown of lncRNA-HIF2PUT inhibited the expression of HIF-2α.

Notes: (A) Quantification analysis by real-time PCR showed that the expression levels of both lncRNA-HIF2PUT and HIF-2α in lncRNA-HIF2PUT siRNA groups were significantly knocked down in DLD-1 and HT29 cell lines. (B) Western blot showed that the HIF-2α protein expression was also markedly decreased. *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.

Figure 3 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.