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Hypoxia Promotes Gastric Cancer Malignancy Partly through the HIF-1 α Dependent Transcriptional Activation of the Long Non-coding RNA GAPLINC

View Article: PubMed Central - PubMed

ABSTRACT

Hypoxia-inducible factor (HIF) activates the transcription of genes involved in cancer progression. Recently, HIF was reported to regulate the transcription of non-coding RNAs. Here, we show that the transcription of a long non-coding RNA (lncRNA), Gastric Adenocarcinoma Associated, Positive CD44 Regulator, Long Intergenic Non-Coding RNA (GAPLINC), is directly activated by HIF-1α in gastric cancer (GC). GAPLINC was overexpressed in GC tissues and promoted tumor migration and invasive behavior. GAPLINC overexpression was associated with poor prognosis in GC patients. Luciferase reporter assays and chromatin immunoprecipitation assays confirmed that HIF-1α binds to the promoter region of GAPLINC and activates its transcription. GAPLINC knockdown inhibited hypoxia-induced tumor proliferation in vivo. Taken together, our results identified a novel role for HIF transcriptional pathways in GC tumorigenesis mediated by the regulation of the lncRNA GAPLINC, and suggest GAPLINC as a novel therapeutic target for reversing chemoradioresistance and prolonging survival.

No MeSH data available.


HIF-1α bound to the HRE of GAPLINC and triggered transcriptional activation. (A) The binding motif of HIF-1α transcription pathway. (B) Effect of HIF-1α on the promoter activities of GAPLINC in MKN45 and SGC7901 cells. Schematic depiction of the different reporter constructs (a and b) used and the luciferase activity. Plasmid activity after normalization with the co-transfected reference vector pRL-TK is shown in the X-axis. Background luminescence from cells transfected with empty pGL3-basic vector was substracted from the sample readings. (C) The ChIP assay indicated that HIF-1α binds to the GAPLINC promoter in MKN45 and SGC7901 cells. Transcription start site (TSS) and putative HIF-1α binding sites are indicated. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a negative control. Visualized bands were considered as positive results and indicated endogenous binding of HIF-1α to DNA fragments (*P < 0.05).
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Figure 6: HIF-1α bound to the HRE of GAPLINC and triggered transcriptional activation. (A) The binding motif of HIF-1α transcription pathway. (B) Effect of HIF-1α on the promoter activities of GAPLINC in MKN45 and SGC7901 cells. Schematic depiction of the different reporter constructs (a and b) used and the luciferase activity. Plasmid activity after normalization with the co-transfected reference vector pRL-TK is shown in the X-axis. Background luminescence from cells transfected with empty pGL3-basic vector was substracted from the sample readings. (C) The ChIP assay indicated that HIF-1α binds to the GAPLINC promoter in MKN45 and SGC7901 cells. Transcription start site (TSS) and putative HIF-1α binding sites are indicated. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a negative control. Visualized bands were considered as positive results and indicated endogenous binding of HIF-1α to DNA fragments (*P < 0.05).

Mentions: We speculated that there may be a direct interaction between GAPLINC and HIF-1α, the most important factor induced by hypoxia. HIF-1α binds the “ACGTG” motif according to JASPAR prediction (Figure 6A) and the “CACGC” sequence (Ren et al., 2014), which is characteristic of hypoxic response elements. Screening of the promoter region of GAPLINC identified two potential binding regions for HIF-1α (−320/−324 and −578/−582). To further investigate the mechanism underlying the HIF-1α mediated regulation of GAPLINC expression, a luciferase assay was performed (Figure 6B). The position of the transcription start site (TSS) was predicted by DBTSS. Putative and wild-type HIF-1α binding sites were constructed. Cotransfection with pEX3-HIF-1α resulted in the activation of the GAPLINC promoter containing two HREs by 4.3- and 3.7-fold in MKN45 and SGC7901 cells, respectively. For the GAPLINC promoter containing one HRE, the activation was 2.5- and 2.8-fold in MKN45 and SGC7901 cells, respectively.


Hypoxia Promotes Gastric Cancer Malignancy Partly through the HIF-1 α Dependent Transcriptional Activation of the Long Non-coding RNA GAPLINC
HIF-1α bound to the HRE of GAPLINC and triggered transcriptional activation. (A) The binding motif of HIF-1α transcription pathway. (B) Effect of HIF-1α on the promoter activities of GAPLINC in MKN45 and SGC7901 cells. Schematic depiction of the different reporter constructs (a and b) used and the luciferase activity. Plasmid activity after normalization with the co-transfected reference vector pRL-TK is shown in the X-axis. Background luminescence from cells transfected with empty pGL3-basic vector was substracted from the sample readings. (C) The ChIP assay indicated that HIF-1α binds to the GAPLINC promoter in MKN45 and SGC7901 cells. Transcription start site (TSS) and putative HIF-1α binding sites are indicated. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a negative control. Visualized bands were considered as positive results and indicated endogenous binding of HIF-1α to DNA fragments (*P < 0.05).
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Figure 6: HIF-1α bound to the HRE of GAPLINC and triggered transcriptional activation. (A) The binding motif of HIF-1α transcription pathway. (B) Effect of HIF-1α on the promoter activities of GAPLINC in MKN45 and SGC7901 cells. Schematic depiction of the different reporter constructs (a and b) used and the luciferase activity. Plasmid activity after normalization with the co-transfected reference vector pRL-TK is shown in the X-axis. Background luminescence from cells transfected with empty pGL3-basic vector was substracted from the sample readings. (C) The ChIP assay indicated that HIF-1α binds to the GAPLINC promoter in MKN45 and SGC7901 cells. Transcription start site (TSS) and putative HIF-1α binding sites are indicated. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a negative control. Visualized bands were considered as positive results and indicated endogenous binding of HIF-1α to DNA fragments (*P < 0.05).
Mentions: We speculated that there may be a direct interaction between GAPLINC and HIF-1α, the most important factor induced by hypoxia. HIF-1α binds the “ACGTG” motif according to JASPAR prediction (Figure 6A) and the “CACGC” sequence (Ren et al., 2014), which is characteristic of hypoxic response elements. Screening of the promoter region of GAPLINC identified two potential binding regions for HIF-1α (−320/−324 and −578/−582). To further investigate the mechanism underlying the HIF-1α mediated regulation of GAPLINC expression, a luciferase assay was performed (Figure 6B). The position of the transcription start site (TSS) was predicted by DBTSS. Putative and wild-type HIF-1α binding sites were constructed. Cotransfection with pEX3-HIF-1α resulted in the activation of the GAPLINC promoter containing two HREs by 4.3- and 3.7-fold in MKN45 and SGC7901 cells, respectively. For the GAPLINC promoter containing one HRE, the activation was 2.5- and 2.8-fold in MKN45 and SGC7901 cells, respectively.

View Article: PubMed Central - PubMed

ABSTRACT

Hypoxia-inducible factor (HIF) activates the transcription of genes involved in cancer progression. Recently, HIF was reported to regulate the transcription of non-coding RNAs. Here, we show that the transcription of a long non-coding RNA (lncRNA), Gastric Adenocarcinoma Associated, Positive CD44 Regulator, Long Intergenic Non-Coding RNA (GAPLINC), is directly activated by HIF-1&alpha; in gastric cancer (GC). GAPLINC was overexpressed in GC tissues and promoted tumor migration and invasive behavior. GAPLINC overexpression was associated with poor prognosis in GC patients. Luciferase reporter assays and chromatin immunoprecipitation assays confirmed that HIF-1&alpha; binds to the promoter region of GAPLINC and activates its transcription. GAPLINC knockdown inhibited hypoxia-induced tumor proliferation in vivo. Taken together, our results identified a novel role for HIF transcriptional pathways in GC tumorigenesis mediated by the regulation of the lncRNA GAPLINC, and suggest GAPLINC as a novel therapeutic target for reversing chemoradioresistance and prolonging survival.

No MeSH data available.