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Cited by 133Cancer, Hypoxia, and MetabolismCancer-related molecular mechanisms researchMetabolism, Diabetes, and Cancer

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LncRNA IDH1-AS1 links the functions of c-Myc and HIF1α via IDH1 to regulate the Warburg effect

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Xiang, Shaoxun, Gu, Hao, Jin, Lei, Thorne, Rick F., Zhang, Xu Dong, Wu, Mian·2018·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1711257115PMC PMC5816142
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Abstract

The oncoprotein c-Myc plays an important role in regulating glycolysis under normoxia; yet, in cancer cells, HIF1α, which is essential for driving glycolysis under hypoxia, is often up-regulated even in the presence of oxygen. The relationship between these two major regulators of the Warburg effect remains to be fully defined. Here we demonstrate that regulation of a long noncoding RNA (lncRNA), named IDH1-AS1, enables c-Myc to collaborate with HIF1α in activating the Warburg effect under normoxia. c-Myc transcriptionally repressed IDH1-AS1, which, upon expression, promoted homodimerization of IDH1 and thus enhanced its enzymatic activity. This resulted in increased α-KG and decreased ROS production and subsequent HIF1α down-regulation, leading to attenuation of glycolysis. Hence, c-Myc repression of IDH1-AS1 promotes activation of the Warburg effect by HIF1α. As such, IDH1-AS1 overexpression inhibited cell proliferation, whereas silencing of IDH1-AS1 promoted cell proliferation and cancer xenograft growth. Restoring IDH1-AS1 expression may therefore represent a potential metabolic approach for cancer treatment.

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Fig. 1. See also Fig. S1. IDH1-AS1 enhances IDH1 enzymatic activity. (A) Whole cell lysates from HeLa and HCT116 cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 were subjected to Western blot analysis. n = 3. (B–D) Whole cell lysates from HeLa and HCT116 cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 were analyzed for IDH1 and IDH2 activity (B), α-KG levels (C), and NADPH/NADP+ ratios (D). IDH1 and IDH2 activity, α-KG levels, or NADPH/NADP+ ratios in cells transduced with shctrl were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (E) Representative flow cytometry histograms of analysis of ROS levels in HeLa cells transduced with shctrl or shIDH1-AS1-1. MFI, mean fluorescence intensity. n = 3. (F) Whole cell lysates from HeLa and HCT116 cells transduced with the psin vertor or psin-IDH1-AS1 were analyzed for IDH1 and IDH2 enzyme activity. The activity of IDH1 and IDH2 in cells transduced with the vector alone was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (G and H) Whole cell lysates from HeLa and HCT116 cells transduced with the psin vertor or psin-IDH1-AS1 were analyzed for α-KG levels (G) and NADPH/NADP+ ratios (H). The α-KG level (G) and NADPH/NADP+ ratios (H) in cells transduced with the vector alone were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (I) Whole cell lysates from HeLa cells transduced with the psin vector alone or psin-IDH1-AS1 were subjected to Western blot analysis. n = 3. (J) In vitro-transcribed sense or antisense IDH1-AS1 was incubated with purified Flag-IDH1 for 1 h before analysis of IDH1 enzyme activity. The activity of IDH1 in samples without incubation with in vitro-transcribed sense or antisense IDH1-AS1 was arbitrarily designated as 1. Values are mean ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (K) Total RNA from HeLa, HCT116, HAFF, and IMR90 cells was analyzed using qPCR for IDH1-AS1 expression. The relative abundance of IDH1 mRNA in HeLa cells was arbitrarily designated as 1. Values are means ± SEMs; n = 3. (*P < 0.05, **P < 0.01, two-tailed paired Student’s t test). (L) Whole cell lysates from HeLa, HCT116, HAFF, and IMR90 cells were analyzed for IDH1 activity. The activity of IDH1 in HeLa cells was arbitrarily designated as 1. Values are means ± SEMs; n = 3. (*P < 0.05, two-tailed paired Student’s t test). (M) c-Myc and IDH1 expression in HeLa, HCT116, HAFF, and IMR90 cells detected using Western blot analysis of whole cell lysates. n = 3. n.s., not significant; shc-Myc, c-Myc shRNA; shctrl, control shRNA.
Fig. 1. See also Fig. S1. IDH1-AS1 enhances IDH1 enzymatic activity. (A) Whole cell lysates from HeLa and HCT116 cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 were subjected to Western blot analysis. n = 3. (B–D) Whole cell lysates from HeLa and HCT116 cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 were analyzed for IDH1 and IDH2 activity (B), α-KG levels (C), and NADPH/NADP+ ratios (D). IDH1 and IDH2 activity, α-KG levels, or NADPH/NADP+ ratios in cells transduced with shctrl were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (E) Representative flow cytometry histograms of analysis of ROS levels in HeLa cells transduced with shctrl or shIDH1-AS1-1. MFI, mean fluorescence intensity. n = 3. (F) Whole cell lysates from HeLa and HCT116 cells transduced with the psin vertor or psin-IDH1-AS1 were analyzed for IDH1 and IDH2 enzyme activity. The activity of IDH1 and IDH2 in cells transduced with the vector alone was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (G and H) Whole cell lysates from HeLa and HCT116 cells transduced with the psin vertor or psin-IDH1-AS1 were analyzed for α-KG levels (G) and NADPH/NADP+ ratios (H). The α-KG level (G) and NADPH/NADP+ ratios (H) in cells transduced with the vector alone were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (I) Whole cell lysates from HeLa cells transduced with the psin vector alone or psin-IDH1-AS1 were subjected to Western blot analysis. n = 3. (J) In vitro-transcribed sense or antisense IDH1-AS1 was incubated with purified Flag-IDH1 for 1 h before analysis of IDH1 enzyme activity. The activity of IDH1 in samples without incubation with in vitro-transcribed sense or antisense IDH1-AS1 was arbitrarily designated as 1. Values are mean ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (K) Total RNA from HeLa, HCT116, HAFF, and IMR90 cells was analyzed using qPCR for IDH1-AS1 expression. The relative abundance of IDH1 mRNA in HeLa cells was arbitrarily designated as 1. Values are means ± SEMs; n = 3. (*P < 0.05, **P < 0.01, two-tailed paired Student’s t test). (L) Whole cell lysates from HeLa, HCT116, HAFF, and IMR90 cells were analyzed for IDH1 activity. The activity of IDH1 in HeLa cells was arbitrarily designated as 1. Values are means ± SEMs; n = 3. (*P < 0.05, two-tailed paired Student’s t test). (M) c-Myc and IDH1 expression in HeLa, HCT116, HAFF, and IMR90 cells detected using Western blot analysis of whole cell lysates. n = 3. n.s., not significant; shc-Myc, c-Myc shRNA; shctrl, control shRNA.CC BY-NC-ND · PubMed Central
Fig. 2. See also Fig. S2. c-Myc inhibits IDH1 enzyme activity. (A) Whole cell lysates from HeLa cells transduced with shctrl or shc-Myc were analyzed for IDH1 and IDH2 activity (Left), α-KG levels (Middle), and NADPH levels (Right). The relative IDH1 and IDH2 activity and α-KG abundance and NADPH/NADP+ ratio in cells transduced with shctrl were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (B) ShRNA knockdown efficiency of c-Myc in HeLa cells as shown in Western blot analysis of whole cell lysates. n = 3. (C) Whole cell lysates from HeLa cells transduced with Flag-vector or Flag-tagged c-Myc were analyzed for IDH1 and IDH2 activity (Left), α-KG levels (Middle), and NADPH levels (Right). The relative IDH1 and IDH2 activity and α-KG abundance and NADPH/NADP+ ratio in cells transduced with Flag-vector were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (D) Overexpression of c-Myc in HeLa cells transduced Flag-tagged c-Myc as measured by Western blot analysis of whole cell lysates. n = 3. (E) Total RNA from HeLa cells transduced with shctrl or shc-Myc was subjected to qPCR analysis of IDH1 and IDH1 mRNA expression. The relative abundance of IDH1 and IDH2 mRNA in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (two-tailed paired Student’s t test). (F) Whole cell lysates from HeLa cells transduced with shctrl or shc-Myc were subjected to Western blot analysis. n = 3. (G) Total RNA from HeLa cells transduced with Flag-vector or Flag-tagged c-Myc was subjected to qPCR analysis of IDH1 and IDH1 mRNA expression. The relative abundance of IDH1 and IDH2 mRNA in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (two-tailed paired Student’s t test). (H) Whole cell lysates from HeLa cells transduced with Flag-vector or Flag-tagged c-Myc were subjected to Western blot analysis. n = 3. n.s., not significant; shctrl, control shRNA.
Fig. 2. See also Fig. S2. c-Myc inhibits IDH1 enzyme activity. (A) Whole cell lysates from HeLa cells transduced with shctrl or shc-Myc were analyzed for IDH1 and IDH2 activity (Left), α-KG levels (Middle), and NADPH levels (Right). The relative IDH1 and IDH2 activity and α-KG abundance and NADPH/NADP+ ratio in cells transduced with shctrl were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (B) ShRNA knockdown efficiency of c-Myc in HeLa cells as shown in Western blot analysis of whole cell lysates. n = 3. (C) Whole cell lysates from HeLa cells transduced with Flag-vector or Flag-tagged c-Myc were analyzed for IDH1 and IDH2 activity (Left), α-KG levels (Middle), and NADPH levels (Right). The relative IDH1 and IDH2 activity and α-KG abundance and NADPH/NADP+ ratio in cells transduced with Flag-vector were arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (D) Overexpression of c-Myc in HeLa cells transduced Flag-tagged c-Myc as measured by Western blot analysis of whole cell lysates. n = 3. (E) Total RNA from HeLa cells transduced with shctrl or shc-Myc was subjected to qPCR analysis of IDH1 and IDH1 mRNA expression. The relative abundance of IDH1 and IDH2 mRNA in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (two-tailed paired Student’s t test). (F) Whole cell lysates from HeLa cells transduced with shctrl or shc-Myc were subjected to Western blot analysis. n = 3. (G) Total RNA from HeLa cells transduced with Flag-vector or Flag-tagged c-Myc was subjected to qPCR analysis of IDH1 and IDH1 mRNA expression. The relative abundance of IDH1 and IDH2 mRNA in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (two-tailed paired Student’s t test). (H) Whole cell lysates from HeLa cells transduced with Flag-vector or Flag-tagged c-Myc were subjected to Western blot analysis. n = 3. n.s., not significant; shctrl, control shRNA.CC BY-NC-ND · PubMed Central
Fig. 3. See also Fig. S3. c-Myc transcriptionally represses IDH1-AS1. (A) Total RNA from HeLa (Left), HCT116 (Middle), and H1299 (Right) cells transduced with shctrl or shc-Myc was analyzed using qPCR for the expression of IDH1-AS1, whereas whole cell lysates were subjected to Western blot analysis. The relative abundance of IDH1-AS1 in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (B) Total RNA and whole cell lysates from P493 cell lysates from P493 cells carrying a Tet-Off c-Myc expression system treated with doxycycline (1 μM) were respectively subjected to qPCR Western blot analysis. The relative abundance of IDH1-AS1 in cells without doxycycline treatment was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). Dox, doxycycline. (C) Total RNA and whole cell lysates from HeLa cells transfected with Flag-vector or Flag-tagged c-Myc at increasing concentrations were subjected to qPCR and Western blot analysis, respectively. The relative abundance of IDH1-AS1 in cells transduced with Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, **P < 0.01; two-tailed paired Student’s t test). (D) Total RNA from HeLa cells cotransduced with shctrl or shMiz1 and Flag-vector or Flag-tagged c-Myc was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (E) Total RNA from HeLa cells cotransduced with GFP-vector or GFP-tagged Miz1 and shctrl or shc-Myc was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (**P < 0.01, ***P < 0.001; two-tailed paired Student’s t test). (F) Whole cell lysates from HeLa cells transduced with Flag-vector, Flag-tagged c-Myc, or Flag-tagged Miz1 were subjected to ChIP assays. ChIP products were analyzed by qPCR using primers directed to the indicated fragments of the IDH1-AS1 promoter. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (G) Schematic illustration of luciferase reporter constructs containing the WT or mutant c-Myc binding site in the promoter of IDH1-AS1. BS, binding site; mut, mutant. (H) HeLa cells transduced with shctrl or shc-Myc were cotransfected with reporter constructs containing the WT (c-Myc-BS) or mutant (c-Myc-mut-BS) c-Myc binding site as illustrated in G and Renilla luciferase plasmids. Transcriptional activity was determined by luciferase assays. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (I) HeLa cells transduced with shctrl or shMiz1 were cotransfected with Flag-vector or Flag-tagged c-Myc and Renilla luciferase plasmids. Transcriptional activity was determined by luciferase assays. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (J) Whole cell lysates from HeLa cells transduced with Flag-vector, Flag-tagged c-Myc, or Flag-tagged c-Myc plus psin-IDH1-AS1 were analyzed for IDH1 activity. The activity of IDH1 in cells transduced with Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (K) Whole cell lysates from HeLa cells transduced with shctrl, shc-Myc, or shc-Myc plus shIDH1-AS1 were analyzed for IDH1 activity. The activity of IDH1 in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). ctrl, control; n.s., not significant; shctrl, control shRNA.
Fig. 3. See also Fig. S3. c-Myc transcriptionally represses IDH1-AS1. (A) Total RNA from HeLa (Left), HCT116 (Middle), and H1299 (Right) cells transduced with shctrl or shc-Myc was analyzed using qPCR for the expression of IDH1-AS1, whereas whole cell lysates were subjected to Western blot analysis. The relative abundance of IDH1-AS1 in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (B) Total RNA and whole cell lysates from P493 cell lysates from P493 cells carrying a Tet-Off c-Myc expression system treated with doxycycline (1 μM) were respectively subjected to qPCR Western blot analysis. The relative abundance of IDH1-AS1 in cells without doxycycline treatment was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). Dox, doxycycline. (C) Total RNA and whole cell lysates from HeLa cells transfected with Flag-vector or Flag-tagged c-Myc at increasing concentrations were subjected to qPCR and Western blot analysis, respectively. The relative abundance of IDH1-AS1 in cells transduced with Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, **P < 0.01; two-tailed paired Student’s t test). (D) Total RNA from HeLa cells cotransduced with shctrl or shMiz1 and Flag-vector or Flag-tagged c-Myc was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (E) Total RNA from HeLa cells cotransduced with GFP-vector or GFP-tagged Miz1 and shctrl or shc-Myc was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (**P < 0.01, ***P < 0.001; two-tailed paired Student’s t test). (F) Whole cell lysates from HeLa cells transduced with Flag-vector, Flag-tagged c-Myc, or Flag-tagged Miz1 were subjected to ChIP assays. ChIP products were analyzed by qPCR using primers directed to the indicated fragments of the IDH1-AS1 promoter. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (G) Schematic illustration of luciferase reporter constructs containing the WT or mutant c-Myc binding site in the promoter of IDH1-AS1. BS, binding site; mut, mutant. (H) HeLa cells transduced with shctrl or shc-Myc were cotransfected with reporter constructs containing the WT (c-Myc-BS) or mutant (c-Myc-mut-BS) c-Myc binding site as illustrated in G and Renilla luciferase plasmids. Transcriptional activity was determined by luciferase assays. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (I) HeLa cells transduced with shctrl or shMiz1 were cotransfected with Flag-vector or Flag-tagged c-Myc and Renilla luciferase plasmids. Transcriptional activity was determined by luciferase assays. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (J) Whole cell lysates from HeLa cells transduced with Flag-vector, Flag-tagged c-Myc, or Flag-tagged c-Myc plus psin-IDH1-AS1 were analyzed for IDH1 activity. The activity of IDH1 in cells transduced with Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (K) Whole cell lysates from HeLa cells transduced with shctrl, shc-Myc, or shc-Myc plus shIDH1-AS1 were analyzed for IDH1 activity. The activity of IDH1 in cells transduced with shctrl was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). ctrl, control; n.s., not significant; shctrl, control shRNA.CC BY-NC-ND · PubMed Central
Fig. 4. See also Fig. S4. IDH1-AS1 interacts with IDH1 and promotes its dimerization. (A) Total RNA from the cytoplasmic, nuclear, or mitochondrial fraction of HeLa cells was subjected to qPCR analysis of IDH1-AS1 expression (Left), whereas total protein from each fraction was subjected to Western blot analysis. The overall relative abundance of IDH1-AS1 cytoplasmic, nuclear and mitochondrial fractions was designated as 100%. Values are means ± SEMs; n = 3. Cyto, cytoplasmic; Mito, mitochondrial; Nucl, nuclear. (B) Whole cell lysates from HeLa cells transduced with Flag-vector, Flag-tagged IDH1, Flag-tagged IDH1R132H, or Flag-tagged IDH2 were subjected to RNA-IP with an anti-Flag antibody. The resulting precipitates were then subjected to qPCR analysis. The relative abundance of IDH1-AS1 in cells transduced with Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). (C) Whole cell lysates from HeLa cells were subjected to RNA-IP using an anti-IDH1 antibody. The resulting precipitates were then subjected to qPCR analysis. Values are mean ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). ND, not detectable. (D) Whole cell lysates from HeLa cells were incubated with in vitro-synthesized, biotin-labeled sense or antisense IDH1-AS1 probes followed by incubation with streptavidin-conjugated beads. The eluted fractions were then subjected to Western blot analysis. Values are means ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). ctrl, control. (E) Whole cell lysates from HeLa cells ectopically expressing GFP-tagged IDH1 and Flag-tagged IDH1 were subjected to IP using an anti-Flag antibody. The resulting precipitates were then subjected to Western blot analysis using an anti-Flag or anti-GFP antibody. n = 3. (F) Whole cell lysates from HeLa cells transduced with Flag-Vector and GFP-IDH1, Flag-IDH1 plus GFP-IDH1, and Flag-Vector plus GFP-IDH1 were subjected to IP using an anti-Flag antibody. The resulting precipitates were then eluted using Flag peptides. Ten percent of the eluents were subjected to Western blot analysis using an anti-Flag or anti-GFP antibody. Ten percent of the eluents were subjected to qPCR analysis of IDH1-AS1 expression. Eighty percent of the eluents were subjected to a secondary phase of IP using an anti-GFP antibody. Ten percent of the resulting precipitates were subjected to Western blot analysis using an anti-Flag or anti-GFP antibody. The rest of the eluents were subject to qPCR analysis of IDH1-AS1 expression. Values are means ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). (G) Whole cell lysates from HeLa cells ectopically expressing Flag tagged-IDH1 or Flag-tagged IDH1R132H mutant were incubated with in vitro-transcribed IDH1-AS1. The resulting precipitates were subjected to either RT-PCR or Western blot analysis. n = 3. (H) Whole cell lysates from HeLa cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 with or without treatment with DSS were subjected to Western blot analysis. n = 3. (I) Whole cell lysates from HeLa cells overexpressing IDH1-AS1 with or without treatment with DSS were subjected to Western blot analysis. n = 3. (J) Whole cell lysates from HeLa cells ectopically expressing Flag-tagged IDH1 were incubated with in vitro-transcribed full-length IDH1-AS1, IDH1-AS1 Fragment 1, IDH1-AS1 Fragment 2, or IDH1-AS1 Fragment 3 as schematically illustrated. The resulting precipitates were subjected to either RT-PCR or Western blot analysis. n = 3. (K) Whole cell lysates from HeLa cells transfected with Flag-tagged IDH1, Flag-tagged IDH1-P1, Flag-tagged IDH1-P2, or Flag-tagged IDH1-P3 as schematically illustrated were subjected to RNA-IP using an anti-Flag antibody, whereas total RNA was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (L) Whole cell lysates from HeLa cells transduced with Flag-tagged IDH1 or Flag-tagged IDH1 with deletion of residue 271–286 (Flag-IDH1Δ271–286) were analyzed for IDH1 activity. The activity of IDH1 in cells transduced with Flag-tagged IDH1 was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (***P < 0.001, two-tailed paired Student’s t test). (M) Whole cell lysates from HeLa cells transfected with Flag-tagged IDH1 or Flag-tagged IDH1 with deletion of residue 271–286 (Flag-IDH1Δ271–286) were subjected to RNA-IP using an anti-Flag antibody, whereas total RNA was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (*P < 0.05, **P < 0.01; two-tailed paired Student’s t test). DSS, disuccinimidyl suberate; IP, immunoprecipitation; shctrl, control shRNA; WB, Western blot.
Fig. 4. See also Fig. S4. IDH1-AS1 interacts with IDH1 and promotes its dimerization. (A) Total RNA from the cytoplasmic, nuclear, or mitochondrial fraction of HeLa cells was subjected to qPCR analysis of IDH1-AS1 expression (Left), whereas total protein from each fraction was subjected to Western blot analysis. The overall relative abundance of IDH1-AS1 cytoplasmic, nuclear and mitochondrial fractions was designated as 100%. Values are means ± SEMs; n = 3. Cyto, cytoplasmic; Mito, mitochondrial; Nucl, nuclear. (B) Whole cell lysates from HeLa cells transduced with Flag-vector, Flag-tagged IDH1, Flag-tagged IDH1R132H, or Flag-tagged IDH2 were subjected to RNA-IP with an anti-Flag antibody. The resulting precipitates were then subjected to qPCR analysis. The relative abundance of IDH1-AS1 in cells transduced with Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). (C) Whole cell lysates from HeLa cells were subjected to RNA-IP using an anti-IDH1 antibody. The resulting precipitates were then subjected to qPCR analysis. Values are mean ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). ND, not detectable. (D) Whole cell lysates from HeLa cells were incubated with in vitro-synthesized, biotin-labeled sense or antisense IDH1-AS1 probes followed by incubation with streptavidin-conjugated beads. The eluted fractions were then subjected to Western blot analysis. Values are means ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). ctrl, control. (E) Whole cell lysates from HeLa cells ectopically expressing GFP-tagged IDH1 and Flag-tagged IDH1 were subjected to IP using an anti-Flag antibody. The resulting precipitates were then subjected to Western blot analysis using an anti-Flag or anti-GFP antibody. n = 3. (F) Whole cell lysates from HeLa cells transduced with Flag-Vector and GFP-IDH1, Flag-IDH1 plus GFP-IDH1, and Flag-Vector plus GFP-IDH1 were subjected to IP using an anti-Flag antibody. The resulting precipitates were then eluted using Flag peptides. Ten percent of the eluents were subjected to Western blot analysis using an anti-Flag or anti-GFP antibody. Ten percent of the eluents were subjected to qPCR analysis of IDH1-AS1 expression. Eighty percent of the eluents were subjected to a secondary phase of IP using an anti-GFP antibody. Ten percent of the resulting precipitates were subjected to Western blot analysis using an anti-Flag or anti-GFP antibody. The rest of the eluents were subject to qPCR analysis of IDH1-AS1 expression. Values are means ± SEMs; n = 3 (**P < 0.01, two-tailed paired Student’s t test). (G) Whole cell lysates from HeLa cells ectopically expressing Flag tagged-IDH1 or Flag-tagged IDH1R132H mutant were incubated with in vitro-transcribed IDH1-AS1. The resulting precipitates were subjected to either RT-PCR or Western blot analysis. n = 3. (H) Whole cell lysates from HeLa cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 with or without treatment with DSS were subjected to Western blot analysis. n = 3. (I) Whole cell lysates from HeLa cells overexpressing IDH1-AS1 with or without treatment with DSS were subjected to Western blot analysis. n = 3. (J) Whole cell lysates from HeLa cells ectopically expressing Flag-tagged IDH1 were incubated with in vitro-transcribed full-length IDH1-AS1, IDH1-AS1 Fragment 1, IDH1-AS1 Fragment 2, or IDH1-AS1 Fragment 3 as schematically illustrated. The resulting precipitates were subjected to either RT-PCR or Western blot analysis. n = 3. (K) Whole cell lysates from HeLa cells transfected with Flag-tagged IDH1, Flag-tagged IDH1-P1, Flag-tagged IDH1-P2, or Flag-tagged IDH1-P3 as schematically illustrated were subjected to RNA-IP using an anti-Flag antibody, whereas total RNA was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (L) Whole cell lysates from HeLa cells transduced with Flag-tagged IDH1 or Flag-tagged IDH1 with deletion of residue 271–286 (Flag-IDH1Δ271–286) were analyzed for IDH1 activity. The activity of IDH1 in cells transduced with Flag-tagged IDH1 was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (***P < 0.001, two-tailed paired Student’s t test). (M) Whole cell lysates from HeLa cells transfected with Flag-tagged IDH1 or Flag-tagged IDH1 with deletion of residue 271–286 (Flag-IDH1Δ271–286) were subjected to RNA-IP using an anti-Flag antibody, whereas total RNA was subjected to qPCR analysis. Values are means ± SEMs; n = 3 (*P < 0.05, **P < 0.01; two-tailed paired Student’s t test). DSS, disuccinimidyl suberate; IP, immunoprecipitation; shctrl, control shRNA; WB, Western blot.CC BY-NC-ND · PubMed Central
Fig. 5. See also Fig. S5. IDH1-AS1 negatively regulates glycolysis through suppressing HIF1α. (A) Whole cell lysates from HeLa cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 were subjected to Western blot analysis. n = 3. (B) HeLa cells transduced with shctrl or shIDH1-AS1-1 were cotransfected with the indicated reporter constructs and Renilla luciferase plasmids. Transcriptional activity was determined by luciferase assays. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). mut, mutant. (C) Whole cell lysates from HeLa and HCT116 cells cointroduced with shctrl or shIDH1-AS1 and shHIF-1α were subjected to Western blot analysis. n = 3. (D) Whole cell lysates from HeLa cells cointroduced with shctrl or shIDH1-AS1-1 and psin-IDH1-AS1 were subjected to Western blot analysis. n = 3. (E) IDH1-AS1 silencing accelerated acidification of the culture medium of HeLa cells. n = 3. (F) Whole cell lysates from HeLa cells transduced with shctrl or shIDH1-AS1-1 with or without treatment with Octyl-α-KG (1 mM) and/or NAC (1 mM) were subjected to Western blot analysis. n = 3. (G and H) Whole cell lysates from HeLa cells transduced with shctrl or shIDH1-AS1-1 with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose (G) and lactate (H) levels. The relative abundance of glucose or lactate in cells transduced with shctrl without treatment was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (I and J) Whole cell lysates from HeLa cells cointroduced with shctrl or shIDH1-AS1-1 and shHIF-1α were analyzed for glucose (I) and lactate (J) levels. The relative abundance of glucose or lactate in cells transduced with shctrl only was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). n.s., not significant; shctrl, control shRNA. (Scale bars, 1 cm.)
Fig. 5. See also Fig. S5. IDH1-AS1 negatively regulates glycolysis through suppressing HIF1α. (A) Whole cell lysates from HeLa cells transduced with shctrl, shIDH1-AS1-1, or shIDH1-AS1-2 were subjected to Western blot analysis. n = 3. (B) HeLa cells transduced with shctrl or shIDH1-AS1-1 were cotransfected with the indicated reporter constructs and Renilla luciferase plasmids. Transcriptional activity was determined by luciferase assays. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). mut, mutant. (C) Whole cell lysates from HeLa and HCT116 cells cointroduced with shctrl or shIDH1-AS1 and shHIF-1α were subjected to Western blot analysis. n = 3. (D) Whole cell lysates from HeLa cells cointroduced with shctrl or shIDH1-AS1-1 and psin-IDH1-AS1 were subjected to Western blot analysis. n = 3. (E) IDH1-AS1 silencing accelerated acidification of the culture medium of HeLa cells. n = 3. (F) Whole cell lysates from HeLa cells transduced with shctrl or shIDH1-AS1-1 with or without treatment with Octyl-α-KG (1 mM) and/or NAC (1 mM) were subjected to Western blot analysis. n = 3. (G and H) Whole cell lysates from HeLa cells transduced with shctrl or shIDH1-AS1-1 with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose (G) and lactate (H) levels. The relative abundance of glucose or lactate in cells transduced with shctrl without treatment was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (I and J) Whole cell lysates from HeLa cells cointroduced with shctrl or shIDH1-AS1-1 and shHIF-1α were analyzed for glucose (I) and lactate (J) levels. The relative abundance of glucose or lactate in cells transduced with shctrl only was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). n.s., not significant; shctrl, control shRNA. (Scale bars, 1 cm.)CC BY-NC-ND · PubMed Central
Fig. 6. IDH1-AS1 inhibition contributes to c-Myc-mediated glycolysis. (A and B) Whole cell lysates from HeLa cells cointroduced with shctrl or shc-Myc and shIDH1-AS1-1 were analyzed for glucose (A) and lactate (B) levels. The relative abundance of glucose or lactate in cells transduced with shctrl only was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (C) shRNA knockdown efficiency of IDH1-AS1 and c-Myc in HeLa cells as tested using qPCR and Western blot analysis, respectively. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (D) Whole cell lysates from HeLa and HCT116 cells cointroduced with shctrl or shHIF-1α and shIDH1-AS1 were subjected to Western blot analysis of the expression. n = 3. (E) Successful overexpression of IDH1-AS1 and c-Myc in HeLa cells as tested using qPCR and Western blot analysis, respectively. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (F and G) Whole cell lysates from HeLa cells cointroduced with the psin vector alone or psin-IDH1-AS1 and Flag-vector or Flag-tagged c-Myc were analyzed for glucose (F) and lactate (G) levels. The relative abundance of glucose or lactate in cells transduced with the psin vector plus Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). shctrl, control shRNA.
Fig. 6. IDH1-AS1 inhibition contributes to c-Myc-mediated glycolysis. (A and B) Whole cell lysates from HeLa cells cointroduced with shctrl or shc-Myc and shIDH1-AS1-1 were analyzed for glucose (A) and lactate (B) levels. The relative abundance of glucose or lactate in cells transduced with shctrl only was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (C) shRNA knockdown efficiency of IDH1-AS1 and c-Myc in HeLa cells as tested using qPCR and Western blot analysis, respectively. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (D) Whole cell lysates from HeLa and HCT116 cells cointroduced with shctrl or shHIF-1α and shIDH1-AS1 were subjected to Western blot analysis of the expression. n = 3. (E) Successful overexpression of IDH1-AS1 and c-Myc in HeLa cells as tested using qPCR and Western blot analysis, respectively. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (F and G) Whole cell lysates from HeLa cells cointroduced with the psin vector alone or psin-IDH1-AS1 and Flag-vector or Flag-tagged c-Myc were analyzed for glucose (F) and lactate (G) levels. The relative abundance of glucose or lactate in cells transduced with the psin vector plus Flag-vector was arbitrarily designated as 1. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). shctrl, control shRNA.CC BY-NC-ND · PubMed Central
Fig. 7. See also Fig. S6. IDH1-AS1 inhibits cell proliferation and shows a tumor-suppressive effect. (A) IDH1-AS1 silencing promoted clonogenicity, which was partially reversed by treatment with α-KG, in HeLa cells. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (B) IDH1-AS1 overexpression inhibited clonogenicity in HeLa cells. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (C) IDH1-AS1 silencing promoted HeLa cell xenograft growth in nu/nu mice. HeLa cells (2 × 106) transduced with shctrl or shIDH1-AS1-1 were xenografted s.c. into flanks of nu/nu mice. Four weeks after injection, mice were killed and photographed and tumors were measured and weighed. Values are means ± SEMs; n = 6 (**P < 0.01, two-tailed paired Student’s t test). (D) IDH1 deficiency promoted HeLa cell xenograft growth in nu/nu mice. HeLa cells (2 × 106) carrying WT IDH1 (WT) or those with IDH1 knocked out (IDH1-KO) were xenografted s.c. into flanks of nu/nu mice. Four weeks after injection, mice were killed and photographed and tumors were measured and weighed. Values are means ± SEMs; n = 6 (**P < 0.01, two-tailed paired Student’s t test). shctrl, control shRNA. (Scale bars, 1 cm.)
Fig. 7. See also Fig. S6. IDH1-AS1 inhibits cell proliferation and shows a tumor-suppressive effect. (A) IDH1-AS1 silencing promoted clonogenicity, which was partially reversed by treatment with α-KG, in HeLa cells. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (B) IDH1-AS1 overexpression inhibited clonogenicity in HeLa cells. Values are means ± SEMs; n = 3 (*P < 0.05, two-tailed paired Student’s t test). (C) IDH1-AS1 silencing promoted HeLa cell xenograft growth in nu/nu mice. HeLa cells (2 × 106) transduced with shctrl or shIDH1-AS1-1 were xenografted s.c. into flanks of nu/nu mice. Four weeks after injection, mice were killed and photographed and tumors were measured and weighed. Values are means ± SEMs; n = 6 (**P < 0.01, two-tailed paired Student’s t test). (D) IDH1 deficiency promoted HeLa cell xenograft growth in nu/nu mice. HeLa cells (2 × 106) carrying WT IDH1 (WT) or those with IDH1 knocked out (IDH1-KO) were xenografted s.c. into flanks of nu/nu mice. Four weeks after injection, mice were killed and photographed and tumors were measured and weighed. Values are means ± SEMs; n = 6 (**P < 0.01, two-tailed paired Student’s t test). shctrl, control shRNA. (Scale bars, 1 cm.)CC BY-NC-ND · PubMed Central

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Metadata

Authors
Xiang, S., Gu, H., Jin, L., Thorne, R. F., Zhang, X. D., & Wu, M.
Affiliations
  • University of Science and Technology of China (CN)
  • Henan University (CN)
Corresponding author
  • Zhang, Xu Dong
  • Wu, Mian
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2018-01-29
Volume
115
Issue
7
Pages
E1465-E1474
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.1711257115
PMC
PMC5816142
OpenAlex
W2793701251
Cited by
129
References
71
Citations by year
2026: 6 · 2025: 16 · 2024: 15 · 2023: 16 · 2022: 15 · 2021: 19 · 2020: 25 · 2019: 11 · 2018: 6
FWCI
4.61
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
National Natural Science Foundation of China (81430065, 31371388)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
long non-coding RNA, c-Myc, HIF-1α, IDH1, Warburg effect, glycolysis, reactive oxygen species, hypoxia, cancer metabolism, cell proliferation
MeSH terms
Animals, Uterine Cervical Neoplasms, Female, Glycolysis, Humans, Isocitrate Dehydrogenase, Ketoglutaric Acids, Mice, Nude, Tumor Cells, Cultured, Transcriptional Activation, Proto-Oncogene Proteins c-myc, RNA, Antisense, Xenograft Model Antitumor Assays, Cell Proliferation, Mice, Hypoxia-Inducible Factor 1, alpha Subunit, RNA, Long Noncoding

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