HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts

Abstract

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named 'hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.

Figure 1

T-UCR-expression induction by hypoxia. (a) Experiment flowchart. (b) RT-qPCR confirmation of T-UCR-expression induction after 48 h under hypoxic conditions in HCT-116 colon cancer cells and hypoxia-simulation conditions by DMOG in the MCF-7 breast cancer cell line. Expression was normalized versus normal conditions (normoxia in HCT-116 and DMSO in MCF-7). Bars indicate the standard deviation from two sample duplicates. Western blot assay performed with HIF-1α-specific antibodies. (c) T-UCR induction measured using RT-qPCR in the VHL-deficient (VHL−) cell line was compared with that in the VHL-proficient (VHL+) 786-O cell line under normoxic conditions. Western blot analysis was performed using HIF-2α-specific antibodies. *P<0.05; **P<0.01; ***P<0.001

Figure 2

Confirmation of a direct association between HIF and T-UCR using ChIP and luciferase assay. (a) Direct recruitment of HIF on T-UCR-expression induction in hypoxia-simulated conditions by DMOG in the MCF-7 breast cancer cell line. We confirmed a direct connection between HIF and T-UCR (uc.475) using ChIP method with HIF-1α antibodies or an IgG control. To enrich genomic fragments that contain hypoxia-response elements (HIF-interacting sites), we amplified the regions represented on the genome graphic using specific primers for pointed locations. The ChIP figures are inverted for a better view of expression induction. NC, negative control; HBB, hemoglobin globin beta, which is supposed to be transcriptional inactive; and carbonic anhydrase IX (CA9), which was used as a positive control in the ChIP experiment. (b) The direct effect of HIF on the uc.475 promoter reporter construct was confirmed by using luciferase assay and was shown as an effect of exogenous pcDNA 3.1-HIF-1α vector constructs (HIF-1α P/A and HIF-1α 3M) on cloned promoter activity. A stronger effect was detected by using luciferase assay normalized using empty pcDNA 3.1 vector. The error bars represent the standard deviation from two independent experiments, each performed in triplicate and measured in duplicate with a luciferase assay. (c) Luciferase activity in HCT-116 and HT-29 cells was measured after transfection with truncated promoter reporter constructs (PT1 and PT2) with a complete deletion of the HIF-1α-binding sites that were present in the originally designed uc.475 promoter reporter construct (WT). Filled circles show the position of the HIF-1α-predicted binding site. ***P-value <0.001 compared with luciferase activity of an empty vector; WT, wild type; PT1, truncated promoter 1; PT2, truncated promoter 2

Figure 3

Uc.475 biological significance under normoxia and hypoxia. (a) The effect of siRNA-targeting uc.475 on cell proliferation was determined under normoxic (left) and hypoxic (right) conditions by using a pool of three siRNAs against uc.475 (siRNA-uc.475). Three independent experiments were performed, and as control scrambled siRNA sequence, siRNA-NEG, was used. (b) FACS analysis was performed 72 and 96 h post transfection with either siRNA-uc.475 or siRNA-NEG in normoxia (left) and hypoxia (right). (c) Western blot analysis to detect differences of uc.475 host gene (OGT) protein expression levels. Hypoxic conditions were confirmed by CA9 induction. Protein-specific bands were normalized to beta-actin. siRNA against OGT pre-mRNA was designed to target OGT-intron 12. *P-value <0.05, **P-value <0.01, ***P-value <0.001

Figure 4

Transcripts produced at the uc.475 genomic locus in an environment deprived of oxygen and/or glucose. (a) The genomic region of OGT from the UCSC Genomic Browser, GRCh37/hg19 with the known transcripts of OGT isoforms 1 and 2. The rounded rectangle represents region containing uc.475. Northern blot-probe locations are indicated. The intron 4 region where uc.475 is located is zoomed in for better view of region of conservation in mammals. (b) RT-qPCR data showing that the uc.475 noncoding transcript and the coding OGT transcript are expressed at similar levels, whereas the ‘hybrid' amplicons between the intron and the two adjacent exons are barely expressed. (c) Glucose- and oxygen-deprivation effects on mRNA expression level of uc.475 and OGT. CA9 mRNA expression was analyzed as a hypoxia-induced gene

Figure 5

Uc.475-transcript induction by hypoxia. Northern blot analysis of MCF-7 RNA identified transcripts of different sizes detected with uc.475 (left) or OGT probes (right). Transcript variants identified by Northern blot signals in MCF-7 cells under normoxia and hypoxia are marked by arrows and visualized by a drawing (middle). The panel PPIA was used as a loading control

Figure 6

Impacts on OGT expression and function after uc.475 inhibition and O-GlcNAcylation change after knocking down uc.475 or OGT. (a) RT-qPCR expression levels of OGT and uc.475 after uc.475 knockdown in HT-29 cells under normoxia and hypoxia. Primers were designed to detect both OGT1 and OGT2 isoforms. Error bars indicate standard deviation. UCR or uc, ultraconserved gene; OGT, O-linked N-acetylglucosamine (GlcNAc) transferase; siRNA, small interfering RNA; HT-29, colon cancer cell line; NOR, normoxia; HYP, hypoxia; PPIA, reference gene cyclophilin A; *P-value <0.05, ***P-value <0.001. (b) RT-qPCR confirmation of OGT gene knockdown using siRNA against OGT. Primers specific for uc.475 and each of the OGT isoforms were used. (c) Western blot performed to detect differences of uc.475 host gene (OGT) protein expression levels after OGT silencing. Hypoxic conditions were confirmed by CA9 induction. Protein-specific bands were normalized to beta-actin as a reference protein. (d) O-GlcNAc protein levels were detected in samples exposed to hypoxic conditions for 72 h or simultaneously transfected with siRNA-targeting either uc.475, OGT pre-mRNA or OGT. All levels were compared with sample transfected with siRNA-NEG. Different concentrations of PUGNAc were added in cell media (40 and 80 μM) to prevent O-GlcNAc from being removed from the proteins. siRNA against OGT pre-mRNA was designed to target OGT-intron 12

Figure 7

Testing the enhancer activity for cloned uc.475-containing region. Uc.475 enhancer activity experiments were performed in normoxia and hypoxia for 48 h. (a) Insert representing a transcript of 2.7-kb region (including uc.475 region) was cloned downstream of the luciferase reporter. (b) Dual-Luciferase reporter assays were performed on U-87 cell line samples exposed to normoxia or hypoxia for 48 h. x axis represents the relative Firefly to Renilla luciferase activity, whereas y axis presents the plasmids. All measurements generated were normalized to the pGL3-TK empty vector. All data shown are the mean of two biological repetitions (at least eight luciferase measurements per experiment). ***P-value <0.001