HIF-2-induced long non-coding RNA RAB11B-AS1 promotes hypoxia-mediated angiogenesis and breast cancer metastasis

Cell culture, transfection, and virus

The culture conditions of MDA-MB-231 (from Rolf Brekken at UT Southwestern in 2015), MDA-MB-468 (from ATCC in 2017), SUM159, MCF-7, T47D, BT474, ZR-75-1, HCC1954, HeLa, HEK293FT, and HEK293T (from Gregg L. Semenza at Johns Hopkins in 2014) cells were described previously (19). Cells within six passages were used for experiments. Human umbilical vein endothelial cells (HUVECs) were cultured in M200PRF medium with low serum growth supplement (Thermo Fisher Scientific) at 37°C in a 5% CO₂/95% air incubator. HUVECs within the first three passages were used for experiments. Hypoxic cells were placed in a modular incubator chamber (Billups-Rothenberg) and flushed with a gas mixture of 1% O₂, 5% CO₂, and balanced N₂. Cells were transfected using PolyJet (SignaGen). Lentiviruses encoding scrambled control (SC) or RAB11B-AS1 short hairpin RNAs (shRNAs) were generated as described previously (19). All cell lines except for HUVECs were annually tested to be mycoplasma-free and have been authenticated by STR DNA profiling analysis in 2016–2017.

RNA extraction, RT-qPCR, and RNA-sequencing (RNA-seq) assays

Total RNA was isolated from the tissues or cultured cells using Trizol (Thermo Fisher Scientific), treated with DNase I (Thermo Fisher Scientific) and then subject to cDNA synthesis with the iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time polymerase chain reaction (qPCR) was performed with iTaq universal SYBR green supermix (Bio-Rad) and normalized to the internal control 18S RNA or RPL13A as described previously (19). The primers used for qPCR are listed in Supplementary Table S1. RNA-seq was performed as described previously (19). The deidentified human breast tumors and benign breast tissues were used and their pathology was verified by a breast cancer pathologist (Y.V.F). The study was approved by the Institutional Review Board at UT Southwestern Medical Center with informed written consent.

5’ and 3’ rapid amplification of cDNA ends (RACEs) assay

5’- and 3’-RACE analyses were performed using the SMARTer RACE 5’/3’ kit (Takara Bio). Total RNA was extracted using the RNeasy micro kit (Qiagen) and treated with DNase. RNA integrity was assessed with the Agilent 4200 TapeStation system. After reverse transcription of total RNA to cDNA, 5’- and 3’-RACE PCRs were performed with the universal and gene-specific primers listed in Supplementary Table S2 and the resulting PCR products were cloned into linearized pRACE vector and sequenced. The nucleotide sequence of RAB11B-AS1 was deposited in the GenBank (MK855053).

Plasmid constructs

Full-length human RAB11B-AS1 cDNA was cloned into pcDNA3.1 or pBluescript II KS (+). RAB11B-AS1 shRNA nucleotides (Supplementary Table S2) were cloned into pLKO.1 vector. HIF-1β single gRNA (sgRNA) nucleotides (Supplementary Table S2) were cloned into lentiCRISPRv2 vector. Wild-type (WT) or mutant RAB11B-AS1 HRE nucleotides (Supplementary Table S2) were cloned into MluI/BglII-linearized pGL2p vector (Promega). All plasmids were verified by DNA sanger sequencing. Other plasmid constructs have been described previously (19).

Luciferase reporter assay

5 × 10⁴ of HEK293T or HeLa cells were seeded onto 48-well plates pre-coated with poly-L-lysine a day prior to transfection. Cells were cotransfected with pGL2p-WT RAB11B-AS1 HRE, pGL2p-mutant RAB11B-AS1 HRE, or pGL2p-VEGFA HRE; pSV40-Renilla; and p3XFLAG-HIF-2α, pcDNA3.1-RAB11B-AS1, or empty vector (EV) and exposed to 20% or 1% O₂ for 24 hours. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega).

Immunoblot assay

Cells were lysed with NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH8.0, 0.5% NP-40, and fresh protease inhibitor) and protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, and visualized by chemiluminescence with ECL prime (GE Healthcare) as described previously (19). The following antibodies were used: anti-HIF-1α (BD Bioscience, Cat.# 610959), anti-HIF-2α (Bethyl Laboratories, Cat.# A700-003), anti-HIF-1β (Novus Biologicals, Cat.# 100-110), RNA polymerase II (Abcam, Cat.# ab817), and anti-β-actin (Proteintech, Cat.# 66009-1-lg).

Chromatin immunoprecipitation (ChIP) assay

Cells were cross-linked with 1% formaldehyde for 10 minutes, quenched in 0.125 M glycine, and lysed with lysis buffer followed by sonication. Then the lysates were incubated overnight at 4°C with anti-RNA Pol II antibody (Abcam, Cat.# ab817), anti-HIF-2α antibody (homemade), anti-HIF-1β (Novus Biologicals, Cat.# 100-110), or IgG (Cell Signaling Technology, Cat.# 2729S). After decrosslinking of the protein-DNA complex and DNA purification, precipitated DNA was subject to library preparation for sequencing or qPCR with primers listed in Supplementary Table S1. Bioinformatics analysis of ChIP-sequencing (ChIP-seq) data was performed as described previously (19). The RNA-seq and ChIP-seq data were deposited at the GEO database (GSE139861).

RNA pulldown assay

The biotin-labeled sense and antisense of RAB11B-AS1 RNA were synthesized in vitro by T7 RNA polymerase (Thermo Fisher Scientific) using pBluescript-RAB11B-AS1 as a template and the biotin RNA labeling mix (Sigma). The RNAs were denatured, refolded in RNA structure buffer (10 mM Tris-HCl, pH 7.0, 0.1 M KCl, and 10 mM MgCl₂), and incubated for 2 hours at 4 °C in the presence of 0.1 μg/μL tRNA, 0.025% BSA, and the pre-cleared lysates, which were extracted from MDA-MB-231 cells exposed to 20% or 1% O₂ for 24 hours in RNA immunoprecipitation buffer (150 mM KCl, 25 mM Tris-HCl, pH7.4, 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT, fresh protease inhibitor, and RNase inhibitor). 0.25% BSA-prebound streptavidin magnetic beads (GE Healthcare) were added for additional 1-hour incubation at 4 °C. The proteins bound to biotin-labeled RAB11B-AS1 RNA were separated by SDS-PAGE and analyzed by immunoblot assay.

Boyden chamber migration and invasion assays

Cell migration and invasion assays were performed as described previously (19). 2 × 10⁴ of cells were suspended in serum-free media and seeded onto the non-coated (migration) or Matrigel-coated (invasion) upper chamber. 10% FBS in the bottom chamber was used as a chemoattractant. Cells were exposed to 20% or 1% O₂ for 16 hours (migration) or 24 hours (invasion), fixed with methanol, and stained with 0.1% crystal violet. Colonies were imaged using microscope and counted in five randomly selected fields at 20x magnification.

In vitro angiogenesis assay

4 × 10⁴ of HUVECs were starved in M200PRF medium without low serum growth supplement for 2 hours, resuspended in fresh conditional media with 1% FBS, and seeded onto a 96‐well plate coated with growth factor‐reduced Matrigel (BD Biosciences). Conditional media were collected by incubating MDA-MB-231-EV or -RAB11B-AS1 cells with serum-free DMEM for 24 hours. HUVECs were imaged using the IncuCyte S3 live-cell analysis system (Sartorius) for 5 hours and the total tube length from five randomly selected fields at 10x magnification was measured by the Wimasis image analysis.

Animal studies

Six-eight weeks old female NOD/SCID IL2Rγnull (NSG) mice were used for animal studies, which has been approved and conducted under the oversight of the UT Southwestern Institutional Animal Care and Use Committee. For the orthotopic xenograft model, 2 × 10⁶ of breast cancer cells suspended in PBS/Matrigel (1:1, Corning) were implanted into the second left mammary fat pad of female NSG mice. Tumor growth was monitored with a caliper every 3 days starting 14 days after implantation and calculated with the formula: volume = 0.52 × length × height × width. Mice were euthanized 32–40 days after cell implantation and primary tumors were harvested. After perfusion with PBS, the whole lungs were inflated with low-melt agarose and analyzed by haematoxylin and eosin (H&E) staining. The whole liver was also collected for H&E staining. Genomic DNA was extracted from the whole lungs and liver and analyzed by qPCR assay with primers for human HK2 gene and mouse and human 18S rRNA. For the tail vein injection model, 1 × 10⁶ of breast cancer cells suspended in 100 μL PBS were injected into the tail vein of female NSG mice. Three weeks later, the lungs were harvested for H&E staining and qPCR analysis with primers for human HK2 gene and mouse and human 18S rRNA. The area of metastatic foci was quantified by ImageJ and normalized to the total area of lung or liver tissues.

Immunohistochemistry (IHC) assay

IHC assay was performed by the Dako Autostainer Link 48 system (Dako) as described (19). The tissue slides underwent deparaffinication, rehydration, and epitope retrieval and were then incubated with the primary antibody: Endomucin (Santa Cruz Biotechnology, 1:50), Cleaved caspase-3 (CC3, Cell Signaling Tchnology, 1:1500), and Ki-67 (Proteintech, 1:1000). Staining was visualized using the EnVision FLEX visualization system (Dako). The endomucin-, Ki-67-, and CC3-positive areas were quantified by ImageJ and normalized to their respective total area.

Statistical analysis

Statistical analysis was performed by Student’s t test with or without Welch’s correction between two groups, and one-way or two-way ANOVA with multiple testing correction within multiple groups. RNA-seq and ChIP-seq were performed in duplicate and other experiments were repeated at least three times. Data were expressed as mean ± SEM. p < 0.05 is considered significant.

RAB11B-AS1 is induced by hypoxia in breast cancer cells in a HIF-2-dependent manner

Our RNA-seq studies (GSE108833) showed that RAB11B-AS1 was a hypoxia-inducible lncRNA in human breast cancer MDA-MB-231 cells. To validate, we performed RT-qPCR in a panel of human breast cancer cell lines, including MDA-MB-231, MDA-MB-468, SUM159, MCF-7, T47D, BT474, ZR-75-1, and HCC1954, exposed to 20% or 1% O₂ for 24 hours. RAB11B-AS1 RNA levels were significantly upregulated by hypoxia in all breast cancer cell lines we tested (Fig. 1A). RAB11B-AS1 is an antisense RNA of the RAB11B gene. To annotate the natural transcript of RAB11B-AS1 in breast cancer cells, we performed 5’- and 3’-RACE and detected a specific 1037-nucleotide RAB11B-AS1 transcript in MDA-MB-231 cells (Fig. 1B; Supplementary Fig. S1). The RAB11B-AS1 transcript had 10 more A nucleotides in the poly(A) tail at its 3’ end but missed 7 nucleotides at the 5’ end as compared with the reference transcript deposited in the GenBank (NR_038237.1, Supplementary Fig. S1). In addition, there were two nucleotide mutations in the exons 2&3 (Supplementary Fig. S1). Together, these data indicate that RAB11B-AS1 is a natural lncRNA and induced by hypoxia in breast cancer cells.

To determine whether HIF is required for hypoxia-induced RAB11B-AS1 in breast cancer cells, we performed RT-qPCR assay in parental, HIF-1α knockout (KO), HIF-2α KO, HIF-1α and HIF-2α double knockout (DKO) MDA-MB-231 cells exposed to 20% or 1% O₂ for 24 hours. HIF-2α KO, but not HIF-1α KO, eliminated hypoxia-induced RAB11B-AS1 expression in MDA-MB-231 cells (Fig. 1C). Similar results were also found in HIF-1α and HIF-2α DKO cells under hypoxia (Fig. 1C). KO of HIF-1α or HIF-2α in MDA-MB-231 cells were confirmed by determining the expression of HIF-1α and HIF-2α (Fig. 1D). Likewise, KO of HIF-1β abolished hypoxia-induced RAB11B-AS1 expression in MDA-MB-231 cells (Fig. 1E and F). These data indicate that HIF-2 is responsible for hypoxia-induced RAB11B-AS1 expression in breast cancer cells.

To unbiasedly identify the HRE on the RAB11B-AS1 gene, we performed ChIP-seq assay in hypoxic MDA-MB-231 cells and found a robust HIF-2α peak annotated with the HRE sequence 5’-ACGTG-3’ at the promoter of the RAB11B-AS1 gene (Fig. 2A). To validate, we performed ChIP-qPCR in MDA-MB-231 cells exposed to 20% or 1% O₂ for 24 hours. HIF-2α was enriched at this HRE, but not on the non-target gene RPL13A (Fig. 2B and C). Hypoxia significantly increased HIF-2α occupancy at the RAB11B-AS1 HRE (Fig. 2B). Similarly, HIF-1β occupancy was detected at the RAB11B-AS1 HRE in hypoxic MDA-MB-231 cells (Fig. 2D). To examine whether this HRE is functional, a 60-base pair DNA fragment containing the HRE was cloned into the upstream of the SV40 promoter and firefly luciferase gene in a pGL2p reporter vector. HEK293T cells were transfected with pGL2p-EV or pGL2p-RAB11B-AS1 HRE, and the control plasmid pSV40-Renilla, and exposed to 20% or 1% O₂ for 24 hours. The RAB11B-AS1 HRE significantly enhanced firefly luciferase activity in HEK293T cells under hypoxia as compared with the EV, but failed to do so in non-hypoxic cells (Fig. 2E), where no HIF-2α protein is present due to its proteasomal degradation, indicating the specificity of the RAB11B-AS1 HRE in response to hypoxia. Mutation of 5’-CGT-3’ into 5’-AAA-3’ within the RAB11B-AS1 HRE completely abolished hypoxia-induced firefly luciferase activity in HEK293T cells (Fig. 2E). Moreover, ectopic expression of HIF-2α significantly increased RAB11B-AS1 HRE-driven firefly luciferase activity in non-hypoxic and hypoxic HEK293T cells (Fig. 2F; Supplementary Fig. S2A). In contrast, HIF-2α KO significantly inhibited hypoxia-induced RAB11B-AS1 HRE activity in HeLa cells (Fig. 2G; Supplementary Fig. S2B). Collectively, these data indicate that RAB11B-AS1 is a direct HIF-2 target gene in human breast cancer cells.

RAB11B-AS1 increases breast cancer cell migration and invasion in vitro

To determine the oncogenic role of RAB11B-AS1 in breast cancer cells, we next generated RAB11B-AS1-overexpressed MDA-MB-231 cells. RT-qPCR assay showed about 14-fold increase in RAB11B-AS1 RNA levels in these cells (Fig. 3A). The Boyden transwell assay showed that ectopic expression of RAB11B-AS1 significantly increased migration and invasion of MDA-MB-231 cells under 20% and 1% O₂ as compared with the EV (Fig. 3B–E). Similar results were also observed in SUM159 cells (Supplementary Fig. S3A–E). These data indicate that RAB11B-AS1 overexpression promotes breast cancer cell migration/invasion in vitro.

To complement gain-of-function studies, we next performed loss-of-function studies by generating two independent RAB11B-AS1 knockdown (KD) MDA-MB-231 cell lines via lentiviral infection. About 80–90% of RAB11B-AS1 KD efficiency was achieved in either of cell lines under hypoxia (Fig. 3F). Hypoxia increased migration of SC MDA-MB-231 cells by more than 1.5-fold, whereas RAB11B-AS1 KD significantly inhibited cell migration under 20% and 1% O₂ (Fig. 3G and H). Similarly, the number of cells invaded through Matrigel was also significantly decreased by RAB11B-AS1 KD under 20% and 1% O₂ (Fig. 3I and J). Likewise, RAB11B-AS1 KD also significantly reduced migration and invasion of SUM159 or MDA-MB-468 cells under 20% and 1% O₂ (Supplementary Fig. S3F–O). Together, gain- and loss-of-function studies indicate that RAB11B-AS1 promotes breast cancer cell motility in vitro.

RAB11B-AS1 promotes tumor angiogenesis and breast tumor metastasis in mice

To investigate the effect of RAB11B-AS1 on breast cancer cell motility in vivo, we implanted EV or RAB11B-AS1-overexpressed MDA-MB-231 cells orthotopically into the mammary fat pad of female NSG mice. Ectopic expression of RAB11B-AS1 in MDA-MB-231 cells did not affect primary tumor growth in mice (Fig. 4A–C). RT-qPCR assay confirmed > 20-fold increase in RAB11B-AS1 RNA levels in RAB11B-AS1 tumors (Fig. 4D). The blood vessel marker endomucin-positive area was significantly increased in RAB11B-AS1 tumors as compared to EV tumors (Fig. 4E and F), whereas the area percentage of staining of Ki-67, a marker for cell proliferation, was comparable in these tumors (Fig. 4E and G). RAB11B-AS1 did not alter the percentage of the cell death marker CC3-positive area in tumors (Supplementary Fig. S4A and B), suggesting that RAB11B-AS1 has no effect on caspase-3-dependent cell death in breast tumors. Notably, H&E analysis in the lungs revealed more spontaneous metastases in mice bearing RAB11B-AS1 tumors than EV tumors (Fig. 4H and I), which was confirmed by analysis of the presence of human genomic DNA in mouse lungs with qPCR (Fig. 4J). Similarly, ectopic expression of RAB11B-AS1 significantly increased MDA-MB-231 cell metastasis to the liver in mice (Fig. 4K–M).

Next, we performed loss-of-function studies in the orthotopic breast cancer mouse model. In line with gain-of-function studies above (Fig. 4A–C), RAB11B-AS1 KD in MDA-MB-231 cells did not affect primary tumor growth in NSG mice (Fig. 5A–C). RT-qPCR assay confirmed robust RAB11B-AS1 KD in tumors (Fig. 5D). IHC analysis showed the density of endomucin-positive blood vessels was significantly decreased in RAB11B-AS1 KD1 or KD2 tumors as compared to SC tumors (Fig. 5E and F). In contrast, Ki-67 staining was comparable between SC and RAB11B-AS1 KD tumors (Fig. 5E and G), in line with gain-of-function studies (Fig. 4E and G). Both H&E and qPCR assays showed decreased spontaneous breast cancer metastasis to the lungs and liver in mice bearing RAB11B-AS1 KD tumors as compared to those with SC tumors (Fig. 5H–M).

To investigate whether RAB11B-AS1 regulates breast cancer cell colonization to increase distant metastasis, we injected SC or RAB11B-AS1 KD1 or KD2 MDA-MB-231 cells into the tail vein of female NSG mice. The lungs were harvested three weeks later. H&E staining detected extensive tumor foci in the lungs from SC tumor bearing mice (Fig. 5N and O). In contrast, fewer breast cancer cells were present in the lungs from mice bearing RAB11B-AS1 KD1 or KD2 tumors (Fig. 5N and O). qPCR assay further confirmed that RAB11B-AS1 KD significantly reduced breast cancer cell colonization in the lungs (Fig. 5P). Collectively, these findings indicate that RAB11B-AS1 increases tumor angiogenesis and promotes breast cancer metastasis to distant organs in mice without affecting primary tumor growth.

RAB11B-AS1 enhances hypoxia-induced angiogenic factors in breast cancer cells through the recruitment of RNA Pol II

To dissect the molecular mechanism of RAB11B-AS1-induced tumor angiogenesis, we investigated whether RAB11B-AS1 regulates the expression of angiogenic factors in breast cancer cells. RNA-seq assay identified 1526 upregulated genes and 1677 downregulated genes in RAB11B-AS1-overexpressed MDA-MB-231 cells under hypoxia (FDR > 0.05, LogCPM > 0, fold change > 1.25, Fig. 6A). Gene ontology analysis of upregulated genes revealed that angiogenesis and response to hypoxia were two of top biological processes (Supplementary Table S3). To validate, we performed RT-qPCR assay in EV and RAB11B-AS1-overexpressed MDA-MB-231 cells exposed to 20% or 1% O₂ for 24 hours. Consistently, ectopic expression of RAB11B-AS1 significantly increased the VEGFA mRNA expression in non-hypoxic and hypoxic MDA-MB-231 cells (Fig. 6B). Interestingly, ANGPTL4 mRNA was also enhanced by RAB11B-AS1 overexpression in non-hypoxic and hypoxic MDA-MB-231 cells (Fig. 6B), despite not being selected as the RAB11B-AS1 target gene according to our selection criteria in RNA-seq (Fig. 6A). Similar results were also observed in SUM159 cells (Supplementary Fig. S5A). Complementary to gain-of-function studies, RAB11B-AS1 KD by either of two shRNAs significantly decreased hypoxia-induced VEGFA and ANGPTL4 mRNAs in MDA-MB-231 and MDA-MB-468 cells (Fig. 6C; Supplementary Fig. S5B). Reduction of VEGFA and ANGPTL4 mRNAs was very modest or little in RAB11B-AS1 KD MDA-MB-231 and MDA-MB-468 cells exposed to 20% O₂ (Fig. 6C; Supplementary Fig. S5B). Other angiogenic factors including ANGPT2, CXCR4, FN1, MMP9, and bFGF were not significantly affected by RAB11B-AS1 KD1 or KD2 (Supplementary Fig. S5C). Ectopic expression or KD of RAB11B-AS1 failed to affect the mRNA levels of RAB11B and RPL13A in MDA-MB-231, MDA-MB-468, or SUM159 cells (Fig. 6B and C; Supplementary Fig. S5). These data indicate that RAB11B-AS1 increases hypoxia-induced angiogenic factors including VEGFA and ANGPTL4 in breast cancer cells.

Next, we studied whether angiogenic factors induced by RAB11B-AS1 in breast cancer cells regulates angiogenesis in vitro. Conditional culture media from EV or RAB11B-AS1-overexpressed MDA-MB-231 cells were collected and incubated with HUVECs. After 5-hour incubation, the length of tubes was significantly increased in HUVECs cultured with RAB11B-AS1 conditional media as compared to EV conditional media (Fig. 6D and E). These data indicate that RAB11B-AS1 increases the expression of angiogenic factors in breast cancer cells to promote angiogenesis in vitro, which supports our in vivo findings (Fig. 4E and F).

RNA Pol II binding to the gene promoter is an essential step in transcriptional regulation. Gene ontology of RAB11B-AS1-upregulated genes identified RNA Pol II-dependent gene regulation as the top biological function, implicating that RNA Pol II may be involved in RAB11B-AS1-mediated gene regulation (Supplementary Table S3). To determine the role of RNA Pol II in RAB11B-AS1-mediated VEGFA and ANGPTL4 expression, we studied whether RAB11B-AS1 interacts with RNA Pol II by RNA pull-down assay. In vitro transcribed biotin-labeled RAB11B-AS1 RNA was incubated in the presence of streptavidin magnetic beads with lysates of MDA-MB-231 cells exposed to 20% or 1% O₂ for 24 hours. Endogenous RNA Pol II was strongly pulled down by RAB11B-AS1 RNA, but barely bound to beads only (Fig. 6F). We further studied whether RAB11B-AS1 regulates RNA Pol II binding to VEGFA and ANGPTL4 genes by RNA Pol II ChIP-qPCR assay. RNA Pol II was enriched at the promoter of VEGFA and ANGPTL4 genes and its enrichment was significantly increased by hypoxia in MDA-MB-231 cells (Fig. 6G and H). RAB11B-AS1 KD significantly reduced RNA Pol II occupancy at the promoter of VEGFA and ANGPTL4 genes in hypoxic MDA-MB-231 cells (Fig. 6G and H), which was consistent with their reduced gene expression in RAB11B-AS KD cells under hypoxia (Fig. 6C; Supplementary Fig. S5). RAB11B-AS1 KD had no effect on RNA Pol II binding to the RPL13A gene in MDA-MB-231 cells (Fig. 6I). These findings indicate that RAB11B-AS1 promotes RNA Pol II binding to VEGFA and ANGPTL4 genes under hypoxia to increase their expression in breast cancer cells.

It has been known that HIF induces VEGFA and ANGPTL4 in cancer cells to promote tumor angiogenesis and metastasis (19). We studied the role of HIF in RAB11B-AS1-mediated VEGFA and ANGPTL4 expression. RNA pull-down assay showed that HIF-1α and HIF-2α both failed to bind to sense or anti-sense of RAB11B-AS1 RNA (Supplementary Fig. S6A). The stability of HIF-1α and HIF-2α proteins is critical for HIF transcriptional activity (16). Ectopic expression or KD of RAB11B-AS1 did not affect HIF-1α and HIF-2α protein levels in MDA-MB-231 cells (Supplementary Fig. S6B and C). We further performed HIF luciferase reporter assay to determine whether RAB11B-AS1 regulates HIF transcriptional activity. HEK293T cells were cotransfected with a HIF reporter plasmid, which contains 2 × VEGFA HRE upstream of the SV40 promoter and firefly luciferase coding gene, and pSV40-Renilla, and RAB11B-AS1 expression vector or EV, and exposed to 20% or 1% O₂ for 24 hours. Ectopic expression of RAB11B-AS1 had no effect on HIF transcriptional activity in non-hypoxic and hypoxic HEK293T cells (Supplementary Fig. S6D). Together, these data indicate that HIF is not directly involved in RAB11B-AS1-mediated VEGFA and ANGPTL4 expression in hypoxic breast cancer cells.

RAB11B-AS1 RNA is upregulated in human breast tumors

To study clinical relevance of RAB11B-AS1, we analyzed its expression in human breast tumors and control benign breast tissues. RAB11B-AS1 RNA levels were significantly higher in human breast tumors as compared to control breast tissues (Fig. 7A). Likewise, VEGFA and ANGPTL4 mRNAs were also upregulated in human breast tumors (Fig. 7B and C). The Pearson correlation analysis revealed that RAB11B-AS1 RNA expression was significantly correlated with VEGFA or ANGPTL4 mRNA expression in human breast tumors (Fig. 7D and E). These data indicate that RAB11B-AS1 RNA is upregulated and correlated with VEGFA and ANGPTL4 mRNAs in human breast tumors.