lncRNA NR2F1‐AS1 promotes breast cancer angiogenesis through activating IGF‐1/IGF‐1R/ERK pathway

Abstract Long non‐coding RNAs (lncRNAs) take various effects in cancer mostly through sponging with microRNAs (miRNAs). lncRNA NR2F1‐AS1 is found to promote tumour progression in hepatocellular carcinoma, endometrial cancer and thyroid cancer. However, the role of lncRNA NR2F1‐AS1 in breast cancer angiogenesis remains unknown. In this study, we found lncRNA NR2F1‐AS1 was positively related with CD31 and CD34 in breast cancer through Pearson's correlation analysis, while lncRNA NR2F1‐AS1 transfection promoted human umbilical vascular endothelial cell (HUVEC) tube formation. In breast cancer cells, lncRNA NR2F1‐AS1 enhanced the HUVEC proliferation, tube formation and migration ability through tumour‐conditioned medium (TCM). In zebrafish model, lncRNA NR2F1‐AS1 increased the breast cancer cell‐related neo‐vasculature and subsequently promoted the breast cancer cell metastasis. In mouse model, lncRNA NR2F1‐AS1 promoted the tumour vessel formation, increased the micro vessel density (MVD) and then induced the growth of primary tumour. Mechanically, lncRNA NR2F1‐AS1 increased insulin‐like growth factor‐1 (IGF‐1) expression through sponging miRNA‐338‐3p in breast cancer cells and then activated the receptor of IGF‐1 (IGF‐1R) and extracellular signal‐regulated kinase (ERK) pathway in HUVECs. These results indicated that lncRNA NR2F1‐AS1 could promote breast cancer angiogenesis through IGF‐1/IGF‐1R/ERK pathway.

biological effects as antisense transcripts on transcriptional regulation, cellular function and many disease including cancer. 10,11 ln-cRNAs take part in numerous cancer-related procedures such as proliferation, apoptosis, stem cell differentiation, metastasis and therapy resistance. [12][13][14][15][16] And one of the underlying mechanisms is that lncRNAs could combine with microRNAs (miRNA) as competing endogenous RNA (ceRNA) to protect the target genes. 17,18 It was reported that lncRNA NR2F1-AS1 regulated hepatocellular carcinoma oxaliplatin resistance, 19 and it was also involved in the progression of endometrial cancer. 20 Moreover, lncRNA NR2F1-AS1 was found to promote proliferation and migration yet suppress apoptosis of thyroid cancer cells through regulating miRNA-338-3p. 21 However, the role of lncRNA NR2F1-AS1 in breast cancer especially in breast cancer angiogenesis is still unknown.
Insulin-like growth factor-1 (IGF-1) is a kind of small polypeptide that could regulate mammary development. 22 Researchers found that IGF-1 also have various biologic effects in cancer development including modulating stem cells, genomic stability, cellular metabolism and angiogenesis. 23 In breast cancer, IGF-1 could induce proliferation and migration, 24,25 hence high expression of IGF-1 was related to increased risk of breast cancer, 26 and IGF-1 was regarded as a promising therapy target in breast cancer. 27 IGF-1 could also act as one of the pro-angiogenetic factors through inducing VEGF or NO in breast cancer microenvironment. 28,29 The receptor of IGF-1 (IGF-1R) with the combination of IGF-1 could exert vital function in cancer such as regulating cancer stem cell, epithelial-mesenchymal transition and tumour microenvironment, 30 which further influence cancer progression, metastasis and angiogenesis. 31 However, the specific mechanism of IGF-1 and IGF-1R regulating breast cancer angiogenesis requires further investigation yet.
In this study, we investigated the promotive effects of lncRNA NR2F1-AS1 on breast cancer angiogenesis both in vitro and in vivo, declaring the potential mechanism through inducing the expression of IGF-1 in breast cancer cells and then activating IGF-1R/ERK pathway in endothelial cells. All the cells were incubated in the humidified incubator at 37°C with 5% CO 2 .

| Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted from human breast cancer cell lines or HUVECs through TRIzol reagent (Invitrogen, USA) and taken into reverse transcription through PrimeScript RT reagent Kit (Takara, Japan) to produce cDNA under manufacturer's instructions. qPCR was conducted through SYBR Green (Takara) and LightCycler480 system (Roche, Switzerland).

| Tumour-conditioned medium (TCM)
Breast cancer cells were seeded in 6-well plates. Then, the culture medium was removed while serum-free DMEM was added. The supernatant was collected after 48-hours culture, centrifuged at 152 g for 10 minutes and filtered with 0.22 μm membrane to get TCM. The TCM was then stored at −80°C for tumour angiogenetic assays in vitro. For the tube formation assay, TCM was concentrated 75-fold with ultrafiltration device (Millipore, USA).

| Tube formation assay
Pre-cooled 96-well plate was coated with 50 μL growth factorreduced Matrigel (BD, Corning, USA) and incubated for 30 minutes at 37°C. Serum-free ECM starved HUVECs were seeded at 2 × 10 4 /well on the gel in 200 μL concentrated TCM (or in 200 μL DMEM containing 2% FBS for transfected HUVECs). Capillary structure was observed continuously within 12-hour period under microscope.

| Wound healing assay
Human umbilical vascular endothelial cells were seeded in 6-well plates to full confluence. Vertical scratches were drawn with pipette tip, after which the culture medium was changed for TCM.
The migration was observed every 24 hours for 3 days under microscope.

| CCK8 proliferation assay
Human umbilical vascular endothelial cells were seeded in 96-well plates to get adherent. Then, the complete medium was removed and TCM was added. CCK8 (Dojindo Laboratories, Japan) assay was performed according to manufacturer's instructions. The absorbance at 450 nm wavelength was measured every day for 5 days with microplate reader (Sunrise, Tecan, Austria).  After incubated in secondary antibody, the protein expression was showed using ECL luminol reagent (Millipore) by Amersham Imager 600 system (AI600, USA).

| Bioinformatics analysis and statistical analysis
The All experiments were carried out for at least three times from biological level. Statistical significance was calculated by two-tailed Student's t test using Prism GraphPad 7.0 software and SPSS 24.0 software. The data with P value <.05 were defined as statistically significant.

| High lncRNA NR2F1-AS1 expression is associated with breast cancer angiogenesis
To explore the effects of lncRNA NR2F1-AS1 on breast cancer, we conducted Pearson's correlation analysis via GEPIA. We found that high expression of lncRNA NR2F1-AS1 was correlated with high level of CD34 (P < .05, R = .34) and CD31 (P < .05, R = .30) in breast cancer, which were two vital markers of endothelial cells ( Figure 1A,B). This indicated that lncRNA NR2F1-AS1 might be associated with angiogenesis in breast cancer. Then, we overexpressed the expression of lncRNA NR2F1-AS1 in HUVECs and in the meantime knock-down of lncRNA NR2F1-AS1 in HUVECs through lentivirus transfection and puromycin selection ( Figure 1C). To explore the role of lncRNA NR2F1-AS1 in angiogenesis, we conducted tube formation assay with above-transfected HUVECs and found that HUVECs overexpressed with lncRNA NR2F1-AS1 formed increased tubes and larger meshes than negative control cells. While after knocking down of lncRNA NR2F1-AS1, the tube formation ability of HUVECs was decreased compared with the control ( Figure 1D). This suggested that lncRNA NR2F1-AS1 might play an important role in breast cancer angiogenesis.

| lncRNA NR2F1-AS1 promotes breast cancer angiogenesis in zebrafish model
To further demonstrate the relationship of lncRNA NR2F1-AS1 and breast cancer angiogenesis in vivo, we labelled the above-trans-   were larger and more, while the MVD was obviously higher compared with that of MCF-7-lenti-Vec-derived tumours ( Figure 4C). Therefore, lncRNA NR2F1-AS1 could promote breast cancer angiogenesis and growth in mouse model.
Among the predicted target genes of miRNA-338-3p, we found that IGF-1 might be closely relevant to the effects of lncRNA NR2F1-AS1 through Pearson's correlation analysis ( Figure 5C), and the predicted binding sites between miRNA-338-3p and IGF-1 through TargetScan software and miRanda software were shown in Tables 1 and 2, respectively. Then, we conducted dual-luciferase reporter gene assay and found that miRNA-338-3p transfection reduced the luciferase activities of wild-type IGF-1 while the luciferase activities of mutated-type IGF-1 maintained the same level after transfecting with miRNA-338-3p ( Figure 5D). This validated that Hence, lncRNA NR2F1-AS1 induced IGF-1 in breast cancer cells could activate the phosphorylation of IGF-1R in HUVECs, which then took effects on angiogenesis. Researchers have found the phosphorylation of IGF-1R led to activation of extracellular signal-regulated kinase (ERK) pathway, 35 which was not only essential for cell growth but also vital for angiogenesis. 36

| D ISCUSS I ON
Tumour angiogenesis is an important procedure for tumour growth and metastasis. 1,2 In our study, we found that lncRNA lncRNAs are regarded to play essential parts in various cancer-related procedure, [12][13][14][15][16] during which sponging miRNA to protect downstream targets is one of the major mechanism. 17,18 lncRNA NR2F1-AS1 could promote drug resistance in hepatocellular carcinoma 19 and was related with progression in endometrial cancer. 20 In thyroid cancer, lncRNA NR2F1-AS1 promoted cell proliferation and migration yet suppress apoptosis through directly binding to miRNA-338-3p, 21 while down-regulation of miRNA-338-3p promoted angiogenesis in hepatocellular carcinoma. 32 In breast cancer, we found miRNA-338-3p, as the target binding miRNA of lncRNA NR2F1-AS1, could probably exert angiogenetic effects according to bioinformatics prediction, which was consistent with the results in hepatocellular carcinoma. Thus, lncRNA NR2F1-AS1 induced breast cancer angiogenesis probably through sponging miRNA-338-3p.
Among the target genes of miRNA-338-3p, we verified that IGF-1, the most promising target on the basis of prediction, could directly bind to miRNA-338-3p. In the meantime, the expression of IGF-1 could be positively regulated by lncRNA NR2F1-AS1, which was the binding competing endogenous RNA (ceRNA) of miRNA-338-3p. Hence, lncRNA NR2F1-AS1 could sponge miRNA-338-3p to protect IGF-1 from suppression. It is reported that secreted IGF-1 could combine with IGF-1R in endothelial cells to promote angiogenesis, 34 and the activation of IGF-1R could further activate ERK pathway. 35 In our study, lncRNA NR2F1-AS1 induced expression of IGF-1 in breast cancer cells, which then increased the phosphorylation of IGF-1R and ERK1/2 in HUVECs.
In conclusion, we revealed the potential angiogenetic effects of lncRNA NR2F1-AS1 in breast cancer. lncRNA NR2F1-AS1 promoted breast cancer angiogenesis both in vitro and in vivo. The underlying mechanism was uncovered that lncRNA NR2F1 sponged miRNA-338-3p to induce IGF-1 in breast cancer cells, which further activated IGF-1R and ERK pathway in HUVECs. Thus, lncRNA NR2F1-AS1 could be a new promising therapy target in breast cancer.

CO N FLI C T O F I NTE R E S T
Authors declare no conflicts of interest for this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.