Introduction

Angiogenesis is a physiological process that form new vessels from pre-existing ones [1, 2], which is critical for solid tumor growth, invasion, and metastasis [1, 3]. Vascular endothelial growth factor A (VEGFA) is one of the most important proangiogenic factors secreted by tumor cells [4, 5]. The expression of VEGFA is directly induced by hypoxia-inducible factor 1-alpha (HIF1α) at the transcriptional level under hypoxia [6, 7]. Bevacizumab, a monoclonal antibody against VEGFA, became the first commercially available anti-angiogenesis drug in multiple solid tumors, although it was prohibited for use for treating metastatic breast cancer by the US FDA in 2011.

MicroRNAs (miRNAs) are small RNA molecules that silences gene expression through binding to the 3′-UTR of gene’s mRNA [8,9,10]. A number of studies suggest that miR-153 inhibits tumor growth and metastasis by targeting the snail family transcription repressor 1 (SNAI1), the zinc finger E-box binding homeobox 2 (ZEB2) [11], the metadherin (MTDH) [12], the ADAM metallopeptidase domain 19 (ADAM19) [13], the AKT serine/threonine kinase 1 (AKT1) [14], the HECT domain E3 ubiquitin protein ligase 3 (HECTD3) [15], and the BCL2 family apoptosis regulator (MCL-1) [d-luciferin (D12505, Bridgen, Bei**g, China) through intraperitoneal injection. After 5 min, it was anesthetized with ketamine and xylazine. The bioluminescent images were collected by using the system of IVIS Lumina XR (Caliper life Sciences, USA). Each tumor tissue was divided into two parts, one is used to purify the total RNA, the other is used for immunohistochemistry. Some tumors were only used in immunohistochemistry for their too small sizes. For the results of qPCR, the sample will be excluded if its value is higher or lower than the mean ± 3 × s.d.

Immunohistochemistry for CD31

The xenograft tumor tissues were fixed in 3.7% formalin solution. The immunohistochemistry assay was performed on 4-μm-thick paraffin sections after the pressure-cooking for antigen retrieval. The anti-CD31 primary antibody (1:400, Abcam, ab28364) was used. Subsequently, we incubated the slides with the anti-mouse/rabbit ultra sensitive polymer system (PV-8000, ZSGB-BIO, Bei**g, China). Signals for all slides were visualized by DAB staining. The slides were mounted after hematoxylin staining. Ten image fields for each slide, except for those are too small, were randomly collected by the persons who did not participate in this study by using microscopy, and the number of microvessel with positive CD31 expression was counted.

Chromatin immunoprecipitation assays

After hypoxia treatment, the diluted DNA–protein complex derived from MCF10A cells was incubated with the rabbit IgG or the anti-XBP1 antibody (Santa Cruz) in 1.5 ml centrifuge tube for 4 h at 4 °C. Then, each tube of sample was added with herring sperm DNA and protein A/G PLUS-Agarose (sc-2003, Santa Cruz), and continued to incubate overnight at 4 °C. Chromosomal DNA was amplified by PCR after purification. The PCR primers for amplifying the interest region on the PTPRN gene promoter (–2238 to –2225 from ATG) were as follows: 5′-GGACTACAGGTACGTGTTAC-3′ and 5′-GAGCCCAGGAGATTGAGA-3′.

Statistical analysis

All data in this study were analyzed by using the software of SPSS 18.0 (SPSS Inc., USA). Each experiment was performed at least three times, and the results are represented as the means ± s.d. The differences between two groups were analyzed by using the two-sided t-test, and the variance is similar between the groups, which are being statistically compared. The value of P smaller than 0.05 were considered statistically significant.