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Original Article In-Vitro Inhibitory Effect of EGFL7-Rnai on Endothelial Angiogenesis in Glioma

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Original Article In-Vitro Inhibitory Effect of EGFL7-Rnai on Endothelial Angiogenesis in Glioma Int J Clin Exp Pathol 2015;8(10):12234-12242 www.ijcep.com /ISSN:1936-2625/IJCEP0014630 Original Article In-vitro inhibitory effect of EGFL7-RNAi on endothelial angiogenesis in glioma Qiang Li1, Ai-Yue Wang2, Qiong-Guan Xu1, Da-Yuan Liu1, Peng-Xiang Xu1, Dai Yu2 1Department of Neurosurgery, Hainan Nongken Hospital, Haikou, Hainan, China; 2Department of Neurology, Haikou Municipal People’s Hospital, Haikou, Hainan, China Received August 17, 2015; Accepted September 23, 2015; Epub October 1, 2015; Published October 15, 2015 Abstract: Objective: To investigate the role and mechanism of epidermal growth factor like domain 7 (EGFL7) in glioma angiogenesis by cell co-culture and RNA interference. Methods: NSCs-HUVECs co-culture system was estab- lished using Transwell culturing techniques. The interactions between glioma and endothelial cells were simulated in-vitro. Cellular expression of EGFL7 in NSCs and HUVEC was targeted and suppressed by lentiviral vector carrying siRNA. The effect of EGFL7 on angiogenesis in glioma in-vitro micro-environment was detected by endothelial cell proliferation, adhesion and tube formation assay. Results: Following EGFL7 gene silencing, expression of EGFL7 in HUVECs was reduced and cell adhesion capability was inhibited significantly. Endothelial cells failed to form a lumen-like structure after EGFL7 gene silencing, shown by the tube formation assay. Conclusion: By regulating en- dothelial cell adhesion, EGFL7 plays a key role in the regulation of glioma angiogenesis. Keywords: Epidermal growth factor like domain 7, angiogenesis, RNA interference Introduction protein, approximately 30 KD, contains a secre- tion signal peptide sequence, and the sequence Gliomas are malignant brain tumors arising has an N-terminus with an EMI structure to reg- from glial cells which are derived from neuroec- ulate intercellular adhesion, two EGF-like struc- toderm, ranking the top among all intracranial tures associated with protein identification, and tumors. In spite of the progress of medical a high conserved C-terminus rich in lysine and technology in recent years, the prognosis of valine. In this study, we co-cultured the primary malignant glioma remains unimproved, with a human neural stem cells and human umbilical mean survival less than 1.5 years. The growth vein endothelial cells to simulate the in-vitro and metastasis of glioma depend on angiogen- interaction between glioma and endothelial esis, which has been considered key character- cells [6-11]. The expression of EGFL7 in these istics of glioma [1]. Not only present in body two types of cells was suppressed by RNA inter- growth and development, angiogenesis is a physiological phenomenon in wound healing, ference (RNAi) and recombinant lentivirus. The ischemia, hypoxia and inflammation, closely role of EGFL7 in glioma angiogenesis and the associated with the occurrence, development mechanism were investigated by detecting the and prognosis of multiple diseases [3, 4]. As endothelial cell adhesion and angiogenesis. the mechanism of tumor angiogenesis is com- plex, it is important to find the key target. Materials and methods Epidermal growth factor like domain 7 (EGFL7) is a recently identified regulatory factor of Cell strains and cell culture endogenous angiogenesis, playing a key role in the formation of tubular structure in embryonic Human umbilical vein endothelial cells (HUVECs) angiogenesis. EGFL7 is highly expressed in liver were purchased from the cell bank of Chinese cancer and malignant glioma [4, 5]. Academy of Sciences, Shanghai, and cultured in full DMEM media containing 1% antibiotics Human EGFL7 gene is located at the end of (Penicillin/Streptomycin, Gibco) and 10% FBS long arm of chromosome 9. The EGFL7 coding (Gibco). Human neural stem cells (NSCs) were EGFL7-RNAi on endothelial angiogenesis isolated from 8-10 weeks human embryo sam- on a low temperature shaker for 10 min. The ples gifted by the Xinqiao Hospital, Third absorbance under 490 nm wavelength was Military Medical University and cultured in detected by a microplate reader and the well DMEM/F12 media (Gibco) containing 1% antibi- containing DMSO was read as blank control in otics, 10% FBS, EFG (10 mg/L), bFGF (10 mg/L) the meantime. NSCs cells were added with 100 and N2 additive. Cells were cultured in an incu- μl 10% SDS in the medium and continued cul- bator with 5% CO2 and saturated humidity at turing for 12 h. The absorbance under 570 nm 37°C. wavelength was detected and the well contain- ing SDS was read as blank control in the Identification of human NSCs meantime. The NSC neurospheres were stained for immu- Construction of pLV3/shRNA/EGFL7 nohistochemistry. NSCs slices were fixed by 4% paraformaldehyde, permeablized by PBS con- Human EFGL7 gene sequence 5’-GCACCTACC taining 0.5% Triton X-100, blocked by 6% goat GAACCATCTATA-3’ was used to synthesize serum, and incubated overnight in 1:20 diluted shRNA primers: 5’-GATCCGTTCTCCGAACGTGT- primary anti-nestin antibody at 4°C. Slides CACGTTTCAAGAGAACGTGACACGTTCGGAGA- were then incubated in 1:200 diluted RBITC ACTTTTTTG-3’ and 5’-AATTCAAAAAAGTTCTCCG- labeled secondary antibody at 37°C for 1 h, AACGTGTCACGTTCTCTTGAAACGTGACACGTTC- and incubated in appropriate amount of diluted GGAGAACG-3’. After annealing, primers were DAPI in darkness for 5 min. Slides were mount- connected with linear shuttle plasmid pLV3. ed using mounting medium containing anti-flu- Positive clones were selected and confirmed orescent quencher and observed using laser by sequencing, named pLV3/shRNA/EGFL7. scanning confocal microscopy. Plasmid pLV3/shRNA/EGFL7 as well as packag- ing plasmid pRsv-REV, pMDlg-pRRE and pMD2G Detection of cellular EGFL7 expression (Tronolab Co.) were extracted using high purity endotoxin free plasmid midi-extraction kit The slides of HUVECs and NSCs were fixed by (NucleoBond Xtra Midi Plus, Macherey-Nagel). 4% paraformaldehyde and permeablized by 293T cells were seeded in 15 ml culture plates PBS containing 0.5% Triton X-100. 3% H2O2 was and cultured overnight until 80-90% conflu- used to remove the endogenous peroxidase ence. Shuttle plasmid and packaging plasmid activity. The slides were blocked in 6% goat were mixed in an appropriate ratio and diluted serum and incubated in 1:20 diluted primary in 1.5 ml serum free DMEM. 300 µl RNAi-Mate anti-nestin antibody at 4°C overnight. Slides (Western Biotechnology Service Center) was were then incubated in 1:200 diluted biotin diluted in 1.5 ml serum free DMEM in another labeled secodary antibody at 37°C for 30 min, tube. The two were mixed after being placed for and in alkaline phosphatase-labeled streptavi- 5 min at room temperature, and placed at room din solution at 37°C for 30 min. DAB substrates temperature for another 20-25 min. Medium of were applied for chromogenic reaction and 293T cells was replaced by serum free medium inbubated in darkness for 10 min. After 1 min and added with transfection mixture dropwise. hematoxylin re-staining and blueing up by water Cells were cultured in the incubator for 4-5 h, rinse, the slides were dehydrated in an alcoho- and replaced by full medium. Incubation was lic gradient, treated with xylene and mounted continued for 72 h. Supernatant was collected with neutral resin. after 72 culture. Cell proliferation by MTT assay Purification of Lenti-shRNA/EGFL7 HUVECs and NSCs were inoculated in a 96-well The recombinant packaging lentivirus after plate at a density of 1×104/well and 1×103/ 293T cell transfection by plasmid was named well, respectively, and cultured for 24 h, 48 h, Lenti-shRNA/EGFL7. The cell culture superna- and 72 h, respectively. Cells were added with tant following transfection was collected, cen- 10 μl and 20 μl 5 mg/ml MTT solution (Sigma), trifuged at 4000 rpm 4°C for 4 min, and filtered respectively and cultured for another 4 h. After using 0.45 µm filter. Per 100 ml filtered super- removal of HUVECs medium, 100 μl DMSO was natant, 50 ml solution containing 2.5 M NaCl added in each well and the plate was shaken and 20% PEG8000 was added and incubated 12235 Int J Clin Exp Pathol 2015;8(10):12234-12242 EGFL7-RNAi on endothelial angiogenesis Figure 1. Detection of nestin expression in human NSCs. Figure 2. Detection of EGFL7 expression in HUVECs and NSCs. Table 1. MTT assay of HUVECs and NSCs at solution were added into a ultracentrifuge tube difference time points from top to bottom, and centrifuged at 20000 HUVECs A1 A2 A3 Mean of A rpm in room temperature for 2 h. The virus belt between 1.30-1.40 g/ml was collected in a dia- 24 h 0.4076 0.4431 0.4469 0.4325 lysis bag, dialyzed in buffer containing 10 mM 48 h 0.6293 0.5917 0.6012 0.6074 Tris-HCl and 2 mM MgCl2 (pH 8.0) overnight at 72 h 0.9145 1.0044 0.9298 0.9496 4°C, collected and stored in aliquots in -80°C NSCs OD1 OD2 OD3 Mean of OD freezer. 24 h 0.3584 0.3673 0.3624 0.3627 48 h 0.3288 0.3296 0.3301 0.3295 Titration of Lenti-shRNA/EGFL7 72 h 0.2906 0.2982 0.2932 0.2940 293T cells were seeded 1×105/well in a 96-well plate and cultured in an incubator for 24 h. The on ice for 1 h to precipitate virus. After centrifu- purified Lenti-shRNA/EGFL7 was diluted 10 gation at 12000 rpm, 4°C for 20 min, the times with full DMEM medium. 100 μl diluted supernatant was discarded. The pellets were virus was added in each well (10-2~10 -6) and resuspended in 10 ml CsCl solution with a con- cultured in an incubator for 24-72 h together centration of 1.10 g/ml (in solvant 20 mM Tris- with blank control.
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