Knockdown of P4HA1 inhibits neovascularization via targeting glioma stem cell-endothelial cell transdifferentiation and disrupting vascular basement membrane

Emerging evidence has demonstrated transdifferentiation process of glioma stem cells (GSCs) into endothelial cells (ECs) in glioma neovascularization. Herein, we focused on screening for genes that were differentially expressed in the transdifferentiation process using microarray analysis. Bioinformatics analysis revealed differential expression of the prolyl 4-hydroxylase subunit alpha-1 (P4HA1) gene. We determined that P4HA1 expression was correlated with histological grade, the level of Ki67 and microvessel density (MVD) in human glioma specimens. Knockdown of P4HA1 inhibited the proliferation, migration and tube formation of GSCs in vitro. In vivo studies revealed that the downregulation of P4HA1 inhibited intracranial tumor growth, prolonged the overall survival time of xenograft mice and suppressed the neovascularization in brain tumors. Moreover, P4HA1 regulates the expression of vascular endothelial growth factor A (VEGF-A), especially an anti-angiogenic isoform-VEGF165b. Additionally, knockdown of P4HA1 inhibited the synthesis of collagen IV, and hence disrupted the structures of vascular basement membranes (BMs) in gliomas. Our study indicates that P4HA1 plays a pivotal role in the process of GSC-EC transdifferentiation and the structural formation of vascular BMs.


Flow cytometry and cell sorting
Flow cytometry was performed using a cell sorter to screen out CD133+ GSCs from the U87MG cell line. Anti-human CD133 antibody (Miltenyi) was used to stain and sort positive GSCs as previously described. 2 Mouse phycoerythrin-IgG1 (Miltenyi) was used as an isotype control.

Cell proliferation
Cell proliferation was determined with a cell counting kit-8 assay (Dojindo).GSCs were initially pretreated with Accutase and then seeded into 96-well plates at a density of 3000 cells per well in 100 µl medium. The cells were cultured for 1-7 days under hypoxic conditions, 10 µl of CCK8 solution was added to each well of the plate for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. All data are presented as the mean ± SD from three experiments.

Cell migration assay
A total of 5×10 4 GSCs were seeded onto the upper compartment of a Transwell plate (24-well, 8 µm pore size, Corning) in 200 µl of serum-free DMEM, and the lower compartment was filled with 600 µl of 5% FBS medium. After incubation in the hypoxic N 2 -O 2 incubator for 8 hours, cells on the upper surface of the membrane were undetectable, and the migrated cells on the lower surface were stained with 1% cresyl violet. The colored cells were counted, and all data represented the mean ± SD from three experiments.

Tube formation assay
The reduced growth factor basement membrane matrix (Geltrex, A1413202, Invitrogen) was thawed overnight at 4°C, and 50 µl/cm 2 /well of Geltrex was placed in 24-well plates without dilution. After incubating at 37°C for 30 minutes, 1.5×10 5 GSCs/well diluted in 1 ml of 10% FBS medium were seeded on the solidified gel. All plates were incubated in the hypoxic N 2 -O 2 incubator for 3 days. Images were randomly captured using a microscope (Olympus, Tokyo, Japan) with 100× magnification. Mesh numbers, mean mesh size, total mesh area, branch numbers, total branching length and total branch length were measured per field with ImageJ software (NIH website).

Establishment of intracranial and subcutaneous GSC tumor models
Four-to six-week-old BALB/c-nu mice were purchased from Beijing HFK Bioscience Ltd. Animal experiments were approved by Experimental Animal Ethics Committee of Beijing Neurosurgical Institute and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. After being anesthetized with isoflurane, mice were stabilized in a stereotactic apparatus (KOPF940). A total of 5 × 10 5 GSCs were injected into the brain to establish intracranial tumor models as previously described. 5 To establish subcutaneous GSCs tumor models, 1×10 6 GSCs in 100 μl PBS were injected directly into the dorsal subcutaneous tissue close to the right forelimb. Subcutaneous tumor size was measured every 3 days with a vernier caliper, and tumor volume was calculated as follows: V = π/6 × Length × Width 2 (mm 3 ).