[In vitro study on expression of tumor stem cell biomarker and transdifferentiation towards endothelial cells of retinoblastoma cells under hypoxia condition].
OBJECTIVE
To investigate the capability of RB cells that represent characteristics of tumor stem cell and trans-differentiate towards endothelial cells under hypoxia microenvironment, as well as their mechanism.


METHODS
Experimental research. RB cell line Y79 was cultured in hypoxia environment with or without rapamycin treatment. Morphological changes, expression of neuron specific enolase (NES) and ATP binding cassette transporters G2 (ABCG2), and expression of HIF-1α protein and mRNA were detected. After been induced toward endothelial cells, expression of CD31 and vWF, up-taking of Dil-acLDL, vasculogenic mimicry (VM) formation in 3D culture and expression of HIF-1α,EphA2 and PI3K were detected. The ANOVA test was performed to compare the differences among groups, and SNK-q test was performed to further comparison.


RESULTS
Y79 cells in normal oxygen group were single or agminated suspending cells. In hypoxia group, part of Y79 cells became adherent. No adherent cells were observed in rapamycin fore-treated group. Positive rates of NES and ABCG2 had significant difference among these three groups (FNES = 698.45, FABCG2 = 864.48, all P < 0.01). Compared with normal group [(98.2 ± 2.5)%, (2.1 ± 2.1)%],NES positive rate [(35.1 ± 3.4)%] was significantly reduced and ABCG2 positive rate [(67.4 ± 3.6)%] was significantly enhanced in hypoxia group (q = 46.11, 50.89; both P < 0.01), no significant changes were observed in rapamycin fore-treated group(P > 0.05). Expressions of HIF-1α protein and mRNA showed significant difference among these three groups (Fprotein = 314.85, FmRNA = 132.01, all P < 0.01). Compared with normal oxygen group (0.165 ± 0.056,0.927 ± 0.715) , HIF-1α protein (1.094 ± 0.077) and mRNA (6.408 ± 0.686) were significantly increased in hypoxia group(q = 31.81, 18.40; both P < 0.01), HIF-1α mRNA (7.219 ± 0.591) was significantly increased but no increased protein expression (0.218 ± 0.061) was observed in rapamycin fore-treated group. After transdifferentiation induction,Y79 cell in hypoxia group expressed CD31 and vWF, acquired the abilities of acLDL up-taking and VM formation. Compared with normal group(0.327 ± 0.108, 0.194 ± 0.033, 0.402 ± 0.068), expression of HIF-1α,EphA2 and PI3K protein (1.440 ± 0.089,0.377 ± 0.056,0.762 ± 0.090) was significant increased in hypoxia group(q = 8.72-23.00, all P < 0.01).


CONCLUSIONS
Under hypoxic condition, part of RB cells can express tumor stem cell markers, transdifferentiate towards endothelial cells and acquire part of endothelial cell function and VM formation capability. HIF-1α through EphA2/PI3K pathway may play an important role in VM formation.
Department of Ophthalmology, the Affiliated Hospital of Qingdao University Medical College,Qingdao 266003, China.
Li-na
LN
Zhang
Gui-qiu
GQ
Zhao
Qian
Q
Wang
Ying-jun
YJ
Niu
Qiang
Q
Xu
Wen-yi
WY
Yang
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D054338:Cell Transdifferentiation; D042783:Endothelial Cells / Q000166:cytology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D036104:Receptor, EphA2 / Q000378:metabolism; D012175:Retinoblastoma / Q000378:metabolism*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D014408:Biomarkers, Tumor; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009363:Neoplasm Proteins; D019869:Phosphatidylinositol 3-Kinases; D036104:Receptor, EphA2; D010751:Phosphopyruvate Hydratase