Influence Of Hypoxia/reoxygenation On The Biology Behaviors Of Ovarian Epithelial Cancer Cell SKOV3 And It's Relationship With HIF-1 α And SNAIL Gene Expressions.

Objectives:To investigate the influence of hypoxia/reoxygenation on the biology behaviors of ovarian cancer cell line SK0V3 and it's relationship with the expression of hypoxia inducible factor 1α (HIF-1α), to study the influence of HIF-1α protein inhibition and E-cadherin (E-cad) protein expression by a molecular target of rapamycin inhibitor, rapamycin in cultured ovarian cancer cell line SKOV3 and to investigate the relationship between HIF-1α and SNAIL gene expression.Materials and Methods:Ovarian cancer cell line SKOV3 were cultured with or without rapamycin under hypoxia 24 hours following by reoxygenation 24,48 hours. The expression of HIF-1α protein and mRNA were checked by western blot and reverse transcription-polymerase chain react ion(RT-PCR) respectively. MTT was used to detecte the ability of cell proliferation. Cell cycle distribution was examined by flow cytometry (FCM). Transwell experiments were used to detecte the ability of cell proliferation and invasion respectively. MMP-2 and MMP-9 activity were compared by Zymography. Expression of MMP-2 mRNA was checked by RT-PCR.Four groups of SKOV3 cells were cultured under different conditions. The expression of HIF-1α and E-cad were assessed by RT-PCR and Western blot. The expression of SNAIL was studied by RT-PCR and real time PCR.To investigate the regulation between SNAIL gene and HIF-1α ,SiRNA of SNAIL and HIF-1α were disigned respectively. CoCl2, an stimulator of HIF-1α and RNA interference technology was used to determine the relationship between HIF-1α and SNAIL.ResultsThe proliferation and invasive ability of SKOV3 both increased significantly cultured hypoxia/reoxygenation 24h. The proliferationability resumed to the level of control with the reoxygenation time prolonged. Flow cytometry analyses showed that SKOV3 cell cultured hypoxia/reoxygenation 24h has an S phase increased and G1 phase decreased. Rapamycin has a G1 phase arrest on SKOV3 cell, as determined by an increase in G1-phase and a decrease in S- phase populations. Transwell and Zymography revealed that the invasive ability of SKOV3 were increase in hypoxia/reoxygenation. Expression of MMP2 and SNAIL mRNA were increase.The expression of HIF-1α and E-cad mRNA and protein were detected in ovarian cancer cells line SKOV3 cultured routinely. The expression of HIF-1α protein increased when cells were exposed to hypoxia and reached peak at hypoxia 16 hour (P=0.005) . The subsequent expression of E-cad reduced significantly after hypoxia and kept declining with the time of hypoxia prolonged. Rapamycin could inhibit the up-regulation of HIF-1 α protein in hypoxia and maintain E-cad expression at a relative high level.The expression of HIF-1α protein and SNAIL mRNA could be inhibited gradually by rapamycin. siRNA of HIF-1α could suppress the expression of SNAIL while siRNA of SNAIL had no influence on HIF-1α protein expression.Conclusion:Hypoxia/reoxygenation could alter the cell cycle distribution, increace the expression of MMP2, regulate the expression of such signal transduction factors as HIF-1α,SNAIL, E-cad, etc. So as to enhance the proliferation ability,improve cell invasiveness,down regulate cell adhesion.HIF-1α protein could be detected in routine cultured SKOV3 cell. The expression of HIF-1α protein and SNAIL mRNA were increase in SKOV3 cell which cultured hypoxia meanwhile the expression of E-cad was decrease. Rapamycin, an inhibitor of HIF-1α could also restrain the up regulation of the SNAIL mRNA, maintain the expression of E-cad. The inhibition of rapamycin on SNAIL might be through HIF-1α pathway and HIF-1α might be an up regulator of SNAIL gene. The HIF-1α -SNAIL pathway might play an important role in ovarian cancer progression.