The Effects And Mechanisms Of Id1 On Biological Behaviour Of Ovarian Cancer Endothelial Progenitor Cells

Backgroud&ObjectiveOvarian cancer is one of the most common cancers in gynecology, which ranks the third among gynecologic tumor. Moreover, it causes more deaths than other types of female reproductive tumor and has a rate of survival for 5 years is about 30%. In recent years, with development of techniques in molecular biology, gene therapy and targeted therapy have been received increasing attention. Although many potential biomarkers of ovarian cancer have been found by proteomics and cDNA microarray, none of them belong to an optimum specific biomarker. Moreover, molecular mechanisms of ovarian cancer in progress and metastasis is poorly known. Metastasis of ovarian cancer is a major causes of poor efficacy treatment and high death rate, As a result, in the current study, we focus on exploring factors related to metastasis of ovarian cancer and effective approachs to prevent and treat ovarian cancer.Tumor angiogenesis is recognized as a critical step in tumor progress through which the small size, located or non-invasive initial tumor gradually develop to big, invasive and metastatic one. A recent study showed that bone marrow-derived EPCs participate in tumor angiogenesis, which accelerate tumor growth. Furthermore Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Anti-angiogenesis as a new strategy in cancer therapy is a research focus in oncology.Inhibitors of differentiation 1 (Id1) belongs to helix-loop-helix (HLH) transcription factors family, it can negtively regulate bioactivity of alkaline bHLH transcription factors, can thus suppress cells differentiation and accelerate cells proliferation. Schindl et al showed that the level of Id1 expression positively related to malignancy degree in ovarian cancer. Lyden et al's study confirmed that Id1 and Id3 played an important role in VEGF signal pathway which related to angiogenesis. In Id1 knocking-out mice, it appeared that tumor growth was significantly inhibited, due to angiogenesis defect. EPCs derived from bone marrow participate in the formation of new blood vessels, thus, it appear that EPCs have closely relationship with Id1. A recent report showed that tumor could-induce high expression of Id1 in EPCs derived from bone marrow but not in other cells, suggesting that Id1 might be a key factor for EPCs. Defect of Id1 in bone marrow could lead to decrease count of EPCs in peripheral blood, it can block tumor angiogenesis, and further suppress development of tumor. EPCs count in peripheral blood has a close relationship with bioactivity and mobilization of EPCs. Thus, Id1 may mediate mobilization and recruitment of EPCs, however, the relative mechanism-is still poorly known.The previous studies showed that many factors including VEGF/EPO/SDF-1, statins and VEGF/SDF-1 gene transfection could significantly accelerate bioactivity of EPCs, So that it could result in a series related episods, VEGF-induced mobilization of bone marrow-derived EPCs resulted in increased EPCs in peripheral blood, accelerating angiogenesis in the ischemic part and improving vessel endothelium repair. The regulatory mechanisms controlling EPCs are very complicated in vitro and in vivo, which involve in many signal pathways. The purpose of the present study was designed to investigate effects of Id1 on proliferation, adhesion, mobilization, and angiogenesis of EPCs and its mechanisms in ovarian cancer, to explore biological function of Id1 in ovarian cancer metastasis. On the basis of that, it could contribute to early diagnose and treat ovarian cancer and prevent its metastasis.Methods1. Levels and clinical behavior of circulating endothelial progenitor cells in human ovarian cancerIn the present study, the numbers of circulating EPCs in the peripheral blood in 25 healty volunteers and 42 patients with ovarian cancer were measured by flow cytometry. Furthermore, by means of a quantitative RT-PCR approach, we measured CD34 and VEGFR2 mRNA of the same patient. Plasma levels of the vascular endothelial growth factor(VEGF) and Matrix metalloproteinase-9 (MMP-9) were quantified by ELISA.2. Expression and bioactivity of Id1 in EPCs with ovarian cancer patientsEPCs were isolated using density gradient centrifugation method and cultured for 7 days. Then adhesion cells were collected. Id1 expression in EPCs with 25 patients with ovarian cancer and 20 healthy control subjects were analyzed by real-time reverse transcription-polymerase chain reaction and western blot. EPC proliferation, migration, adhesion and tube formation were detected by MTT, transwell chamber, adhesion assays and Matrigel assays.3. Lentivirus-mediated silencing of Id1 gene in human EPCsDouble-stranded DNA containing the interference sequences were synthesized according to the structure of a pGCSIL-GFP viral vector and then inserted into a linearized vector. The positive clones were identified as lentiviral vectors that expressed human Id1 short hairpin RNA (shRNA). We transfected human EPCs, pGCSIL-GFP with Id1 specific RNAi lentiviral vectors as well as controls. Id1 silenced cells were screened by SKOV-3. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of Id1 mRNA and protein, respectively. MTT assay, transwell migration assay, in vitro adhesion assay, tube formation assay were used to assess the functional effects of Id1 silencing on EPCs proliferation, migration, adhesion and tube formation.4. The mechanisms of Id1 on ovarian cancer endothelial progenitor cells biological behaviourCirculating EPCs cultures were from ovarian cancer patients and healthy control subjects. Integrin a4 expression was analyzed by real-time reverse transcription-polymerase chain reaction and western blot. Expressions of PI3K/Akt were analyzed by western blot. EPC proliferation, migration, adhesion and tube formation were detected by MTT, transwell chamber, adhesion assays and Matrigel assays.Results1. The number of EPCs/ml in the peripheral blood of pre-treatment and post-treatment ovarian cancer patients were higher than that of healthy controls (P<0.01 and P<0.05 respectively). Treatment significantly reduced the number of EPCs/ml of peripheral blood in patients (P<0.05). There were higher levels of EPCs in StageⅢandⅣpatients than those in stageⅠandⅡpatients (P<0.05). Residual tumor size more than 2cm showed significantly higher levels of EPCs than those of residual tumor size less than 2cm (P<0.05). Pretreatment mRNA levels of CD34 were not significantly increased in ovarian cancer patients, whereas VEGFR2 expression was increased(P<0.05). The VEGF and MMP9 protein concentrations for pretreatment ovarian cancer patients were higher than those of healthy controls. Treatment significantly reduced plasma protein levels of VEGF and MMP9 in patients (P<0.05). Plasma VEGF and MMP9 protein concentrations and circulating EPCs levels were correlated in pretreatment ovarian cancer patients (P<0.01).2. After 7 days of culture, ex vivo expanded EPCs derived from peripheral blood of healthy human volunteers and ovarian cancer patients exhibited spindle-shaped morphology. EPCs were characterized as adherent cells double positive for Dil-Ac-LDL uptake and lectin binding based on their appearance in a fluorescent microscope. A total of 90% of adherent cells showed uptake of Dil-Ac-LDL and lectin binding after 7 days of culture. The endothelial phenotype of expanded EPC was further characterized by expression of endothelial markers such as von willebrand factor, CD31, and VEGFR2. Positive vWF, CD31, and VEGFR2 were found on the cells, according to the immunofluorescence assay. We used real-time RT-PCR to examine mRNA expression of Idl in EPCs, and western blot analysis revealed a higher Idl protein expression in human ovarian cancer EPCs than in healthy controls(P<0.01). The EPCs of ovarian cancer patients showed increased migration and adhesion to fibronectin and endothelial cells. Statistical analyses revealed that ovarian cancer enhanced EPC proliferation, migration, adhesion and tune formation (P<0.01).3. To confirm the role of Id1 in EPCs from ovarian cancer patients, we performed gene-silencing experiments. EPCs were transfected with lentiviral-Id1-siRNA vector. The negative control containing pGCSIL/U6 mock vector only was denoted NC-GFP-LV. After the Id1-RNAi-LV construct was transfected into EPCs, Id1 mRNA expression levels in transfected cells were compared with those in nontransfected and control-transfected (NC-GFP-LV) EPCs by quantitative RT-PCR. Cells with Id1-RNAi-LV transfection showed a 80% reduction in the level of Id1 expression. To further confirm the specificity of Id1-RNAi-LV-mediated Id1 silencing, Id1 protein expression was determined by Western blot. Id1 protein expression in cells transfected with Id1-RNA-LV decreased significantly compared with that of control cells. These results indicated that lentivirus-mediated RNAi was an effective way of modulating Id1 expression in cultured EPCs. After the Id1-RNAi-LV construct was transfected into EPCs, the cells were cultured 7 days and we then performed EPCs proliferation, migration, adhesion and tube formation analysis. Id1-RNAi-LV markedly reduced EPCs functions. Cells transfected with Idl-RNAi-LV displayed less proliferation, migration and adhesion abilities compared with non-transfected control cells. Cells transfected with NC-GFP-LV, on the other hand, exhibited no change in cell proliferation, migration, adhesion and tube formation abilities, compared with non-transfected control EPCs. These data indicated that Id1 was crucial in the biological behaviour of EPCs in ovarian cancer.4. To explain the effect of Id1 on migration toward peripheral blood and recruitment to tumor tissues, we explored the expression of integrin a4 on the EPC surface. In keeping with the mRNA results, western blots showed an increase in integrin a4 protein expression in EPCs(P<0.01). After the Id1-RNAi-LV construct was transfected into EPCs, integrin a4 mRNA expression levels in transfected cells were compared with those in nontransfected and control-transfected (NC-GFP-LV) EPCs by quantitative RT-PCR. Cells with Id1-RNAi-LV transfection showed a reduction in the level of integrin a4 mRNA expression (P<0.05). Integrin a4 protein expression was then determined by Western blot. Integrin a4 protein expression in cells transfected with Id1-RNA-LV decreased significantly compared with that of control cells (P<0.05). These results indicate that the effect of Id1 on adhesion and angiogenesis is associated with activation of integrin a4.To explore which signaling pathways might participate in Id1-mediated cell mobilization and recruitment in EPCs, we investigated whether PI3K/AKT pathway involving the progress by using pharmacological inhibitors. Elevated AKT-Ser473 phosphorylation was observed in EPCs, compared with control cells (P<0.01), whereas AKT-Ser473 phosphorylation was completely abolished by LY294002. Id1 and integrinα4 expression were also strongly decreased by incubation with LY294002 (P<0.05). EPCs from ovarian cancer had increased levels of cell proliferation, migration, adhesion and tube formation, which were strongly decreased by incubation with LY294002 (P<0.05). These results indicate that Id1-induced EPCs biological behaviour is mediated by the PI3K/AKT pathway. Taken together, these data strongly suggest that Id1 induced migration and adhesion of EPCs is mediated by an increase in integrin a4 expression, which involves the PI3K/Akt pathway.Conclusions1. Our results demonstrated that EPCs could work as a potential biomarker to monitor disease progression and angiogenesis or response to treatments in ovarian cancer patients.2. Human peripheral blood EPCs were successfully isolated and confirmed. The expression of Id1 in ovarian cancer EPCs was significantly increased compared to healthy ones. Furthermore, proliferation, migration and adhesion of ovarian cancer EPC were increased compared to healthy subjects ones.3. Lentivirus vectors of targeting human Idl gene for RNAi were successfully constructed. Three targeting sites of Id1-1, Id1-2 and Id1-3 were successfully cloned. Lentivirus vectors were packaged and their titers were detected successfully, the site Id1-3 targeting Id1 had the best interfering results. Compared with the normal human EPCs, Those EPCs that infected with Id1-3 or Id1-NC lentivirus appeared more capacity in the proliferation, migration, adhension and tube formation. It suggests that it may serve as a new gene-modified cell therapy carrier, Id1 silencing EPCs could reduce the Id1 expression as well as decrupt endothelial dysfunction. These indicate that Id1 is important for EPCs migration and adhesion. 4. We showed that Idl induced migration and recruitment of human EPCs via the PI3K and Akt signaling pathway which involves in activation of integrin a4. Our data also provide further insights into the understanding of neovascularization. These findings raise the possibility that therapeutic strategies which dedicate to inhibit EPCs angiogenesis, might enhance the efficacy of certain cytotoxic anti-angiogenesis ovarian cancer therapies, at the same time, and reduce the risk of ovarian cancer metastasis.