| FSH plays an important role in ovarian epithelial carcinogenesis. However, the molecular mechanisms involved in FSH role for the development and progression of ovarian cancer are unknown. OCT4 plays crucial roles in the pathogenesis of human malignancies. Our previous study showed that the OCT4 mediates FSH-induced inhibition of epithelial ovarian cancer cells apoptosis. However, the role of OCT4 in FSH-induced invasion of ovarian cancer has not been investigated in detail. Therefore, the purpose of the current study was to determine whether the effect of FSH on ovarian cancer invasion is mediated by OCT4 mechanism. In this study, we show that FSH induces the EMT and invasive phenotype in epithelial ovarian cancer cells. We also found that FSH stimulation enhanced the expression of OCT4 at both the m RNA and protein levels in a dose- and time-dependent manner. In addition, FSH treatment increased the expression of FSHR. Knockdown of FSHR inhibited FSH-stimulated OCT4 expression. Pretreatment with U0126, a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, neutralized the enhanced expression of OCT4 and Snail by FSH-induced. We further showed that the activated expression of Snail and N-cadherin, the suppressed expression of E-cadherin and cell morphological change by FSH stimulation can be blocked by OCT4 specific small interfering RNA. Moreover, our results showed that OCT4 mediates FSH-induced an increase in the ability of migration and invasion in ovarian cancer. Taken together, our work reveals that OCT4 plays an essential role in FSH-induced EMT and invasion in epithelial ovarian cancer, which may be a potential therapeutic target for ovarian cancer patient.Part I. FSH induces the EMT and invasive phenotype in epithelial ovarian cancer cellsObjective:Human epithelial ovarian cancer cell lines Hey and Ho8910 were treated with FSH. A morphological transition was observed in Hey and Ho8910 cells stimulated with FSH. The protein and m RNA levels of epithelial marker E-cadherin and mesenchymal characteristics N-cadherin were assessed.Methods:Human epithelial ovarian cancer cell lines Hey and Ho8910 were treated with different concentrations of FSH. A morphological transition from a cobblestone-like shape to fibroblastic-spindle appearance was observed in Hey and Ho8910 cells stimulated with FSH. The protein and m RNA levels of epithelial marker E-cadherin and mesenchymal characteristics N-cadherin were assessed through western blotting and RT-q PCR.In addition, a significant increase in cell invasion was demonstrated in ovarian cancer cells treated with FSH(Con and 50 m IU/ml) through transwell assay.Results:(1) We found that the protein and m RNA levels of epithelial marker E-cadherin were dramatically downregulated after FSH treatment in dose-dependent manner. On the contrary, the expression of mesenchymal characteristics N-cadherin was obviously upregulated in the same way. When FSH concentration is 50 m IU/ml, FSH has most significant roles in regulating EMT-related proteins.FSH may induce epithelial ovarian cells epithelial mesenchymal transformation from molecular expression level.(2) A morphological transition from a cobblestone-like shape to fibroblastic-spindle appearance was observed in Hey and Ho8910 cells stimulated with FSH(Con and 50 m IU/ml). FSH can induce epithelial ovarian cells epithelial mesenchymal transformation from the morphological point of view.(3) In addition, a significant increase in cell invasion was demonstrated in ovarian cancer cells treated with FSH(Con and 50 m IU/ml) through transwell assay. FSH can induce epithelial ovarian cells epithelial mesenchymal transformation and invasion from the aspects of function.Conclusions:Collectively, these data indicate that FSH plays a role in inducing EMT and promoting invasion in oepithelial ovarian cells.Part II. FSH treatment upregulates FSHR and OCT4 expression, and FSHR is required for FSH-induced OCT4 expression in ovarian cancer cellsObjective:To investigate that the genes regulated by FSH may be involved in the EMT and progression.in epithelial ovarian cells Hey and Ho8910.Methods:FSHwith different concentrations(0, 5, 25, 50, 100 m IU/ml) stimulated epithelial ovarian cancer cell lines and Hey Ho8910. FSHwith 50 m IU/mlstimulated epithelial ovarian cancer cell lines Hey and Ho8910 by different time points(0, 0.5, 1, 3, 6, 12, 24 h).The protein expression level of OCT4 was assesses through western blotting.Similarly, FSH with different concentrations(0, 5, 25, 50, 100 m IU/ml) stimulated epithelial ovarian cancer cell lines and Hey Ho8910. The m RNA and protein levels of FSHR expression in Hey and Ho8910 cells were assessed by q RT-PCR and western blotting. We knock down the expression of FSHR using RNA interference assay.The m RNA and protein levels of FSHR and OCT4 expression in Hey and Ho8910 cells were examined by q RT-PCR and western blotting.Results:(1) In certain range of concentration and time, FSH could significantly increase OCT4 protein expressionin epithelial ovarian cancer cells Hey and Ho8910 in both a dose- and time-dependent manner. When FSH concentration is 50 m IU/ml, FSH has most significant roles in upregulating the expression of OCT4.(2) In certain range of concentration, FSH could significantly increase the m RNA and protein levels of FSHR expression in epithelial ovarian cancer cells Hey and Ho8910 in a dose-dependent manner. When FSH concentration is 50 m IU/ml, FSH has most significant roles in upregulating the expression of FSHR.(3) We knock down the expression of FSHR using RNA interference assay, si RNA mediated depletion of FSHR could block the effects of FSH on OCT4 expression levels.Conclusions:In certain range of concentration and time, FSH could significantly increase OCT4 protein expression in epithelial ovarian cancer cells Hey and Ho8910 in both a dose- and time-dependent manner.In certain range of concentration, FSH could significantly increase the m RNA and protein levels of FSHR expression in epithelial ovarian cancer cells Hey and Ho8910 in a dose-dependent manner, si RNA mediated depletion of FSHR could block the effects of FSH on OCT4 expression levels.Part III. ERK1/2 is involved in FSH-induced the expression of OCT4 and Snail in epithelial ovarian cancer cellsObjective:To investigate the molecular signaling involved in FSH-induced the expression of OCT4 and Snail in epithelial ovarian cancer cells Hey and Ho8910.Methods:Epithelial ovarian cancer cells Hey and Ho8910 were divided into five groups, respectively. ovarian cancer cells Hey and Ho8910 were treated with Con, FSH, inhibitors against PI3K/Akt(LY294002)+FSH,ERK1/2(U0126)+FSH and ROS(NAC)+FSH. The m RNA and protein levels of OCT4 and Snail expression were examined through western blotting and RT-q PCR analysis.Results:(1) Western blotting and RT-q PCR analysis showed that the enhanced expression of OCT4 by FSH-induced was attenuated when ovarian cancer HO8910 and Hey cells were pretreated with ERK1/2(U0126)-specific inhibitors, but not when pretreated with PI3K/Akt(LY294002)- or ROS(NAC)-specific inhibitors.(2) RT-q PCR and Western blotting results showed treatment with the ERK1/2 inhibitor U0126 blocked the FSH-induced upregulation of Snail, but the PI3K/Akt inhibitor LY294002 and ROS inhibitor NAC had no effect on Snail expression level.Conclusions:These results demonstrate that ERK1/2 is involved in FSH-induced the expression of OCT4 and Snail in epithelial ovarian cancer cells Hey and Ho8910.Part IV. OCT4 is required for FSH-induced downregulation of E-cadherin and upregulation of N-cadherin and Snail expressionin epithelial ovarian cancer cells Hey and Ho8910Objective:To investigate the involvement of OCT4 in FSH-induced the suppression of E-cadherin expression and the activation of N-cadherinand Snail in epithelial ovarian cancer cells Hey and Ho8910.Methods:Epithelial ovarian cancer cells Hey and Ho8910 were divided into four groups, respectively. Cells transfected with Si-Con or si RNA targeting OCT4 to knock down OCT4 were treated with Con or FSH. The m RNA and protein levels of E-cadherin, N-cadherinand Snail expression were examined through western blotting and RT-q PCR analysis.Cells morphology was microscopically assessed.Results:(1) The results of RT-q PCR and western blotting analysis showed that OCT4 si RNA treatment significantly diminished FSH-induced Snail m RNA levels. Similarly, treatment with OCT4 si RNA attenuated FSH-induced increases in Snail protein levels. Taken together, these results demonstrated that OCT4 is required for FSH-induced Snail expression in ovarian cancer cells.(2) Cells were transfected with si RNA targeting OCT4 to knock down OCT4. Treatment with FSH induced a morphological change in Hey and Ho8910 cells, from a cobblestone-like morphology to fibroblastic-spindle appearance, whereas the morphology of OCT4-depleted cells remained unchanged.(3) OCT4 si RNA treatment attenuated both basal and FSH-induced E-cadherin downregulation and N-cadherin upregulation in m RNA and protein levels. These data indicate that OCT4 is necessary for the expression of E-cadherin and N-cadherin by FSH-induced.Conclusions:These results demonstrate that OCT4 is required for FSH-induced downregulation of E-cadherin and upregulation of N-cadherin and Snail expression in epithelial ovarian cancer cells Hey and Ho8910.Part V.OCT4 mediates FSH-induced migration and invasionin epithelial ovarian cancer cells Hey and Ho8910Objective:To examine whether OCT4 is involved in FSH-induced migration and invasion in epithelial ovarian cancer cells Hey and Ho8910.Methods:Epithelial ovarian cancer cells Hey and Ho8910 were divided into four groups, respectively. Cells transfected with Si-Con or si RNA targeting OCT4 to knock down OCT4 were treated with Con or FSH. Wound healing assays and transwell invasion assays were used to estimate the role of FSH on ovarian cancer cells migration and invasion.Results:(1) Wound healing assays were used to estimate the role of FSH on ovarian cancer cells migration. FSH treatment induced a significant increase in Hey and Ho8910 cells migration. FSH-induced cell migration was attenuated in cells treated with OCT4 si RNA. Taken together, these results demonstrated that OCT4 mediates FSH-induced migration in ovarian cancer cells.(2) Transwell invasion assays were used to estimate the role of FSH on ovarian cancer cells invasion. FSH treatment induced a significant increase in Hey and Ho8910 cells invasion. FSH-induced cell invasion was attenuated in cells treated with OCT4 si RNA. Taken together, these results demonstrated that OCT4 mediates FSH-induced invasion in ovarian cancer cells.Conclusions:These results demonstrate that OCT4 mediates FSH-induced migration and invasion in epithelial ovarian cancer cells Hey and Ho8910. |
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