Effect Of 1α,25 Dehydroxvitamin D On Stemness During Malignant Transformation Of Mouse Ovarian Surface Epithelial Cells

Objective:Epithelial ovarian cancer is one of the common malignant ovarian tumors,and about 90%of it originates from ovarian surface epithelial（OSE）cells.The model on malignant transformation of mouse ovarian surface epithelial（MOSE）cells has been successfully established in our laboratory.The three transitional states were distinguished by both in vitro and in vivo growth criteria,including early passages（ME,P10-P30）,intermediate passages（MI,P31-P80）,and late passages,（ML,P90-P120）.It has been reported that Epithelial-Mesenchymal Transition（EMT）and stemness contribute to spontaneously oncogenic transformation of MOSE cells.And the classical Wnt signaling pathway could maintain characteristics of cancer stem-like cells（CSCs）by regulating proliferation and self-renewal.The vitamin D might have crucial role in the prevention and therapy of cancer.We previously demonstrated that1α,25hydroxyvitamin D（1,25（OH）₂D₃）,the activity of vitamin D,could inhibit the migration and invasion of both human ovarian cancer cell SKOV-3 and MOSE cells.And we also found that it could postpone spontaneously neoplastic transformation of MOSE cells by regulating EMT.In present study,1,25（OH）₂D₃ was used to continuously treat MOSE cells during process of malignant transformation of MOSE cells to investigate whether 1,25（OH）₂D₃ affects stemness of MOSE cells.We explored the effect of vitamin D on the characteristics of cancer stem-like cells during spontaneous malignant transformation of ovarian epithelial cellsMethods:The main work of this study includes three sections.（1）1,25（OH）₂D₃reduces the stem cell-like properties during malignant transformation of MOSE cells.MOSE cells in early passages was treated with or without 10nM 1,25（OH）₂D₃until late passages.The proliferation of MOSE cells were determined by plating efficiency.The proportion of stem-like cells and transcription level of pluripotent stem-related genes in MOSE cells were analyzed by fluorescence activated cells sorting（FACS）and Real-time qPCR,respectively.The self-renewal ability was measured by sphere forming rate and limiting dilution assay.The survival curves for late stage of MOSE cells were analyzed to evaluate irradiation sensitivity of X-ray.（2）The effect of1,25（OH）₂D₃ on the stemness of ovarian cancer stem-like cells.Cancer Stem-like Cells were isolated from malignant transformation MOSE cells treated with 10 nM1,25（OH）₂D₃ using FACS,and were cultured in serum-free medium with 10 nM1,25（OH）₂D₃.Real-time qPCR was used to analyze the expression of Notch1,Notch2,and KLF4.The self-renewal ability of CSCs was measured by sphere forming rate and limiting dilution assay.Tumorigenicity in vivo were compared between CSCs and1,25（OH）₂D₃-treated CSCs.（3）The effect of VDR/β-catenin signaling pathway on stemness of MOSE cells treated with 1,25（OH）₂D₃.After MOSE cells were continuously administrated with 10nM 1,25（OH）₂D₃,mRNA expressions of VDR,β-catenin,c-Myc,and CyclinD1 were detected by Real-time qPCR.The co-localization ofβ-catenin with VDR were examined by immunofluorescence and immunoprecipitation.Furthermore,transcription level of VDR andβ-catenin in CSCs were determined by Real-time qPCR.Results:1.1,25（OH）₂D₃ reduces the stem cell-like properties during spontaneous malignant transformation of MOSE cells.In the process of spontaneously malignant transformation,MOSE cells displayed"pebble shape"in early passage,and they transformed into"spindle shape"in late stage.10nM 1,25（OH）₂D₃ could postpone this change of morphology during oncogenic transformation.The colony formation assay showed that proliferation of MOSE cells was significantly inhibited by 1,25（OH）₂D₃（p<0.05）.Both functional marker（side population,SP）and surface markers（CD44,CD117）of stem cell were downregulated by 1,25（OH）₂D₃ during spontaneous malignant transformation of MOSE cells（p<0.05）.Real-time qPCR assay showed that1,25（OH）₂D₃ markedly decreased the mRNA expression of NANOG（p>0.05）,but increased one of SOX2（p<0.05）.The sphere forming rate in 1,25（OH）₂D₃-treated group was significantly inhibited,compared with control group.Limiting dilution assay suggested that the number of late stage MOSE cells generating one tumor sphere was dramatically increased from 431 to 1299 after treated with 1,25（OH）₂D₃.These results indicated that the self-renewal capacity of MOSE cells were suppressed by 1,25（OH）₂D₃.Moreover,we found that survival rate of MOSE cells irradiated by X-ray was markedly higher than that of MOSE cells combined irradiation with 1,25（OH）₂D₃,indicating that1,25（OH）₂D₃ increased radiosensitivity of MOSE cells.Therefore,The results indicated that 1,25（OH）₂D₃ suppresses the stem cell-like properties during spontaneously malignant transformation of MOSE cells.2.The effect of 1,25（OH）₂D₃ on the stemness of ovarian cancer stem-like cells.The cancer stem-like cells（side population,SP and CD44+/CD117+）and non-cancer stem-like cells（non-side population,NSP and CD44-/CD117-）were isolated by FACS,respectively.Compared to control group,1,25（OH）₂D₃ significantly reduced the mRNA expressions of Notch1 and KLF4 in SP cells（p<0.05）.The sphere forming rate of SP cells was suppressed by 1,25（OH）₂D₃（p<0.05）.Both sphere forming and limiting dilution assay suggested that 1,25（OH）₂D₃ significantly decreased the self-renewal capacity of SP cells.Moreover,we found that survival rate of CD44+/CD117+cells irradiated with X-Ray,was higher than that of CD44+/CD117+cells treated by1,25（OH）₂D₃,indicating that 1,25（OH）₂D₃ may increase the sensitivity of ovarian cancer stem-like cells to X-ray.After 100 CD44+/CD117+cells were transplanted into ovary of nude mice,the incidence of tumor was dramatically decreased from 100%in control group to 40%in vitamin D-treated group.In addition to,ascites and metastatic tumor were also decreased in vitamin D group,suggesting that vitamin D reduced tumorigenicity of CSCs.These results illustrated that vitamin D reduced stemness of ovarian cancer stem-like cell in both vitro and vivo.3 The effect of VDR/β-catenin signaling pathway on the stemness after MOSE cells treated with 1,25（OH）₂D₃.Real-time qPCR assay showed that 1,25（OH）₂D₃reduced the expression of VDR,but promoted expression ofβ-catenin of MOSE cells in late passages（p<0.05）.Immunofluorescence and immunoprecipitation have shown that1,25（OH）₂D₃ promoted VDR gathered to the nucleus,and inhibitedβ-catenin translocation from cytoplasm to nucleus.Moreover,the direct interaction between VDR andβ-catenin were strengthened by 1,25（OH）₂D₃.Furthermore,the transcription level of c-Myc and CyclinD1,downstream gene of Wnt pathway,were reduced after MOSE cells treated with 1,25（OH）₂D₃.These results indicated that 1,25（OH）₂D₃ suppressed stem cell-like properties MOSE cells through regulating VDR/β-catenin signaling pathway.Conclusions:1.1,25（OH）₂D₃ reduces the stem cell-like properties during spontaneous malignant transformation of MOSE cells.2.1,25（OH）₂D₃ suppressed the stemness through decreasing the mRNA expressions of Notch1 and KLF4,self-renewal capacity,and increasing radiosensitivity of ovarian cancer stem-like cells.Meanwhile,Vitamin D decreased tumorigenicity and metastasis of ovarian cancer stem-like cells in vivo.3.1,25（OH）₂D₃ suppressed stem cell-like properties of MOSE cells by regulating VDR/β-catenin signaling pathway.