| Object Existing researches showed that 90% of ovarian cancer originated in Ovarian Surface Epithelial Cell(OSEC). Many studies have found that Vitamin D can inhibit occurrence and development of a variety of tumors, and improve the prognosis of cancer. Our laboratory has been successfully established the spontaneous malignant transformation of Mouse Ovarian Surface Epithelial Cell(MOSEC). And Epithelial-Mesenchymal transformation(EMT) has been involved in this process. We investigate that the effect of vitamin D on malignant transformation of MOSEC by regulating EMT. In this study, MOSEC is cultured and administrated with the active Vitamin D,1,25(OH)2D3,The proliferation and migration of MOSEC was detected, and the expression of m RNA and protein related-EMT is determined. The change key enzymes of the vitamin D metabolism is analyzed to explore effective dose of 1,25(OH)2D3. To explore the role of 1,25(OH)2D3 in MOSEC spontaneous malignant transformation and its possible mechanism. Method(1) 1,25(OH)2D3 postpones the process of malignant transformation of MOSEC. The MOSEC was cultured, and was treated with 1nmol/L of 1,25(OH)2D3 from passage 20(P20) to P120 in vitro. The proliferation and migration of the MOSEC were determined by plate colony, soft agar colony, transwell and wound healing assay in the phase of MOSEC malignant transformation. The effect of 1,25(OH)2D3 on the survival rate of MOSEC treated with paclitaxel was deterred by CCK-8 kit. The tumorgenesis in vivo was analyzed by Nude mice intraperitoneal injected with MOSEC administrated with 1,25(OH)2D3..(2) 1,25(OH)2D3 postpones the process of malignant transformation of MOSEC by regulating EMT. The morphology was observed in the process of MOSEC treated with 1,25(OH)2D3 or negative control ethanol, respectively. The expression of EMT-related proteins E-cadherin, N-cadherin, Snail, β-catenin and Vimentin was determined by Western-blotting and Immunofluorescence. Furthermore, Real-time Q-PCR was used to find the EMT-related genes express, for example SPP1, COL3A1, MMP3 and ESR1.(3) The impact of the key enzyme in the vitamin D metabolism on the malignant transformation process of MOSEC. The expression of m RNA of CYP27B1, CYP24A1 and VDR was determined by real time PCR. The effective dose of 1,25(OH)2D3 was analyzed in the phases of malignant transformation of MOSEC. All statistical analyses were performed with SAS9.1. Further clarify the possible mechanisms of 1,25(OH)2D3 retary the MOSEC malignant transformation.Result1. 1,25(OH)2D3 postpones the process of malignant transformation of MOSEC(1) Proliferation in vitro. According to the effect of 1,25(OH)2D3 on morphology, proliferation and migration capability of MOSEC, the process of malignant transformation was divided into three phases, Ⅰphase(P20-p60),Ⅱphase(P70-P90) and Ⅲphase(P100-P120). The plate colony result shows 1,25(OH)2D3 increased the cloning diameter at Ⅰand Ⅱphase(P<0.0005). But colony formation rate of MOSEC was inhibited in all three phases(P<0.05). The soft agar colony assay reveals that 1,25(OH)2D3 inhibited colony formation rate of MOSEC inⅠ and Ⅲ phase, but promoted that at Ⅱphase(P<0.05).(2) In vivo tumorigenicity Compare with control group cells, metastatic focus was reduced(5.1561 g vs 1.2335g)in nude mice injected, MOSEC treaded with 1,25(OH)2D3. the amount of ascites was decreased(13ml vs 4ml. Moreover, 1,25(OH)2D3 suppressed the expression of N-cadherin, MMP3 and CYP24A1 in ascites cells. These results suggest that the tumorigenesis in vivo is decreased by 1,25(OH)2D3.(3) MOSEC sensitivity to paclitaxel The survival rate are decreased in MOSEC treated with paclitaxel and 1,25(OH)2D3, respectively. However, MOSEC administrated by 1,25(OH)2D3 have a lower viability than that in the control group(P <0.05). Experimental results show that 1,25(OH)2D3 enhances the sensitivity of MOSEC to paclitaxel.(4) Migration in vitro. In spontaneous malignant transformation of MOSEC, 1,25(OH)2D3 had no effect on migration ability in P60 and P70 cells(P> 0.05), but it inhibited any other passages. Furthermore weakened the ability of P40 cells in wound healing at 12, 24 and 48 h, respectively(P<0.05). Therefore, 1,25(OH)2D3 inhibits migration of MOSEC in vitro.2. 1,25(OH)2D3 postpones the process of malignant transformation of MOSEC by regulating EMT.(1) Cell morphology changes. In sponsous malignant transformation process, MOSEC displayed epithelial cells-like "pebble shape" in I phase, then it was transformed into mesenchymal cell-like "spindle shape" in II and III phases. 1,25(OH)2D3 inhibited this conversion in Ⅰand Ⅲ phase, but promoted this process in Ⅱ phase.(2) The expression of EMT markers protein. The result of western-blotting revealed that 1,25(OH)2D3 inhibited the expression of N-cadherin, Snail and β-catenin,, while promoted the expression of E-cadherin, which are all related to EMT. It also showed the 1,25(OH)2D3 inhibited translation of β-catenin from cytoplasm to nucleus. These indicated MOSEC by regulation proteins related-EMT.(3) Expression of EMT-related genes. 1,25(OH)2D3 promoted expression of MMP3 and ESR1, but inhibited the expression of SPP1, in I phase of malignant transformation of MOSEC. In Ⅱ phase, 1,25(OH)2D3 increased the expression of SPP1, COL3A1, but decreased the expression of MMP3 and ESR1. And in Ⅲ phase, 1,25(OH)2D3 inhibited EMT through inhibiting expression of SPP1 and MMP3 and promoting expression of ESR1. These results illustrated that 1,25(OH)2D3 regulated different EMT-related genes in three phases of malignant transformation of MOSEC, which may be one of the reasons 1,25(OH)2D3 increased proliferation of MOSEC in phase II.3. 1,25(OH)2D3 postpones malignant transformation of MOSEC related to key enzymes of Vitamin D metabolism. The result of real-time Q-PCR assay showed that expression of CYP27B1, CYP24A1 and vitamin D receptor(VDR)all displayed a normal distribution curve and reached a maximum at P70-MOSECⅡphase in the spontaneous malignant transformation of MOSEC. The expression of CYP24A1 was dramatically increased in MOSEC treated with 1,25(OH)2D3(P<0.05), reaching 15,000 times at phase II. However, CYP24A1 express growth rate are different in treatment groups,P60 and P90 were significantly higher than other passages, In other words, Expression in phase Ⅱ is higher than phaseⅠand Ⅲ. Compared with the control MOSEC, The expression of CYP27B1 and VDR was significantly increased in MOSEC at phase I, which resulted in the increased effective doses of 1,25(OH)2D3 in this period. The effective doses of 1,25(OH)2D3 was increased in phase III for the same reason. However, higher expression of CYP24A1, and lower expression of CYP27B1 and VDR lead to the decreased effective doses of 1,25(OH)2D3in phase II. These results indicated that the effective doses of 1,25(OH)2D3 are different in three phases of malignant transformation, although MOSEC was cultured in vitro by the same concentration of 1,25(OH)2D3.Conclusion1. Active vitamin D postpones the process of malignant transformation of MOSEC by inhibiting the proliferation and migration of cells in phases Ⅰ and Ⅲ in vitro, and enhancing the sensitivity to paclitaxel. In the meanwhile, 1,25(OH)2D3 decreased tumorigenicity and metastasis of MOSEC in phasesⅡin vivo. However, 1,25(OH)2D3 regulated different EMT-related genes in three phases of malignant transformation of MOSECthis study also showed that 1,25(OH)2D3 promotes the proliferation of MOSEC in phase Ⅱ.2. 1,25(OH)2D3 inhibits the progress of EMT by up-regulating E-cadherin, down-regulating N-cadherin, Snail and β-catenin in malignant transformation of MOSEC. However, 1,25(OH)2D3 regulated different EMT-related genes in three phases of malignant transformation of MOSEC.3. The effective doses of 1,25(OH)2D3 were different due to expression key genes of vitamin D metabolism(CYP27B1 and CYP24A1) and VDR in spontaneous malignant transformation of MOSEC. The effective doses of 1,25(OH)2D3 were increased for the reason of higher expression of CYP27B1 and VDR in phases I and III. They were relatively decreased in phase II, because of drastically increased expression of CYP24A1 in this phase. |
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