| Objective:Ovarian cancer is one of the most common gynecological malignant tumor with a high case fatality rate,due to its high recurrence rate and metastasis.Over the past decade,many studies showed that active vitamin D[1α,25-dihydroxy-vitaminD3,1α,25(OH)2D3]inhibited the proliferation,migration and invasion of malignant tumor cells.However,our previous and others study found that CYP24A1,the enzyme that catalyzes the inactivation of 1α,25(OH)2D3,was dramatically increased after cells were treated with active vitamin D.In addition,CYP24A1 has been shown to be highly expressed in numerous cancers,including lung,cervix,breast and colon cancers,as compared with surrounding normal tissues.Some studies have indicated that CYP24A1 may be a candidate oncogene.In this project,we investigated whether the CYP24A1 could play the role as an oncogene.We overexpressed CYP24A1 level in two ovarian cancer cell lines to determine the effects of CYP24A1 on proliferation,migration and invasion.The pull down-mass spectrometry assay was performed to discover the proteins which directly interacted with CYP24A1 to elucidate the mechanism by which these cells undergo EMT(Epithelial-mesenchymal transition).This study provides new targets and strategies for clinical prevention and treatment of ovarian cancer.Methods:The study was divided into three parts.(1)The overexpression CYP24A1 enhanced the proliferation,migration and invasion of ovarian cancer cells.The differentially expressed CYP24A1 between ovarian tumors and normal tissues was analyzed by TCGA database.The expression of CYP24A1 in ovarian cancer tissues,normal tissues and benign adenoma tissues was detected by human ovarian cancer tissue-array.Two ovarian cancer cells were stably transfected with either with an empty lentiviral vector or with the CYP24A1 overexpression vector.Over-expression of mRNA and protein levels of CYP24A1 were confirmed using Real-Time PCR and Western-blotting,respectively.CCK-8 and colony formation assays were performed to detect the proliferation of ovarian cancer cells,as indicated by proliferation rate(=(the absorbance in 24 and 48 hours-the absorbance in 0 hour)/the absorbance in 0 hour)and proliferation ability(absorbance values were used to show the number of alive cells),respectively.Wound healing assay was performed to detect the effect of overexpression CYP24A1 on migration of ovarian cancer cells.The migration ability was indicated by the migration index(=(the initialized width of scratch)-(the final width of the scratch)]/(the initialized width of the scratch).Transwell assay was used to assess the effect of overexpression CYP24A1 on the invasion of ovarian cancer cells,as indicated by the invasion ability(absorbance values were used to show the number of cells which passed through the basement membrane of the transwell chamber).(2)Knockdown CYP24A1 inhibited the proliferation,migration and invasion of ovarian cancer cells.CCK-8 and colony formation assays were used to evaluate the effect of knockdown CYP24A1 on the proliferation of ovarian cancer cell lines.Wound healing assay was used to assess the effect of knockdown CYP24A1 on migration of ovarian cancer cells.Transwell assay was performed to detect the effect of knockdown CYP24A1 on the invasion of ovarian cancer cells,as indicated by the invasion ability(the area of effective cells was used to show the number of cells which passed through the basement membrane of the transwell chamber).(3)The CYP24A1 promoted EMT process by directly interacting with FUS.We have detected the proteins that interact directly with Flag-CYP24A1 by pull down-spectrometry assays,and found that the RNA-binding protein Fused in Sarcoma(FUS)interacted directly with Flag-CYP24A1.It was reported that FUS promoted the miR-200c-mediated inhibition of ZEB1,suppressing EMT.Co-immunoprecipitation(Co-IP)and Immunofluorescence were used to confirm the direct interaction between CYP24A1 and FUS.Yeast two-hybrid was used to discover the interaction site between CYP24A1 and FUS.We also dectecd the interaction between CYP24A1 or its mutants and FUS through pull down experiments.Real-Time PCR was used to determined the level of miR-200c in ovarian cancer cells which were overexpressed or knockdown CYP24A1.Western-blotting was also performed to detect the expression of FUS,ZEB1,Snail,E-cadherin and Vimentin in overexpression/knockdown CYP24A1 ovarian cancer cells to elucidate the mechanism of CYP24A1 by which these cells undergo EMT.Results:1.Overexpression CYP24A1 promotes proliferation,migration and invasion of ovarian cancer cells.The results of TCGA database showed that the expression of CYP24A1 in ovarian tumors was higher than that in normal tissues(p>0.05).But the results from tissue microarray showed that the expression of CYP24A1 in ovarian cancer tissues was remarkably higher than that in normal and benign adenoma tissues(p<0.01).The expression of both mRNA and protein levels of CYP24A1 were significantly increased in SKOV-3 and OVCAR-8 after stably transfected with the CYP24A1 overexpression vector.The colony formation assay displayed that the ovarian cancer cells which were overexpressed CYP24A1 grew faster than the negative control group which was transfected with negative vector(p<0.01).The CCK-8 assay also showed that the proliferation rate was significantly increased in the overexpression group after seeding at 24 and 48 hours(p<0.05).The wound healing assay showed that the migration index was insignificantly different between the overexpressed CYP24A1 and negative control group after cells were scratched for 12 hours and 24 hours,but it was significantly decreased in the negative control group after scratching at 48 hours(p<0.05).Transwell assay also showed that the invasive ability in the overexpression CYP24A1 cells was significantly stronger than that in the negative control group(p<0.01).These results indicated that the overexpression CYP24A1 promoted the proliferation,migration and invasion of ovarian cancer cells2.Knockdown CYP24A1 inhibits proliferation,migration and invasion of ovarian cancer cells.Colony formation assay showed that ovarian cancer cells in knockdown the CYP24A1 grew significantly slower than those in the negative control group(p<0.01).The CCK-8 assay also showed that the proliferation rate in knockdown group was significantly reduced after seeding at 24 and 48 hours(p<0.01).Wound healing assay displayed that the migration index in knockdown CYP24A1 ovarian cancer cells was significantly lower than that in the negative control cells at 24 and 48 hours after scratching(p<0.05).Transwell assay also showed that the invasion ability was significantly reduced in the knockdown CYP24A1 cells,compared with the negative control group(p<0.05).These results implied that knockdown CYP24A1 inhibited the proliferation,migration and invasion of ovarian cancer cells.3 CYP24A1 directly interacts with FUS to inhibit EMT process in ovarian cancer cells.(1)CYP24A1 directly interacts with FUS.The results from pull down-mass spectrometry showed that Flag-CYP24A1 was directly interacted with FUS which is an RNA-binding protein.It was reported that FUS promoted the inhibitory effect of miR-200c on ZEB1.The result from Co-IP demonstrated that there was a direct interaction between Flag-CYP24A1 and FUS in ovarian cancer cells.The results from Yeast two-hybrid and pull down experiments further showed that the amino acid 87-239 domain of CYP24A1 is necessary for binding to FUS,since only the mutant that lacked this region of CYP24A1 exhibited loss of binding to FUS.We also determined that amino acid 237 of CYP24A1 is critical for binding to FUS,because this mutant displayed dramatical reduce of binding to FUS.Immunofluorescence assay also displayed that Flag-CYP24A1 were colocalized with FUS in the nucleus and its periphery in only OVCAR-8 cells,not SKOV-3 cells.These results indicated that the 87-239 amino acids of CYP24A1 was needed for directly binding to FUS,and the 237 amino acid of CYP24A1 was critical.(2)Effect of CYP24A1 on the expression of FUS and miR-200c/ZEB 1 in ovarian cancer cells.Western-blotting showed that overexpression CYP24A1 significantly reduced the expression of FUS in OVCAR-8 cells(p<0.05),while the knockdown CYP24A1 increased the level of FUS in OVCAR-8 cells(p<0.05).The results from Real-Time PCR showed that overexpression CYP24A1 reduced the level of miR-200c in OVCAR-8 cells(p>0.05),while knockdown CYP24A1 insignificantly increased the expression of miR-200c(p<0.05).These results revealed that the overexpression CYP24A1 could reduce the expression of FUS and miR-200c.Molecular Cell publishes reported of novel results in 2018 that FUS promoted the inhibition of miR-200c on the expression of the EMT key transcription factor ZEB1,since miR-200c and ZEB1 are conserved miRNA-mRNA pairs.The study published on Oncogene also reported that miR-200c has an inhibitory effect on the expression of ZEB1.Therefore,we continue to detect the effects of CYP24A1 on level of ZEB1.Western-blotting showed that ZEB1 was significantly increased in the overexpressed CYP24A1 OVCAR-8 cells(p<0.05),while the expression of ZEB1 was significantly decreased in the knockdown CYP24A1 OVCAR-8 cells(p<0.05).Collectively,these results hinted that CYP24A1 reduced the expression of FUS,and increased the level of ZEB1 by attenuating the inhibitory of miR-200c on ZEB1.(3)Effect of CYP24A1 on the expression of EMT-related proteins in ovarian cancer cells.The increased expression of ZEB1 suggested that the effect of CYP24A1 on migration and invasion may be associated with the progression of EMT.Therefore,we continued to explore the effects of CYP24A1 on level of other EMT-related proteins.Western-blotting experiments showed that the expression of Snail,EMT transcription factor,was insignificantly changed after overexpressioned CYP24A1.However,the level of E-cadherin was significantly decreased(p<0.05),and the expression of Vimentin,mesenchymal cell marker,was slightly but insignificantly increased.Conversely,the expression of Snail was significantly decreased in knockdown CYP24A1 OVCAR-8 cells(p<0.05),but no SKOV-3 cells(p>0.05).Meanwhile,the expression of E-cadherin was markedly increased(p<0.05),and the level of Vimentin was significantly decreased in the knockdown CYP24A1 ovarian cancer cells(p<0.05).These resulted suggested that knockdown CYP24A1 inhibited EMT by upregulating E-cadherin and downregulating Vimentin and Snail.Together,these results indicated the 87-239 amino acid domain of CYP24A1 interacted directly with FUS which attenuates the inhibitory of miR-200c on the expression of ZEB1,therfore promoted EMT progression by downregulating E-cadherin and upregulating Vimentin and Snail.Conclusion:1.The level of CYP24A1 in ovarian tumors is higher than that in normal tissues and benign adenoma tissues.Overexpression CYP24A1 could promote the proliferation,migration and invasion of ovarian cancer cells.2.Knockdown CYP24A1 could inhibit the proliferation,migration and invasion of ovarian cancer cells.3.The 87-239 amino acids domain of CYP24A1 directly interacted with FUS,attenuating the inhibitory of miR-200c on expression of ZEB1,and promoted the EMT process by downregulating the E-cadherin and upregulates Vimentin and Snail.These results reveal that CYP24A1/FUS/miR-200c/ZEB1 pathway involves in the progression of EMT in ovarian cancer cells. |
PDF Full Text Request