Vitamin D Metabolic Enzyme CYP24A1 Prompts Lung Cancer Metastasis Through FUS/miR200c/ZEB1 Pathway

Objective:Cancer is still a major public health problem.In China,the incidence and mortality of lung cancer accounted for 18.1%and 24.1%of the total tumors respectively,which far exceeds the world’s average=.The non-small-cell lung carcinoma cancer,including squamous-cell carcinoma,non-small-cell lung carcinoma,large cell cancer,accounts for more than 80%of lung cancer cases,and is is the main cause of high mortality of lung cancer.Preclinical research indicates that the active metabolite of vitamin D,1α,25（OH）₂D₃,might have a potential as anticancer agents because their treatment has the anti-proliferative and pro-apoptotic effect.CYP24A1,a member of the cytochrome P450 enzyme family,is a vitamin D-inactivating enzyme,which catabolizes 1α,25（OH）₂D₃ It has been reported that the inactivation or mutation of CYP24A1 may be the cause of idiopathic infantile hypercalcemia.In addition,there were also studies indicated the overexpression of CYP24A1 were associated with poor prognosis of patients with esophageal,colorectal,prostate,breast and other cancers.Our previous studies using an ovarian cancer cell model suggested that CYP24A1 could interact directly with FUS to influence the inhibitory effect of miR200c on ZEB1.In the present study,we investigated whether CYP24A1 impacted on the proliferation and invasion of lung cancer through mediating FUS/miR200c/ZEB1 pathway.Methods:This research is divided into three parts.（1）CYP24A1 promotes proliferation and metastasis of lung cancer cells:A stable A549-shCYP24A1 model was constructed by knocking down CYP24A1 with lentivirus transfection in A549 cells with high endogenous CYP24A1 expression.In Calu-1 cells with low endogenous CYP24A1 expression,a stable overexpressed（OE）-CYP24A1 model was constructed by lentivirus transfection.Real-time PCR and Western blotting were used to verify the efficiency of CYP24A1 knock-down and overexpression effects of CYP24A1.CCK-8 and plate colony formation were used to detect survival and proliferation of lung cells,respectively.The effects of CYP24A1 on the migration and invasion of lung cancer cells were determined by Transwell migration and invasion assays.Sixteen BALB/C nude mice at approximately 5 weeks of age were subcutaneously injected into the mice with 100μl cell suspensions containing 10⁷ cells.The mice injected with A549 cells were used as Scramble tumor controls（N=6）,mice injected with shCYP24A1-1（N=5）and shCYP24A1-2（N=5）cells were used as CYP24A1-knockdown tumors.The longest diameter（a）and the shortest diameter（b）of the subcutaneous tumor were measured weekly.The animals were sacrificed 30 days post-injecting.The volume of tumor was calculated as formula:1/2（length ×width2）.For intravenous injections,1×10⁷ tumour cells labeled with luciferase were suspended in 100 μL of PBS and injected into the lateral tail vein of mice.The metastasis of lung cancer cells,including Scramble group（N=4）,shCYP24A1-1 group（N=3）and shCYP24A1-2 group（N=3）,was observed by Imaging System.All mice were sacrificed 40 days post injection,after which lungs were removed and fixed in 10%formalin.Immunohistochemistry assay was used to evaluate the expression of Ki67,CYP24A1.（2）CYP24A1-mediated FUS/miR200c/ZEB1 pathway promotes EMT process in human lung cancer cells:Immunofluorescence assay was used to detected the colocalization between CYP24A1 and FUS in cells co-transfected by both CYP24A1 and FUS vector.Western blotting was used to determine the expression of EMT related proteins including E-cadherin,Vimentin,Snail,ZEB1,and RNA-binding protein FUS in lung cells and tumor tissues.Real-Time PCR was used to detect the effect of CYP24A1 on the levels of miR200c in lung cells.（3）CYP24A1 promotes the malignant transformation of immortalized human lung bronchial epithelial cells:Using lentiviral infection,a stable cell model of overexpressed CYP24A1 was established in immortalized human pulmonary bronchial epithelium BEAS-2B cells.The overexpression of CYP24A1 was verified by Real-time PCR and Western blotting.The OE-CYP24A1 BEAS-2B cells were cultured continuously in vitro,and the cellular morphology was observed under an inverted microscope every 10 generations.CCK-8 assay was used to examine effect of CYP24A1 on 1 BEAS-2B cells.The expression of EMT-related proteins,such as E-cadherin,ZEB1,Snail,and RNA-binding protein FUS in BEAS-2B cells was detected by Western-blotting,and the miR200c levels in BEAS-2B cells was detected by Real-Time PCR.Results:1.CYP24A1 promotes proliferation and metastasis of lung cancer cellsTo investigate the effect of CYP24A1 expression on the proliferation,migration and invasion of lung cancer cells,a cell line model with knock-down or over-expressed CYP24A1 was constructed.The results of Real-Time PCR and Western-blotting showed that the expression of CYP24A1 mRNA（p<0.01）and protein（p<0.01）in A549 cells with high expression of endogenous CYP24A1 were significantly decreased after transfection with ShCYP24A1 lentivirus,the mRNA level（p<0.01）and protein expression（p<0.01）of Calu-1 cells with low endogenous expression were significantly increased after over expressing CYP24A1（OE-CYP24A1）lentivirus.The results showed that the CYP24A1 knock-down and over-expression lung cancer cell models were successfully established.Compared with negative control（scramble）cells,the proliferation rate and clone formation rate of lung cancer cells were significantly decreased in CYP24A1 knockdown cells（p<0.01）,and the migration（p<0.01）and invasive ability（p<0.01）of lung cancer cells were also decreased.In contrast,high expression of CYP24A1 enhanced the migration（p<0.05）and invasion（p<0.001）of lung cancer cells compared with the empty control cells.These results suggested that increased expression of CYP24A1 could promote cell proliferation and invasion in vitro.Moreover,compared with the scramble control group,the subcutaneous tumor volume（p<0.05）and the tumor weight（p<0.05）were significantly decreased,the numbers of pulmonary nodules and the relative bioluminescent value of transplanted cells decreased in the CYP24A1 knockdown group.In addition,the expressions of CYP24A1 and Ki67 in tumor tissue were also significantly decreased in the knock-down group,suggesting that the decreased expression of CYP24A1 significantly inhibited the tumorigenicity and metastasis of lung cancer cells in vivo.Taken together,these results demonstrated that the proliferation,migration and invasion of lung cancer cells were significantly inhibited by reduced CYP24A1 in vivo and in vitro,whereas the migration and invasion of lung cancer cells were significantly enhanced by overexpressed CYP24A1.2.CYP24A1-mediated FUS/miR200c/ZEB1 pathway promotes EMT process in lung cancer cellsIn our previous project,we found the interaction between CYP24A1 and FUS using RNA pull down-mass spectrometry and co-immunoprecipitation assay.Recent data in Molecular Cell suggested that FUS promoted the miR200c mediated degradation of ZEB1.However,it remains unknown if the CYP24A1/FUS/miR200c/ZEB1 signaling pathway could be involved in the EMT process in lung cells.In Calu-1 cells of overexpressed CYP24A1,FUS was found to be colocalization with CYP24A1 around the nucleus,but not in cytoplasm.The results from Western-blotting showed that overexpression of CYP24A1 dramatically increased the expression of FUS（p<0.01）and ZEB1 protein（p<0.01）,compared with empty vector control.Inversely,knockdown of CYP24A1 significantly decreased the expression of FUS（p<0.05）,compared with scramble control.In addition,knock-down CYP24A1 significantly increased miR200c expression（p<0.01）,and increased CYP24A1 expression inhibited miR200c levels（p<0.05）.Moreover,compared with the scramble control tumors,FUS expression was increased,and ZEB1 expression was decreased in subcutaneous tumor tissues in the knockdown CYP24A1 group.These data indicated that CYP24A1 mediated the inhibitory effect of miR200c on ZEB1 by regulating FUS expression.In addition,we found that the expressions of E-cadherin in shCYP24A1-1 and shCYP24A1-2 cells were higher than that in the scramble control cells（p<0.05）,and the expressions of Vimentin was decreased by a 0.23-fould reduction in shCYP24A1-1 group（p<0.05）.The Snail expressions in a shCYP24A1 cells were also decreased due to reduction of CYP24A1（p<0.05）.In contrast,the expressions of Snail and Slug in Calu-1 cells were increased（p<0.05）,when CYP24A1 was overexpressed.The expressions of E-cadherin were decreased by 0.18-fold（p<0.05）,and Vimentin was increased（p<0.05）due to overexpression of CYP24A1 in Calu-1 cells.Taken together,these results indicated that CYP24A1 mediated the EMT process in lung cancer cells via FUS/miR200c/ZEB1 pathway.3.CYP24A1 promotes malignant transformation of immortalized human lung bronchial epithelial cells.To explore whether CYP24A1 can be acted as a potential oncogene,we established overexpressed CYP24A1 in BEAS-2B cells an immortalized human lung bronchial epithelial cells,in which the levels of CYP24A1 mRNA（p<0.05）and protein（p<0.01）were significantly increased,compared with the empty control cells.The cellular morphology in overexpressed CYP24A1 BEAS-2B cells gradually changed from short stick and round shape to long shuttle shape during continuous culture in vitro.The overexpression of CYP24A1 could significantly increase the proliferation rate of BEAS-2B cells（p<0.05）.Similarly,over-expressed CYP24A1 insignificantly increased the expression of FUS protein（p>0.05）,except for the 2nd generation BEAS-2B cells（p<0.05）.Moreover,the levels of miR200c in BEAS-2B-OE-CYP24A1 cells decreased significantly（p<0.01）.And the expressions of ZEB1 and Snail were insignificantly increased,E-cadherin was insignificantly decreased in BEAS-2B-OE-CYP24A1 cells（p>0.05）.These results suggested that overexpression of CYP24A1 promoted the proliferation and EMT phenotype of immortalized human lung bronchial epithelial cells,which may be associated with upregulating FUS and inhibiting miR200c levels.Conclusion:1.Overexpression of CYP24A1 could enhance proliferation,migration and invasion of lung cells.2.The degradation of ZEB1 by miR200c was enhanced by decreased CYP24A1 expression.The expression of Snail and Vimentin was decreased and E-cadherin was increased due to knockdown CYP24A1.These results indicated that CYP24A1 could mediate EMT progression in lung cancer cells via FUS/miR200c/ZEB1 pathway.3.Overexpressed CYP24A1 could promote Mesenchymal-like phenotype,and enhanced cell proliferation in immortalized human lung bronchial epithelial cells.FUS may be associated with the miR200c-mediated degradation of ZEB1 due to decreased expression of CYP24A1.