Effect Of Induced CYP24A1 Of Vitamin D Catabolic Enzyme On The Proliferation And Migration Of Ovarian Cancer Cells

Objective:Ovarian cancer is one of the most common malignant tumors in the female reproductive system.It has the characteristics of strong invasion,strong migration and high recurrence rate.In recent years,studies on vitamin D have shown that 1α,25-dihydroxy-vitaminD₃（the active form of vitamin D,1α,25（OH）₂D₃）has the functions of inhibiting the proliferation and promoting differentiation of tumor cells.The preliminary studies of our group have found that 1α,25（OH）₂D₃ could inhibit the proliferation of malignant transformation of mouse ovarian surface epithelial cells.With the extension of processing time,however,the inhibition ability of 1α,25（OH）₂D₃ was weakened.At the same time,the CYP24A1 gene increased thousands times in this process.The 24-hydroxylase encoded by the CYP24A1 gene is the key enzyme in the metabolic process of vitamin D,which can make 1α,25（OH）₂D₃ turn to extremely low active metabolites,thus maintaining the stability of the blood calcium concentration.The expression level of CYP24A1 in normal tissue is low.However,the results of research showed that the expression of CYP24A1 in tumor tissue was increased.In order to investigate whether the induced CYP24A1 antagonized the antitumor effect of1α,25（OH）₂D₃,this study established a stable CYP24A1 low expression and overexpressed ovarian cancer cell lines.We analyzed effects of induced CYP24A1 on the proliferation and migration of ovarian cancer cells and sensitivity to chemotherapeutic drugs.And the gene chip technology was used to analyze the changes in the expression of the CYP24A1 co-expression genes.Then we explored the molecular mechanism of the induced CYP24A1 to influence the antitumor activity of1α,25（OH）₂D₃.Methods:The study was divided into two parts.（1）The effect of induced CYP24A1 on the 1α,25（OH）₂D₃ inhibiting proliferation and migration of ovarian cancer cells.Establish the ovarian cancer cell lines with stable low expression and overexpression of CYP24A1.Plate clone formation assay detected the effect of1α,25（OH）₂D₃ on the proliferation of ovarian cancer cells with different CYP24A1 expression levels.The effect of 1α,25（OH）₂D₃ on migration of ovarian cancer cells with different CYP24A1 levels was detected by scratch test.CCK-8 assay was used to detect cell viability,and analyzed the effect of induced CYP24A1 on the sensitivity of ovarian cancer cells to the combined action of 1α,25（OH）₂D₃ and carboplatin.（2）The mechanism of CYP24A1 affecting the anti-cancer effect of 1α,25（OH）₂D₃.Using the CYP24A1 promoter sequence to transform the luciferase report system,adding1α,25（OH）₂D₃ treatment factors,to analyze the change of the activity of CYP24A1 promoter.Gene chip technology was used to analyze the differentially expressed genes in SKOV-3 cells after treating with 1α,25（OH）₂D₃.Further analyzed the genes which were co-expressed with CYP24A1 and verified by qRT-PCR.Then the GO and KEGG Pathway enrichment analysis were carried as well as the functional analysis to look for possible mechanism of induced CYP24A1 influencing the antitumor activity of1α,25（OH）₂D₃.Results:1.The effects of knockdown CYP24A1 on the ability of 1α,25（OH）₂D₃to inhibit proliferation and migration of ovarian cancer cells.We successfully established the SKOV-3 and OVCAR-8 ovarian cancer cell lines with the CYP24A1 gene stable low expression or overexpression.After 1α,25（OH）₂D₃ treatment,the proliferation ability of the CYP24A1 gene knockdown group was significantly lower than that of the negative control group（p<0.001）,and the proliferation ability of the CYP24A1 overexpression group was significantly higher than that of the negative control group（p<0.05）.The colony formation assay showed that in the SKOV-3 cells the clone-forming ability of the CYP24A1 knockdown group was significantly lower than that of the negative control group（p<0.001）.And the 1α,25（OH）₂D₃ in the negative control group decreased the clone formation rate by 59.78%,while 1α,25（OH）₂D₃reduced the clone formation rate of the CYP24A1 knockdown group by 76.82%.The results of scratch test showed that 1α,25（OH）₂D₃ did not significantly change the cell migration index in the negative control group,but 1α,25（OH）₂D₃ decreased the cell migration index in the CYP24A1 knockdown group significantly（p<0.05）.This indicated that after CYP24A1 knockdown,it could enhance the inhibitory effect of1α,25（OH）₂D₃ on proliferation,and the CYP24A1 gene overexpression showed resistance to the inhibition of proliferation of 1α,25（OH）₂D₃.And reducing CYP24A1 expression could not only reduce the migration capacity of ovarian cancer cells,but also increase the inhibition ability of 1α,25（OH）₂D₃ to cell migration.2.The effects of CYP24A1 expression level on the sensitivity of ovarian cancer cells to 1α,25（OH）₂D₃ combined with carboplatin.CCK-8 assay results showed that ovarian cancer cells viability decreased significantly after treating with 1α,25（OH）₂D₃for 48 hours,and cell viability decreased with the prolongation of treatment time,which was time dependent.P<0.05).When carboplatin was used alone to treat ovarian cancer cells,cell viability decreased with the increase of carboplatin concentration,and showed a dose-dependent effect（p<0.001）.We selected low concentration carboplatin（20 mg/L）combined with 1α,25（OH）₂D₃（100 nmol/L）for 48 hours to observe the effect of combined effect on chemosensitivity of ovarian cancer cells.The results showed that1α,25（OH）₂D₃ enhanced the anti-proliferation effect of carboplatin（p<0.001）,similar to the inhibition effect of the 80 mg/L carboplatin alone treatment group.And we also observed that the cells in the CYP24A1 knockdown group could further reduce the cell viability of the combined action.3.The effects of 1α,25（OH）2D3 on the activity of CYP24A1 promoter.The results of luciferase activity assay showed that 1α,25（OH）₂D₃ could significantly increase the activity of CYP24A1 promoter in SKOV-3 cells.And this effect was mainly related to the treatment time of 1α,25（OH）₂D₃,and was not related to the concentration of 1α,25（OH）₂D₃.However,in the CYP24A1 knockdown group,the mRNA of CYP24A1 still was induced high expression after treating with 1α,25（OH）₂D₃.It indicated that knocking down CYP24A1 did not reduce the induction of CYP24A1 by1α,25（OH）₂D₃.4.1α,25（OH）₂D₃ induced CYP24A1 associated gene expression profile analysis.The results of this part showed that 1α,25（OH）₂D₃ made the 105 gene expression changes significantly in the SKOV-3 cells,among which 85 genes were up-regulated and 20 genes down regulated.Moreover,after functional enrichment of differentially expressed genes,we found that the main involvement of these genes involved in GO biological processes such as positive regulation of cell proliferation,cell adhesion,transcription regulation of DNA templates.And differentially expressed genes participated in TGF-βsignaling pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,ect.In addition,we also analyzed 39 differentially expressed genes,which were co expressed with CYP24A1.It was proved that CYP24A1 may affect the expression of differently expressed genes such as KIT,TRPV6,IGFBP3,GRK5,IGFBP1,thus affecting the process of inhibiting cell proliferation and migration by1α,25（OH）₂D₃.