The Mechanisms Of FOXD1 Modulating Serous Ovarian Cancer Proliferation And Metastasis

Despite advances in the diagnosis and treatment of ovarian cancer in the past decades,the incidence and mortality of ovarian cancer have not been significantly reduced.The reason is as follows:First,the ovaries are deep in the pelvis,and the early symptoms of ovarian cancer are atypical.When the disease produces noticeable symptoms,most patients have already progressed to advanced stages;in the second place,there is no barrier around ovary,the spread of cancer cells is much earlier than most cancers;thirdly,although platinum-based chemotherapy for advanced patients after cytoreductive surgery can achieve good results,the recurrence rate and drug-resistance rate of relapsing patients are high.With the development of molecular biology in recent years,people gradually realized that ovarian cancer is a polygenic disease that caused by activation of oncogene or inactivation of tumor suppressor genes.Therefore,exploring the factors that affect the occurrence and development of ovarian cancer at molecular level is a promising strategy.Forkhead box D1(FOXD1)is a transcription factor and belongs to forkhead box family.It is primarily involved in the development of the kidney and retina during embryogenesis.Recent studies have reported that FOXD1 is involved in the regulation of metastasis and proliferation of glioma and lung cancer.This study focuses on the role and mechanism of FOXD1 in serous ovarian cancer.At present,there are no reports in this area at home and abroad.This research mainly includes the following four parts:1.The expression of FOXD1 in serous ovarian cancer and its clinical significance.2.The effect of FOXD1 on proliferation of ovarian cancer cells and the underlying mechanism.3.The effect of FOXD1 on metastatic of ovarian cancer cells and the underlyingmechanism.4.The study on the upstream regulation mechanism of FOXD1.Part I:THE EXPRESSION OF FOXD1 IN SEROUS OVARIAN CANCER AND ITS CLINICAL SIGNIFICANCEBACKGROUND and OBJECTIVE:Jiang et al.analyzed the mRNA expression profile of three data sets(GSE14001,GSE15578,and GSE12172)in the GEO database.The results suggested that the FOXD1 expression of ovarian cancer tissues is significantly lower than that of normal tissues.The purpose of this study was to detect the expression of FOXD1 in serous ovarian cancer tissues and its clinical significance.MATERIALS and METHODS:Western blot was used to detect the expression of FOXD1 in high-grade serous ovarian carcinoma(HGSOC,n=8)and normal fallopian tube epithelium(n=7),and then qRT-PCR was employed to further verify FOXD1 expression in 20 cases of HGSOC tissue and 11 cases of normal fallopian tube epithelium tissue.We analyzed the expression of FOXD1 at mRNA level in GSE9891 dataset and evaluated its impact on clinical outcomes.In addition,the expression of FOXD1 in tissue microarrays(including 120 cases of HGSOC tissue)was detected by immunohistochemical staining,and its effect on clinical prognosis was then calculated.RESULTS:The results of Western blot showed that the expression of FOXD1 in the normal fallopian tube tissue was significantly higher than that in HGSOC tissue.The qRT-PCR results were consistent with Western blot results.In the GSE9891 dataset(n=276),both the overall survival and progression-free survival of FOXD1 high-expression group were significantly longer than FOXD1 low-expression group.There are 152 cases of HGSOC patients in GSE9891 dataset,and both the overall survival and progression-free survival of HGSOC patients with higher FOXD1 expression were longer.Immunohistochemical staining was used to detect the expression of FOXDI in 120 cases of HGSOC patients.The result showed that the overall survival of HGSOC patients with high expression of FOXD1 was prolonged.CONCLUSION:The downregulation of FOXD1 is related to the bad prognosis in HGSOC patients.Part ?:THE EFFECT OF FOXD1 ON PROLIFERATION OF OVARIAN CANCER CELLS AND THE UNDERLYING MECHANISMBACKGROUND and OBJECTIVE:According to previous reports,FOXD1 can influence the proliferation of cancer cells in lung,breast and brain cancers.The results of the Part I showed that FOXD1 may act as a tumor suppressor gene in serous ovarian cancer.The purpose of this study was to observe the effect of FOXD1 on the proliferation of ovarian cancer cells and explore the underlying regulatory mechanisms.MATERIALS and METHODS:We established stably transfected cell lines with upregulated or downregulated FOXD1 by means of lentivirus infection.The effect of FOXD1 on the proliferation of ovarian cancer cells was determined by cloning assay.To further verify the effect of FOXD1 on the proliferation of ovarian cancer cells in vivo,the xenograft mouse model was employed to examine the effects of FOXD1 on tumor growth.Flow cytometry was applied for cell cycle analysis.Western blot was used to detect the relationship between the expression of cell cycle-associated proteins and FOXD1 expression.The transcription factor binding site was predicted by JASPAR.After the potential binding sites were obtained,the activity of each potential binding site was verified by dual-luciferase reporter assay.In addition,we also used chromatin immunoprecipitation to further verify that FOXD1 binds to these binding sites.Rescue assay was used to verify that the proliferation inhibition effect of FOXD1 was mainly caused by P21 overexpression.RESULTS:We found that increased FOXD1 significantly inhibited the colony forming ability of ovarian cancer cells in vitro and decreased FOXD1 enhanced colony forming ability of ovarian cancer cells.The xenograft mouse model showed that overexpression of FOXD1 significantly inhibited the growth of ovarian cancer cells in vivo.Cell cycle analysis showed that FOXD1 induced cell cycle arrest at G1 phase.Western blot showed that there was a positive correlation between FOXD1 expression and P21 expression in ovarian cancer cell lines.JASPAR predicted that there were three potential FOXD1 binding sites in the promoter of P21.The dual-luciferase reporter assay showed two of these three binding sites showed binding activity.Subsequently,we further validated that FOXD1 directly bind to these two binding sites by means of chromatin immunoprecipitation.Rescue assay confirmed that FOXD1 inhibited the proliferation of ovarian cancer cells by modulating P21 expression.We also verified that FOXD1 inhibited the proliferastion of P53-null H1299 cells.CONCLUSION:FOXD1 inhibits the proliferation of ovarian cancer cells by promoting the expression of P21 in a p53-independent way.Part III:THE EFFECT OF FOXD1 ON METASTASIS OF OVARIAN CANCER CELLS AND THE UNDERLYING MECHANISMBACKGROUND and OBJECTIVE:Ovarian cancer is often called the silent killer.Most ovarian cancer patients have few symptoms at early stage and diagnosed at late stages with meatstasis.Therefore,it is of great significance to study the mechanisms that affect the metastasis of ovarian cancer.According to previous studies,FOXD1 is involved in the regulation of glioma cell metastasis,but its specific regulatory pathways are not indicated.Another purpose of this study is to explore the impact of FOXD1 on ovarian cancer metastasis and explore the underlying molecular mechanisms.MATERIALS and METHODS:The effect of FOXD1 on invasion and migration of ovarian cancer cells was examined by wound healing assay and Transwell assay.A xenograft model in mice was employed to assess the metastatic abilities of the A2780 cells.Subsequently,the changes of EMT-related genes were detected by Western blot and qRT-PCR.JASPAR was then used to predict the FOXD1 binding sites of genes regulated by FOXD1.The activity of potential binding site was verified by dual-luciferase reporter assay.In addition,we also use chromatin immunoprecipitation to further verify that FOXD1 binds to these binding sites.RESULTS:Compared with the control group,the invasion and migration ability of ovarian cancer cells with FOXD1 overexpression was significantly weakened,the invasion and migration ability of the cells with FOXD1 knockdown was significantly enhanced.The xenograft model showed that FOXD1 also suppressed the metastasis ability of ovarian cancer cells in vivo.Western blot results showed that in the FOXD1 overexpression group,E-cadherin was significantly elevated,N-cadherin and ZEB2 was significantly reduced.The trend of changes in E-cadherin,N-cadherin,and ZEB2 in the FOXD1 knockdown group was just opposite to that in the overexpression group.JASPAR showed that there are three potential FOXD1 binding sites in the promoter of ZEB2.With the dual-luciferase reported assay,we found out that one of the binding sites has binding activity.Subsequently,the directly binding between FOXD1 protein and P21 promoter was confirmed by chromatin immunoprecipitation.CONCLUSION:FOXD1 inhibits the metastatic ability of ovarian cancer cells by targeting ZEB2.Part IV:THE STUDY ON THE UPSTREAM REGULATION MECHANISM OF FOXD1BACKGROUND and OBJECTIVE:Based on the above results,the expression of FOXD1 is decreased in ovarian cancer tissues,and decreased FOXD1 is associated with poor prognosis.In addition,FOXD1 inhibits the proliferation and metastasis of ovarian cancer cells.We believe that FOXD1 acts as a tumor suppressor gene in ovarian cancer.But the reason why FOXD1 is downregulated in HGSOC patients is still unclear.Therefore,we need to further explore the specific mechanisms that lead to the decreased expression of FOXD1 in cancer tissues.MATERIALS and METHODS:We used the microRNA target gene prediction databases TargetScan and miRanda to predict miRNAs that may inhibit FOXD1 expression.The qRT-PCR technique was used to detect the expression level of miRNA in HGSOC tissues.Subsequently,miRNA was manipulated by transfection of miRNA mimics and inhibitors and then the expression of FOXD1 was detected by Western blot and qRT-PCR.The dual-luciferase reported assay were used to detect whether miR-30a-5p or miR-200a-5p directly regulates FOXD1.RESULTS:The results of TargetScan and miRanda showed that both miR-30a-5p and miR-200a-5p have a binding site at the 3 'untranslated region of FOXD1 mRNA.Compared with normal fallopian tube epithelium,the expression of miR-30a-5p and miR-200a-5p in HGSOC patients was significantly increased.After transfection of miR-30a-5p or miR-200a-5p mimics,the expression of FOXD1 showed a significant decrease at both mRNA and protein levels.Dual-luciferase reported assay results show that either miR-30a-5p or miR-200a-5p can bind to the 3' untranslated region of FOXD1 mRNA and inhibit its expression.FOXD1 targeted by miR-30a-5p or miR-200a-5p.CONCLUSION:In ovarian cancer cells,both miR-30a-5p and miR-200a-5p directly inhibit the expression of FOXD1.