The Study On The Biological Function And Mechanism Of Splicing Factor DDX23 In Serous Ovarian Cancer

Ovarian cancer is the most lethal gynecological malignancy.According to the recently released Chinese cancer data covering a population of 380 million,there were 57,200 new cases of ovarian cancer and 27,200 deaths in China in 2016.The latest statistics from the American Cancer Society(ACS)showed that in 2021,there were 21,410 new cases and 13,770 deaths of ovarian cancer.The mortality of ovarian cancer among female cancer patients ranked fifth.Globally,the five-year relative survival rate for ovarian cancer generally ranged from 30%to 50%.The clinical symptoms of early-stage ovarian cancer lack specificity,and high-accuracy molecular diagnostic methods for early-stage ovarian cancer are limited.Therefore,more than 80%of patients with ovarian cancer are diagnosed at an advanced clinical stage.Serous ovarian cancer(SOC)is the most common subtype of ovarian cancer with a high degree of malignancy.Currently,the first-line treatment for ovarian cancer is cytoreductive surgery combined with systemic chemotherapy,but most advanced patients will still experience recurrence and chemotherapy resistance after achieving complete remission.With the development of molecular medicine,the application of anti-angiogenic drugs,poly ADP-ribose polymerase inhibitors(PARPi),immunotherapy and other new therapies have changed the traditional treatment mode of ovarian cancer and significantly improved the survival rate of patients.Despite advances in the diagnosis and treatment of ovarian cancer,some patients still relapse within a short period of time.Therefore,we still need to further study the molecular expression pattern and pathogenesis of ovarian cancer.RNA splicing is a gene regulation mechanism widely existing in eukaryotes.It is a process in which the spliceosome,under the cooperation of splicing factors,removes introns and joins exons to produce mature mRNA.Splicing factors play important roles in various aspects of RNA processing,including transcription,RNA splicing and RNA decay,etc.Abnormal expression of splicing factors is associated with malignant progression and immune dysregulation of various tumors.The splicing factor DDX23,a member of the DEAD-box RNA helicase family,is involved in the formation of U4/U6-U5 tri-snRNP and B complex.Therefore,DDX23 plays a crucial role in spliceosome assembly and RNA splicing.Missense alterations in DDX23 can cause atypical neurodevelopment.Meanwhile,abnormal DDX23 expression has been implicated in glioma and hepatocellular carcinoma progression.At present,the expression pattern and clinical significance of DDX23 in SOC have not been characterized.The biological function and related mechanism of DDX23 in SOC have not been elucidated to date.In response to the above scientific questions,we conducted the following systematic studies.Part Ⅰ:Expression and clinical significance of splicing factor DDX23 in serous ovarian cancerObjectiveTo explore the expression profile of DDX23 in SOC.To clarify the influence of DDX23 on the clinicopathological characteristics and survival prognosis of SOC patients.MethodsBased on the previous research results of our group,combined with the data from GeneCards,CPTAC and TCGA databases,we screened out the splicing factor DDX23 and analyzed its expression in SOC.The DDX23 mRNA expression in SOC and normal fallopian tube(FT)samples was detected by qRT-PCR.IHC analysis was performed using tissue microarrays(TMAs)containing SOC and FT specimens to explore the expression pattern of DDX23.Combined with clinical data,the correlation between DDX23 expression and clinicopathological characteristics such as age,ascites,and CA125 level of SOC patients was analyzed.The Kaplan-Meier method was used to analyze the survival prognosis of patients.Results1.DDX23 is highly expressed in SOCRNA splicing-related pathways are abnormally activated in SOC.DDX23 is one of the core splicing factors that is significantly highly expressed in SOC.CPTAC data showed that DDX23 protein was highly expressed in various tumor types,and the protein expression level of DDX23 was significantly increased in ovarian cancer compared with normal ovarian tissue.TCGA data and qRT-PCR experiments showed that the mRNA expression level of DDX23 in SOC was significantly higher than that in control tissue.2.Correlation between DDX23 expression and clinicopathological features of SOC patientsIHC staining revealed significantly higher DDX23 expression in SOC specimens.High DDX23 expression was positively correlated with poor overall survival(OS),and had no correlation with the age,ascites,CA125 level and other factors.Cox proportional hazard regression analysis indicated that DDX23 expression was an independent high-risk factor for OS,besides advanced FIGO stage.3.High DDX23 expression is associated with poor prognosis in SOC patientsKaplan-Meier survival analysis in our cohort confirmed that patients in the high DDX23 expression group had a shorter OS than those in the low expression group.The Kaplan-Meier plotter online data were consistent with our results.ConclusionRNA splicing-related pathways are abnormally activated in SOC.DDX23 is a core splicing factor that is significantly highly expressed in SOC.The expression of DDX23 is correlated with OS of SOC patients,and the high expression of DDX23 indicates poor prognosis of patients.DDX23 is expected to be an effective marker for predicting the prognosis of SOC patients.Part Ⅱ:The effect of splicing factor DDX23 on malignant biological behavior of serous ovarian cancerObjectiveTo investigate the effect of splicing factor DDX23 on the malignant biological behavior of ovarian cancer cells,such as proliferation,migration,invasion,and tumor growth in vivo.MethodsDDX23 knockdown and overexpression ovarian cancer cell lines were established by lentiviral infection.MTT assays and clonogenic assays were used to detect the effect of DDX23 on the proliferation of ovarian cancer cells in vitro.Flow cytometry was used to analyze cell cycle progression.G1 phase arrest related markers and tumor aggressiveness related markers were detected by Western Blot.Transwell assays and wound healing assays were carried out to detect the effect of DDX23 on the migration and invasion of ovarian cancer cells.Subcutaneous and abdominal tumorigenesis assays were used to detect the tumorigenic ability of ovarian cancer cells in vivo.Ki-67 expression of in xenograft tumors was detected by IHC staining.Results1.DDX23 promotes the proliferation of ovarian cancer cells in vitroIn MTT assays,compared to the negative control(NC)groups,DDX23 silencing inhibited the growth of ovarian cancer cells and the inhibition was most evident in the last day,whereas DDX23 overexpression promoted cell proliferation.Similarly,in clonogenic assays,DDX23 knockdown reduced the colony formation ability,whereas DDX23 overexpression increased the colony forming ability of ovarian cancer cells.2.DDX23 maintains ovarian cancer cell cycle progressionCell cycle analysis revealed that compared to NC group,DDX23 silencing could increase the percentage of cells in the G1 phase while decreasing the percentage of cells in S phase in three ovarian cancer cell lines.We measured G1 phase arrest related markers by Western Blot and the results showed that DDX23 knockdown decreased the expression of CCND1 and CDK4,but increased p21 expression.DDX23 knockdown inhibited cell proliferation through G1 phase arrest.3.DDX23 promotes migration and invasion of ovarian cancer cells in vitroIn transwell assays,knockdown of DDX23 significantly reduced the number of ovarian cancer cells passing through the basement membrane of Transwells,and reduced cell migration and invasion;while overexpression of DDX23 enhanced the migration and invasion of ovarian cancer cells.In wound healing assays,at 12h post-scratch,ovarian cancer cells with DDX23 knockdown migrated less than NC group.We measured tumor aggressiveness related markers by Western Blot and the results showed that DDX23 knockdown decreased the expression of Slug and N-cadherin,but increased E-cadherin expression.4.DDX23 promotes the tumor growth of ovarian cancer cells in vivoIn subcutaneous tumorigenesis assays,DDX23 silencing could apparently inhibit the growth of xenograft tumors.IHC staining showed that Ki-67 expression was decreased in xenograft tumors of DDX23 knockdown mice group.In abdominal tumorigenesis assays,DDX23 silencing could apparently inhibit the abdominal tumorigenesis ability of ovarian cancer cells.ConclusionDDX23 can promote the proliferation,migration and invasion of ovarian cancer cells.DDX23 is required for cell cycle progression of ovarian cancer cells.DDX23 knockdown inhibits cell proliferation through G1 phase arrest.DDX23 affects the tumorigenic ability of ovarian cancer cells in vivo,and the decreased function of DDX23 weakens the tumorigenic ability of ovarian cancer cells in vivo.Part Ⅲ:The mechanism of splicing factor DDX23 promoting the malignant progression of serous ovarian cancerObjectiveTo explore the upstream transcriptional regulatory mechanism of DDX23 overexpression in SOC.To screen the downstream differentially expressed genes(DEGs)related to DDX23 function.To explore the downstream regulatory mechanism of DDX23 promoting the malignant progression of SOC.To search for the key executor in DDX23-induced malignant phenotype of SOC.MethodsUsing gene co-expression analysis,TCGA DEGs analysis,JASPAR and Cistrome website,E2F1 was screened as an upstream transcript factor of DDX23.The potential binding sites of E2F1 on DDX23 promoter were predicted.The effects of E2F1 on the mRNA and protein expression of DDX23 were detected by qRT-PCR and Western Blot.The wild-type and mutant-type plasmids of DDX23 promoter were constructed using pGL4.26 vector.The effect of E2F1 on the transcriptional activity of DDX23 was detected by dual-luciferase reporter gene assays.The ChIP assays were used to verify the direct binding of E2F1 to DDX23 promotor.RNA-seq was performed in DDX23 knockdown and control A2780 cells.DEGs were identified by RNA-seq analysis.Using gene co-expression analysis,TCGA DEGs analysis and RNA-seq data,FOXM1 was screened as a key downstream gene of DDX23.TCGA and CPTAC data were used to analyze the expression of FOXM1 in ovarian cancer and normal ovarian tissues.The regulatory effect of DDX23 on FOXM1 expression was detected by qRT-PCR and Western Blot.The effects of FOXM1 on the proliferation and migration of ovarian cancer cells were detected by MTT and Transwell assays.The effect of DDX23 on the expression of FOXM1 transcripts was detected by qRT-PCR experiments.Rescue experiments were performed by overexpressing FOXM1 in DDX23 knockdown ovarian cancer cells.Western Blot,cell proliferation,migration assays and subcutaneous tumorigenesis experiment were used in rescue experiments.Results1.E2F1 Activated DDX23 Transcription in Ovarian Cancer CellsCo-expression analysis revealed that the mRNA expression of E2F1 and DDX23 were positively correlated in ovarian cancer.E2F1 was predicted to bind to DDX23 promoter based on Cistrome Data Browser data.E2F1 expression was up-regulated in ovarian cancer based on TCGA data.qRT-PCR and Western Blot experiments showed that knockdown of E2F1 in ovarian cancer cells inhibited the expression of DDX23 at both mRNA and protein levels,while overexpression of E2F1 promoted the mRNA and protein expression of DDX23.DDX23 knockdown had no effect on E2F1 expression at both mRNA and protein levels.Luciferase assays showed that E2F1 overexpression increased the luciferase activity in HEK293T cells transfected with the DDX23 promoter wild-type plasmid,but not in cells with mutant-type plasmid.The ChIP-PCR assays confirmed that E2F1 could bind to the DDX23 promoter region directly.2.Identification of DEGs Involved in DDX23 Function by RNA-seqThe changes in the transcriptome with DDX23 knockdown were analyzed and a total of 4115 DEGs were identified(1.5FC,P<0.05).There were 1921 upregulated genes and 2194 downregulated genes.Using gene co-expression analysis,TCGA DEGs analysis and RNA-seq data,17 genes were screened and a well-known oncogene FOXM1 was focused.DDX23 was shown to be positively associated with the expression of FOXM1.FOXM1 was upregulated in ovarian cancer at both mRNA and protein levels based on data from TCGA and CPTAC.3.DDX23 Regulated the Production of the Main Oncogenic Transcript of FOXM1The FOXM1 expression decreased at both mRNA and protein levels after DDX23 knockdown.The FOXM1 expression increased at both mRNA and protein levels after DDX23 overexpression.FOXM1 silencing inhibited the proliferation and migration capacity of ovarian cancer cells,while ectopic expression of FOXM1 enhanced their proliferation and migration potential.FOXM1 transcripts mainly include FOXM1A,FOXM1B and FOXM1C.Both FOXM1B and FOXM1C have transcriptional activity,while FOXM1A does not.qRT-PCR experiments showed that FOXM1C expression was much higher than the other two transcripts in ovarian cancer cells.FOXM1C expression was dramatically decreased after DDX23 knockdown,whereas FOXM1A and FOXM1B expression did not change significantly.4.FOXM1 Mediated DDX23-driven Malignant Progression of Ovarian Cancer CellsOverexpression of FOXM1 could rescue the reduced cell proliferation and migration induced by DDX23 silencing.In subcutaneous tumorigenesis experiments,overexpression of FOXM1 could rescue the reduced tumorigenic ability of ovarian cancer cells in vivo.ConclusionDDX23 was a direct transcriptional target of E2F1 in ovarian cancer cells.E2F1 could bind to the DDX23 promoter and activated DDX23 transcription.DDX23 regulated the production of FOXM1C,the main oncogenic transcript of FOXM1.DDX23 silencing reduced the production of FOXM1C,thereby attenuating the malignant progression of ovarian cancer.FOXM1 was a key executor in DDX23-induced malignant phenotype of SOC.Highlights(1)Based on the abnormal activation of the RNA splicing pathway in SOC,this project screened out the highly expressed core splicing factor DDX23 in SOC.In this study,the expression and clinical significance of DDX23 in SOC patients were systematically studied for the first time using the tissue microarrays and clinical data from our center.This study demonstrated that up-regulation of DDX23 is associated with poor prognosis of patients,and DDX23 is expected to be an effective indicator for predicting the prognosis of SOC patients.(2)This study is the first to clarify the cancer-promoting role of the splicing factor DDX23 in SOC.DDX23 is essential for maintaining the malignant phenotype of SOC.(3)This study is the first to clarify the upstream transcriptional regulation mechanism of the up-regulation of splicing factor DDX23 in SOC.DDX23 is the direct transcriptional activation target of E2F1.In this study,FOXM1,a key downstream executor of DDX23-induced SOC malignant phenotype,was screened,and the downstream regulatory mechanism of DDX23-induced SOC malignant progression was elucidated.Limitations(1)In this study,the function of DDX23 has not been deeply studied in the hot topics such as targeted drug resistance and tumor immune microenvironment in SOC.At present,we have formulated a follow-up experimental plan to continue these topics and conduct researches on the above-mentioned directions.(2)Small molecule inhibitors or related antisense oligonucleotides(AS Os)targeting DDX23 have not yet been developed.Therefore,in vivo tumor suppression experiments cannot be performed using patient-derived tumor xenograft(PDX)models.(3)This study only investigated the regulatory effect of DDX23 on FOXM1 expression.As an important core splicing factor,DDX23 has diverse and complex regulatory modes.DDX23 may directly or indirectly regulate more target genes.In the future,RIP-seqa CLEP-seq and other experiments can be performed to find more downstream key target genes of DDX23 and study the corresponding RNA binding motifs.