Genomic Characteristics Of Epithelial Ovarian Cancer And Screening,Validation And Mechanism Of EPS8L1

Background:Ovarian cancer is one of the three major malignant tumors of the female reproductive system,and its mortality rate ranks the first among gynecological tumors.Epithelial ovarian cancer is the most common pathological type of ovarian cancer,which has the poorest prognosis.The causes of this result are occult onset and lack of effective early diagnosis strategies.Currently,the role of biomarkers in early diagnosis and prognosis of epithelial ovarian cancer is still very limited.Therefore,exploration the mechanism of epithelial ovarian cancer and found new biomarkers,would be helpful for the early diagnosis of epithelial ovarian cancer.The Next-Generation Sequencing?NGS?is a milestone in the history of the development of Sequencing technology,which could sequence millions of molecules simultaneously.This technology has been widely used in genome Sequencing,the transcriptome Sequencing and gene expression regulation in the areas of research.Liquid biopsy is a new technology,which could capture the blood circulating tumor cells?CTC?,plasma cell-free DNA?cfDNA?and exosomefor.It can be used in disease diagnosis,therapeutic evaluation and prognosis judgement of prognosis.This emerging technology is developing rapidly thanks to the NGS technology and precision medicine.Supported by the NGS technology,the mode of research is changing from traditional hypothesis to data-driven,which greatly reduced the randomness and blindness.In this study,we analyzed the genomic characteristics of epithelial ovarian cancer patients,screened the differentially expressed genes in the tissues of patients,and explored their regulatory effects and mechanisms on the malignant biological behavior of ovarian cancer cells.It is important to understand the tumorigenesis of ovarian cancer and discover new biomarkers of ovarian cancer.Chapter1 Genomic characteristics of epithelial ovarian cancer.Objective:To recognize the genomic expression characteristics of epithelial ovarian cancerMethods:1.The patients with epithelial ovarian cancerwho met the requirements of the study were enrolled.The clinical data of the patients were collected.The peripheral blood of the patients was collected before surgery,and the tissues?tumor and contralateral normal ovarian tissues?were collected during surgery?comprehensive staging surgery for epithelial ovarian cancer includes:hysterectomy,bilateral ovaries,bilateral fallopian tubes,omentum resection+retroperitoneal lymph node dissection?renal vein level?+resection of any gross suspicious lesions?.DNA/RNA in tissue and DNA in buffy coat were extracted.2.Exome and transcriptome sequencing were performed on HiSeq 3000 from Illumina.DNA extracted from normal ovarian tissue was used as control samples in patients with unilateral tumor.DNA extracted from buffy coat was used as control in patients with bilateral tumors.Bioinformatics analysis and Sanger sequencing were carried out.3.GO cluster analysis,KEGG and Reactome enrichment analysis were performed for differentially expressed genes.Results:1.A total of 31 cases of epithelial ovarian cancer were enrolled,including 18 cases of stage ??58.07%?,2 cases for stage ??6.45%?and the rest of the 11 cases of?-? period.Case collection began in February 2016 and was followed up to December 2019 with a median follow-up of 41 months.11 patients died and 7 relapsed after treatment.The results showed that most epithelial ovarian cancer patients were diagnosed in the advanced stage,with poor therapeutic effect and poor prognosis.2.A total of 1598 somatic SNVs?single nucleotide variants,SNV?were detected in 31 patients,among which 5 mutation sites of TP53,FAM83H-AS11,KRTAP4-3,FBXW10 and ZNF814 genes were detected in more than 2 patients.3.463 potential pathogenic sites were found,which were distributed on 437 genes.The average mutation sites of each gene were 1.06?1-9?.There are 9 mutation sites in TP53,among which is the newly discovered SNV?TP53:NM₀01126115:exon3:c.C346T:p.R116W?.4.Of the 437 genes,117 had differential expression in transcription level.The results of GO cluster analysis showed that embryonic organ development,embryonic organ morphogenesis and the cilium were the functional group with the most enriched genes.The enrichment results of KEGG pathway showed that MAPK and PI3K-Akt pathways were the main enrichment pathways.In the analysis of Reactome pathway,hemostasis,collagen formation,cell surface interaction at the vessel wall and degradation of the extracellular matrix were the most important gene enrichment pathways.Chapter2 Screening and validation of EPS8L1 geneObjective:To screen and validate the differentially expressed genes,which were related to the occurrence and development of epithelial ovarian cancerMethods:1.Transcriptome data were analyzed,and differential genes were screened.2.qRT-PCR and Western blot were used to verify the expression of differentially expressed genes in ovarian cancer and normal tissue samples.3.Immunohistochemical staining of tissue microarray was used to detect the expression of differential gene in ovarian cancer and normal tissue samples.4.cfDNA was extracted from peripheral blood.The concentration of cfDNA was detected by Qubit fluorescence quantifier and the levels of differential genes in cfDNA were detected by qPCR.5.Detect the expression of the differentially gene by qRT-PCR and Western Blot,and analyze the relationship between the expression and clinicopathological characteristics.Results:1.31 Tumor samples were compared with 10 Normal ovarian samples?Tumor vs Normal?,a total of 4963 differentially expressed genes were found by RNA-Seq,and a total of 4028 differentially expressed genes were found in 10 pairs of paired samples?Tumor vs Normal_paired?.Finally,EPS8L1,a gene possibly involved in the tumorigenesis of epithelial ovarian cancer,was selected for further study after comprehensive consideration of literature review,gene function and potential impact on tumor.2.The results ofqRT-PCR showed that the expression of EPS8L1-mRNA in ovarian cancer tissues increased to 3.17 times compared with that in normal ovarian tissues?p?0.001?.Western blot results showed the expression of EPS8L1-protein in ovarian cancer tissues increased to 2.79?p?0.001?.3.The results of immunohistochemical staining showed that EPS8L1 was localized in the cytoplasm.Among the 123 cases of ovarian malignant tumor tissues,120?97.55%?were positive,among which 41?33.33%?were strongly positive.Among the 60 cases of normal ovarian tissue,11 cases?18.3%?showed positive EPS8L1 staining,all of which were weakly positive,while the other 49 cases were negative.The positive rate in the tumor group was significantly higher than that in the normal ovarian group?p?0.05??4.The results of cfDNA testing suggested that the cfDNA concentrations and EPS8L1-cfDNA relative levels in patients with advanced stage??+??,lymph node metastasis and distant metastasis were higher than that in the patients with early stage??+??.5.Further analysis of clinicopathological characteristics showed that the expression of EPS8L1-mRNA was not correlated with gender,age,tumor size and pathological type of patients,but correlated with tumor grade,lymph node metastasis and FIGO stage.Kaplan-meier's survival analysis chart was drawn based on follow-up information,which suggested that the survival time of patients with high EPS8L1-mRNA expression was significantly lower than that of patients with low EPS8L1-mRNA expression?P?0.05?.Chapter3 The effects of EPS8L1 on malignant biological behavior of epithelial ovarian cancer cellsObjective:To investigate the effect of EPS8L1 on malignant biological behavior of epithelial ovarian cancer cells.Methods:1.The basic expression of EPS8L1 in normal ovarian epithelial cells and different ovarian cancer cells was detected by qRT-PCR and Western blot.2.Lentivirus was used to establish stable transfected EPS8L1 knockdown?EPS8L1-Si?and EPS8L1 overexpressing?EPS8L1-OE?ovarian cancer cells.3.The proliferation activity of ovarian cancer cells was detected by CCK-8 kit.4.The clonality of ovarian cancer cells was detected by clone formation assay.5.Apoptosis of ovarian cancer cells was detected by flow cytometry.6.The invasion and migration of ovarian cancer cells were detected by Transwell assay.7.The expression of EMT-related?epithelial mesenchymal transformation,EMT?proteins was detected by Western blot.Results:1.qRT-PCR and Western blot results showed that the expression of EPS8L1 in ovarian cancer cells?3AO,SKOV3,CAOV3,A2780,OVCAR3,HO8910?was significantly higher than that in normal ovarian epithelial cells?IOSE80?.Among these 6 ovarian cancer cells,the expression level of EPS8L1 in SKOV3 was the highest,so SKOV3 was selected for follow-up assay.2.Knocking down EPS8L1 significantly inhibited the malignant biological behavior(proliferation,clonality,anti-apoptosis,invasion,migration and EMT of SKOV3 in ovarian cancer cells.However,the overexpression of EPS8L1 has the opposite effect.Chapter4 To explore the molecular mechanism of EPS8L1 regulating the malignant biological behavior of epithelial ovarian cancer cells,based on MAPK and PI3K-Akt signaling pathwaysObjective:Based on the first part,the mechanism of EPS8L1 regulated the malignant biological behavior of epithelial ovarian cancer cells.Methods:1.The RT2ProfilerTM PCR Array was used to examine the effect of EPS8L1 on MAPK and PI3K/Akt signaling pathway.2.The differential gene expression of MAPK and PI3K/Akt signaling pathway effected of EPS8L1 overexpression/knockdown were detected byW estern Blot.3.The activators and inhibitors were used to explore that EPS8L1 effected proliferation,clonality,apoptosis,migration,invasion and EMT of SKOV3 through MAPK and PI3K-Akt pathways.Results:1.ProfilerTM PCR Array showed that the significantly differential genes affected by EPS8L1 in MAPK pathway were CDKN2D,CDKN2A,ETS1 and GRB2.The significantly differential genes affected by EPS8L1 in PI3K/Akt pathway were RAF1,ADAR and EIF4E.Overexpression of EPS8L1 significantly promoted the activation of MAPK and PI3K/Aktpathways,while knocking down of EPS8L1 had an opposite effect on MAPK and PI3K/Akt pathways.2.Activators of MAPK and PI3K/Akt pathway?EGF,IGF-1?could significantly reverse the inhibition of malignant biological behavior of ovarian cancer cells caused by knocking down EPS8L1.However,MAPK and PI3K/Akt pathway inhibitors?U0126-EtOH,PI-103?significantly eliminated the promotion effect of EPS 8L1-overexpression on malignant biological behavior.Chapter5 The effect of EPS8L1 on tumorigenesis of ovarian cancer cells in vivo and its mechanismObjective:To investigate the effect of EPS8L1 on tumorigenesis of ovarian cancer cells in nude mice and its mechanismMethods:1.Establish nude-mouse transplanted tumor model with EPS8L1 knockdown and EPS8L1 overexpression.Investigate the effect of EPS8L1 on tumorigenesis in vivo by tumor formation in nude mice.2.The effect of EPS8L1 on apoptosis was detected by TUNEL staining.3.MAPK and PI3K/Akt activator were injected to nude-mice in EPS8L1 knockdown group to explore the effect of EPS8L1 on ovarian cancer cell tumorigenesis through MAPK and PI3K/Akt pathway.Results:1.Subcutaneous xenograft tumorigenic ability of nude mice showed that knockdown EPS8L1 significantly inhibited the tumorigenic ability and tumor growth of SKOV3 cells in nude-mice,while overexpression of EPS8L1 had the opposite effect.2.Knockdown of EPS8L1 significantly increased the apoptosis of subcutaneous tumor cells in nude-mice,while overexpression of EPS8L1 showed the opposite result.3.Intraperitoneal injection of MAPK and PI3K/Akt activators significantly restored the growth of subcutaneous tumors and eliminated the inhibitory effect of knockdown EPS8L1 on the growth of subcutaneous transplanted tumors in nude-mice.Summary and conclusion1.Epithelial ovarian cancer patients have specific SNV characteristic and abnormal gene expression profiles.MAPK and PI3 A-Akt pathways are the main enrichment pathways of differential genes.2.EPS8L1 was the gene related to the occurrence and development of epithelial ovarian cancer.It is highly expressed in epithelial ovarian cancer tissues and correlated with tumor grade,lymph node metastasis,clinical stage and prognosis.The expreesion of EPS8L1-cfDNA in peripheral blood was also related to the occurrence and development of epithelial ovarian cancer,which could be expected as a new biomarker of epithelial ovarian cancer.3.EPS8L1 could promote malignant biological behavior of epithelial ovarian cancer cells in vitro and vivo through MAPK and PI3K/Aktpathways.In summary,we believe that EPS8L1 is a high-response gene of epithelial ovarian cancer,which has important reference value for the diagnosis,progress and prognosis assessment of ovarian cancer.It is worth for further study.