Screening Of Biomarkers In High-grade Serous Ovarian Cancer And The Mechanism Of Metallothionein Regulating The Occurrence And Development Of Serous Ovarian Cancer

Part Ⅰ Identification of molecular biomarkers for diagnosis and prognosis of high-grade serous ovarian cancerObjectiveDue to the lack of early diagnosis strategies and effective treatments,ovarian cancer is still the leading death of gynecological malignancies.Most patients present with advanced disease at the time of diagnosis because of the insidious onset and rapid progression.Early diagnosis of ovarian cancer can significantly improve patient prognosis.Hence,it is of great significance to identify key diagnostic and prognostic biomarkers specific for ovarian cancer.Since high-grade serous ovarian cancer(HGSOC)is the most common and most malignant type of ovarian cancer and accounts for the majority of deaths,we selected HGSOC as the research object,aiming to identify potential biomarkers for early diagnosis and prognosis of HGSOC.MethodsSix datasets(GSE14001,GSE18520,GSE26712,GSE27651,GSE40595,and GSE54388)that met the inclusion criteria were downloaded from the GEO database.Differentially expressed genes(DEGs)between HGSOC and normal ovarian surface epithelium samples were screened via integrated analysis.Functional enrichment analysis were performed on DEGs through DAVID,including GO functional annotation and KEGG pathway enrichment analysis.After using STRING to build a protein-protein interaction(PPI)network,the PPI network was visually analyzed through Cytoscape to identify hub genes and the functional modules in the PPI network.Then the functional enrichment analysis were performed on the key module 1.The Kaplan-Meier Plotter database was utilized to evaluate the prognostic value of these ten hub genes in HGSOC.The expression level of these hub genes was not only confirmed by the Oncomine database,but also validated by qRT-PCR and Western Blot using clinical samples.Results1.This study successfully screened out 103 DEGs,which included 28 upregulated DEGs and 75 downregulated DEGs;2.Enrichment analysis showed that the up-regulated DEGs were mainly related to cell proliferation and cell division,while the down-regulated DEGs were mainly enriched in Wnt signaling pathway and multiple metabolism-related pathway;3.After building the PPI network,ten hub genes were identified一EPCAM,ALDH1A1,ZWINT,BUB1B,NEK2,DLGAP5,MELK,CEP55,CKS2,and KDR,only ALDH1A1 and KDR were downregulated in HGSOC;4.Seven hub genes were partitioned into the key module 1,which was significantly enriched in DNA replication and cell cycle pathways;5.Survival analysis showed that expression of MELK,CEP55,and KDR were significantly correlated with overall survival of HGSOC patients.ConclusionBased on the results of bioinformatic analysis,MELK,CEP55,and KDR might become promising biomarkers for diagnosis and prognostic evaluation of HGSOC.Part Ⅱ MT1G promotes the development of serous ovarian cancer through PI3K/AKT pathwayObjectiveOvarian cancer has the highest mortality rate among gynecological malignancies.Serous ovarian cancer is the most common type of ovarian cancer.Metallothionein(MT)is a cysteine-rich protein with low molecular weight that plays important roles in maintaining metal ion homeostasis,detoxification of heavy metals,and anti-oxidative stress.MT family include at least 11 functional members,which can be divided into four isoforms:MT1(MT1 A,MT1B,MT1E,MT1F,MT1G,MT1H,MT1M,and MT1X),MT2(MT2A),MT3,and MT4.More and more studies has shown that MTs were closely related to carcinogenesis,drug resistance and prognosis.This study aimed to determine the expression level and prognostic value of each MT isoform in serous ovarian cancer,to explore the biological functions and potential molecular mechanisms of MT1G in the development of serous ovarian cancer,and to provide new insights for targeted treatment of ovarian cancer.MethodsMT mRNA expression of various MT isoforms in serous ovarian cancer and normal ovarian tissues was detected using qRT-PCR.MT protein expression was detected inserous ovarian cancer,normal ovarian tissues,and normal fallopian tube fimbria by Western Blot and immunohistochemistry.Based on the clinical samples and TCGA database,the correlation of MT expression with clinicopathological characteristics in serous ovarian cancer was analyzed.Differential expression of MT isoforms between serous ovarian cancer and normal ovarian tissues was validated using Oncomine database.Combined with qRT-PCR results,MT 1G was selected as the subject of this study for its significant differential expression.The effects of MT1G on ovarian cancer cell growth and proliferation were determined through plate clone formation experiment,CCK-8 and EdU cell proliferation assays.The roles of MT1G in cell cycle distribution and apoptotic rate were measured using flow cytometry assays.The effects of MT1G on the cell migration ability were examined by cell scratch test and Transwell assay.Western Blot was used to detect the changes in the expression of downstream target genes of PI3K/AKT signaling pathway when MT1G expression was regulated.Furthermore,the role of MT1G overexpression on tumor growth was validated through in vivo experiments.Kaplan-Meier Plotter database was used to analyze the effects of MT expression on the overall survival and progression-free survival of patients with serous ovarian cancer.Results1.mRNA expression levels of MTIA,MT1F,MT1G and MT2A in serous ovarian cancer tissues were significantly higher than normal ovarian tissues.Oncomine database analysis showed that MT1G expression was the highest in serous ovarian cancer and the difference was also the most significant.2.MT protein expression in serous ovarian cancer tissue was significantly higher than normal ovarian tissues and normal fallopian tube fimbria.The immunohistochemical localization of MT was mainly in the cell cytoplasm.3.Clinicopathological characteristics of serous ovarian cancer:The median age at the time of diagnosis was over 50 years old.The majority of patients presented with stage Ⅲon initial consultation.More than 90%of patients had high-grade serous ovarian cancer.The rate of lymph node metastasis of serous ovarian cancer was high.Most patients with serous ovarian cancer could not achieve RO resection at primary surgery.4.Expression of MT in high-grade serous ovarian cancer tissues was significantly higher than in low-grade serous ovarian cancer tissue.5.MT1G overexpression enhanced cancer cell proliferation,clonal formation and migration,while MT1G knockdown had the opposite effects.Flow cytometry assays showed that overexpression of MT1G could promote the G1/S phase transition to accelerate the cell cycle and inhibit the apoptosis of serous ovarian cancer cells.6.PI3K/AKT signaling pathway could be activated by MT1G overexpression and inhibited after interfering with MT1G expression,which indicated that MT1G might affect the development of serous ovarian cancer by regulating the PI3K/AKT signaling pathway.7.In vivo experiments showed that the volume and weight of subcutaneous tumors in the MT1G overexpression group were significantly greater than those in the control group;similarly,the tumor growth rate in the MT1G overexpression group was also considerably faster when compared with the control group.MT1G overexpression group had greater tumor burden compared to the control group when cancer cells were intraperitoneally injected.8.High expression of MT1M and MT2A significantly shortened overall survival and progression-free survival of patients with serous ovarian cancer.In addition,high expression of MT1E,MT1F,MT1G,MT1H,MT3 and MT4 also had the same effect on the progression-free survival of patients.ConclusionMTs were mostly highly expressed in serous ovarian cancer.MT1G might promote the development of serous ovarian cancer by regulating the PI3K/AKT signaling pathway.High expression of MTs might predict poor prognosis of patients with serous ovarian cancer.MT might be used as a potential therapeutic target for ovarian cancer.Novelty of this study1.This study aimed to demonstrate the roles and mechanism of MT in the development of serous ovarian cancer.It comprehensively analyzed the expression level and prognostic value of various MT isoforms in serous ovarian cancer for the first time,and discussed the effects of MT1G on biological behavior of serous ovarian cancer cell2.This study reported that MT1G might participate in the progression of serous ovarian cancer via regulating the PI3K/AKT pathway for the first time,providing a new insight for targeted therapy of ovarian cancer.Limitations of this study1.MT isoforms had different expression in various cancer types.For example,MT expression in hepatocellular carcinoma and thyroid carcinoma was demonstrated to be downregulated because of promoter methylation.However,this study had not yet clarified the mechanism of the aberrant expression of MT in serous ovarian cancer.2.Previous studies had shown that MT was involved in the acquisition of chemotherapy resistance.Hence,the relationship between MT and chemotherapy resistance still needs to be discussed in the future.