| Objective: Ovarian cancer is the gynecological malignant tumor with the highest mortality.Nearly 80% of patients were in advanced stage at the time of discovery,and the 5-year survival rate was only 30-40%.Recurrence and metastasis of ovarian cancer are difficult to treat.N6-methyladenosine(m6A methylation)is the most extensive and abundant modification of messenger RNA(mRNA).It participates in the occurrence and development of cancer by regulating cell proliferation,metastasis,immune response,etc.The methylation of m6 A is a dynamic and reversible process.Many cancers show the imbalance of m6A-related gene expression and the change of m6 A level.Therefore,the study of methylation and demethylation of m6 A will help to further clarify the pathogenesis of ovarian cancer and provide evidence for finding effective therapeutic targets.FTO(Fat mass and obesity-associated protein)belongs to non-heme Fe2+/ α-The alk B protein family of ketoglutarate-dependent oxygenase is the first m6 A demethylase discovered by humans,which plays a role in promoting or inhibiting cancer in different cancers.The function and mechanism of FTO in ovarian cancer are not completely clear.Epithelial-mesenchymal transition(EMT)is an important driving factor of tumor malignant biological behavior,and is closely related to tumor cell metastasis.Recent studies have found that m6 A can affect tumor progression by regulating EMT.Therefore,this study aims to explore the role of m6 A demethylase FTO in ovarian cancer,as well as its effect on EMT of ovarian cancer cells and its molecular mechanism.Methods: 1.M6 A dot blot array was used to detect the level of m6 A in ovarian cancer tissue and normal ovarian tissue.TCGA combined with GTEX database,CPTAC database,qRT-PCR and Western blot were used to analyze the expression level of FTO in ovarian cancer tissue and normal ovarian tissue.The expression of FTO in normal ovarian tissue,ovarian benign tumor,ovarian borderline tumor and epithelial ovarian cancer was detected by immunohistochemistry,to analyze the relationship between the expression level of FTO and the clinicopathologic parameters of ovarian cancer patients.2.After constructing ovarian cancer cells with knockdown FTO expression,over-expression of FTO,and over-expression of m6 A demethylase activity functional site mutation FTO,the knockdown and over-expression efficiency were detected by qRT-PCR,and the level of m6 A in ovarian cancer cells was detected by m6 A dot blot.CCK-8 experiment,EdU experiment,Transwell invasion experiment and wound healing experiment were used to detect the effect of FTO expression level and its enzyme activity on the proliferation,invasion and migration of ovarian cancer cells,and Western blot experiment was used to study the effect of FTO expression level on epithelial-mesenchymal transition related indicators.The effect of overexpression of FTO on the growth rate,size and weight of ovarian cancer cells transplanted subcutaneously in nude mice was studied through the subcutaneous tumorigenesis experiment in female nude mice.The effect of FTO on the metastasis of ovarian cancer cells in vivo was studied by constructing a female nude mouse peritoneal metastasis model.3.Western blot was used to detect the expression of several key EMT enzymes in ovarian cancer cells of the FTO overexpression group and the control group,and identified that the expression of SNAI1 was regulated by FTO.The effects of overexpression of wild-type and demethylase site mutant FTO on SNAI1 mRNA expression were detected by qRT-PCR.Me RIP experiment and qRT-PCR were used to identify whether there was a m6 A modification site on SNAI1 mRNA,and the change of m6 A modification level on SNAI1 mRNA after overexpression of FTO.4.After actinomycin D(ACD)treatment,the effect of overexpression of FTO on the stability of SNAI1 mRNA was detected by qRT-PCR.IGF2BP1/2/3 knockdown ovarian cancer cells were constructed,and the level of SNAI1 mRNA was detected by qRT-PCR.After actinomycin D(ACD)treatment,qRT-PCR was used to detect the effect of IGF2BP2 knockdown on the stability of SNAI1 mRNA,and the effect of IGF2BP2 knockdown on the stability of SNAI1 mRNA after knockdown and overexpression of FTO.RIP test and qRT-PCR were used to prove the binding of IGF2BP2 protein to SNAI1 mRNA,and the change of SNAI1 mRNA level of IGF2BP2 binding after FTO overexpression was detected.We leveraged the RMBase website(http://rna.sysu.edu.cn/rmbase/)to query the m6 A site on human SNAI1 mRNA that may bind to IGF2BP2.RIP assay and qRT-PCR were used to identify the major m6 A binding sites of IGF2BP2 and SNAI1 mRNA in ovarian cancer cells.5.Interfere with the expression of IGF2BP2 and SNAI1 in ovarian cancer cells with low FTO knockdown to conduct the rescue test,detect the protein expression level of FTO,IGF2BP2,SNAI1 and EMT-related indicators by Western blot,and detect the proliferation,invasion and migration ability of ovarian cancer cells by CCK-8 test,EdU test,Transwell invasion test and wound healing test.Results:Part 1: The results of m6 A dot blot showed that compared with normal ovarian tissue,the mRNA level of epithelial ovarian cancer tissue increased.The results of bioinformatics analysis,qRT-PCR and Western blot showed that FTO was down-regulated in epithelial ovarian cancer.The immunohistochemical results showed that the expression level of FTO in ovarian cancer tissue was significantly lower than that in normal ovarian tissue,while the expression of FTO in benign ovarian tumor and borderline ovarian tumor did not change significantly.Clinicopathological analysis showed that the low expression of FTO was related to the increase of FIGO stage and greater omentum metastasis in patients with epithelial ovarian cancer.Part 2 : After overexpression of FTO,the level of m6 A in ovarian cancer cells decreased,the ability of proliferation,invasion and migration of ovarian cancer cells significantly decreased,EMT weakened,the growth rate,size and weight of subcutaneous ovarian cancer cell transplanted tumor in nude mice reduced,and the metastasis of ovarian cancer cells in the abdominal cavity of nude mice decreased.However,overexpressing the functional site mutation of FTO demethylase activity has no significant effect on the m6 A level of ovarian cancer cells and the proliferation,invasion and migration ability of ovarian cancer cells.Knock down FTO can increase the level of m6 A in ovarian cancer cells,significantly enhance the ability of proliferation,invasion and migration,and promote EMT.Part 3 : Western blot test detected the protein expression of several major EMT-related transcription factors after overexpression of FTO,and found that the protein expression of SNAI1 in FTO overexpression group decreased.qRT-PCR results showed that the expression of SNAI1 mRNA in ovarian cancer cells with FTO overexpression was significantly reduced,while the expression of SNAI1 in the cells with demethylase site mutation FTO overexpression was not significantly changed.The results of Me RIP experiment indicated that there was m6 A modification on SNAI1 mRNA of ovarian cancer cells,and the level of m6 A modification of SNAI1 decreased after overexpression of FTO.The actinomycin D(ACD)experiment showed that the expression curve of SNAI1 mRNA in the FTO overexpression group moved down and the half-life decreased significantly compared with the control group,indicating that the stability of SNAI1 mRNA was reduced by FTO overexpression.The result of qRT-PCR showed the change of SNAI1 mRNA after IGF2BP1/2/3 knockdown,and the result showed that the change of SNAI1 mRNA expression was the most obvious after IGF2BP2 knockdown.The ACD experiment showed that the stability of SNAI1 mRNA decreased after IGF2BP2 knockdown.In addition,the low expression of IGF2BP2 in FTO-knockout cells can reduce the mRNA stability of SNAI1.In contrast,knockdown of IGF2BP2 in FTO overexpression cells did not change the mRNA stability of SNAI1.Next,RIP experiment and qRT-PCR results showed that IGF2BP2 protein was bound to SNAI1 RNA.After overexpression of FTO,the level of SNAI1 mRNA bound by IGF2BP2 decreased.Through the query on the RMBase website,we found that the m6 A site on human SNAI1 mRNA that may bind to IGF2BP2 is located in the 3’UTR region of SNAI1 mRNA.The results of RIP test and qRT-PCR showed that IGF2BP2 was mainly bound to the m6 A site # 1(chr20;48604663)on SNAI1 mRNA.The rescue experiment of cell function test showed that compared with the negative control group,the migration,invasion,proliferation and cell viability of ovarian cancer cells with FTO knockdown were enhanced,and the knockdown of IGF2BP2 in ovarian cancer cells with FTO knockdown reduced the migration,invasion,proliferation and cell viability of cells.The knockdown of SNAI1 in ovarian cancer cells with FTO knockdown reversed the increase of migration,invasion,proliferation and cell viability.In addition,the results of Western blot showed that the knockdown of IGF2BP2 reversed the expression of SNAI1 and the enhancement of EMT induced by knockdown of FTO.Conclusion: 1.In epithelial ovarian cancer,the level of m6 A increased and the expression of FTO decreased,and the low expression of FTO was related to the increase of FIGO stage and greater omentum metastasis in patients with epithelial ovarian cancer.2.FTO inhibits the proliferation,invasion,migration,epithelial-mesenchymal transition of ovarian cancer cells in vitro,and the growth and intraperitoneal metastasis of ovarian cancer subcutaneously transplanted tumor in nude mice,and this effect depends on its m6 A demethylase activity.3.The down-regulation of FTO in ovarian cancer cells increased the level of m6 A on SNAI1 mRNA,promoted the combination of IGF2BP2 and SNAI1 mRNA,resulting in increased stability of SNAI1 mRNA,increased expression of SNAI1,and promoted EMT,growth and metastasis of ovarian cancer cells.FTO can inhibit the proliferation,invasion and migration of ovarian cancer by regulating the expression of SNAI1. |
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