| Objective: Choroidal melanoma(CM)is the most common primary intraocular malignant tumor in adults,which is prone to metastasis and often affects multiple organs such as the liver,lungs,and bones.Even with early active treatment and followup,about 50% of CM patients still die from tumor metastasis [1],which not only leads to the loss of visual function but also a serious threat to the patient’s life.The metastasis mechanism of CM is still unclear,therefore,in-depth study of the relevant molecular mechanisms during CM transfer is of great significance for understanding the disease and exploring new therapeutic targets.N6 methyladenine(m6A)is one of the most common forms of RNA modification in eukaryotes,and this modification process remains relatively stable with the participation of methyltransferase,demethylase,and reader protein.The disorder of m6 A methylation modification process is involved in the occurrence and development of various cancers,and has attracted much attention in the study of tumor mechanisms.Scientists have discovered that m6 A methylation modification plays a complex and important role in CM.IGF2BP2,as a classic m6 A reader protein,can regulate the translation,editing,and stability of target RNA by binding to m6 A modified methyl groups.Research has found that IGF2BP2 is upregulated in many tumors and plays an important role in the occurrence and development of tumors.However,its expression and function in CM have not been reported yet.The E2 F family,including E2F1-E2F8,is a well-known transcription factor family that plays an important role in maintaining genomic integrity,regulating various physiological processes such as cell proliferation,differentiation,and apoptosis.E2F2 not only affects cell division and proliferation,but also plays a role in angiogenesis,extracellular matrix remodeling,and tumor cell migration and invasion.In recent years,E2F2 has received much attention in the field of tumor research,but its expression and function in CM have not been reported yet.According to bioinformatics analysis,it is predicted that the expression level of E2F2 in CM is higher than that in normal choroidal tissue,and high expression of E2F2 is associated with poorer survival prognosis in CM.Through searching public databases and bioinformatics websites,it was found that E2F2,as a transcription factor,has a specific binding site with the HSPA8 gene promoter region,which can promote HSPA8 transcription.The two are reliably positively correlated in CM.HSPA8,as a potentially significant downstream factor of E2F2 and a member of the heat shock protein family,is upregulated in various tumors and participates in the activation of various signaling pathways such as MAPK(RAF/MEK/ERK).It can be seen that E2F2 plays a complex and important role in CM,and is expected to become a new therapeutic target for CM.In the preliminary research of our research group,RNA transcriptome sequencing was performed on CM cells,and it was found that E2F2 m RNA was methylated with m6 A.This result suggests that the implementation of E2F2 function is related to the regulation of reader proteins.Exploring the expression,function,and regulatory relationship of E2F2 and IGF2BP2 in CM under the background of m6 A methylation modification,and gaining a deeper understanding of the mechanism of E2F2 on CM metastasis.While supplementing the research on m6 A methylation modification in CM,the aim is to explore new therapeutic targets for this tumor.Methods:1.Retrieve the TCGA public database and combine bioinformatics analysis to predict the expression of IGF2BP2 in different clinical stages and histopathological types of CM,as well as the differences in patient survival prognosis under different levels of IGF2BP2 expression.Collect eye tissue samples obtained from patients diagnosed with CM and undergoing enucleation surgery in our hospital between January 2000 and December 2022,as well as choroidal tissue samples obtained from patients undergoing eye content removal surgery due to non tumor diseases such as eye trauma or eye atrophy.The expression of IGF2BP2 protein in CM tissue and normal choroidal tissue was detected through immunoblotting and immunohistochemical techniques,respectively.After successfully silencing and overexpressing IGF2BP2 on CM cells,in vitro cell experiments were conducted to investigate the changes in migration and invasion ability of CM cells.At the same time,immunoblotting experiments were used to detect the changes in the expression levels of epithelial mesenchymal transition(EMT)related proteins in CM cells after silencing and overexpressing IGF2BP2.CM cells that successfully overexpressed IGF2BP2 were injected into the tail vein to establish a lung metastasis model in nude mice,and the changes in tumor cell metastasis ability in nude mice were observed.2.Retrieve the TCGA public database and UALCAN bioinformatics website to predict the expression of E2F2 in different clinical stages and histopathological types of CM,as well as the differences in patient survival prognosis at different E2F2 m RNA transcription levels.Collect eye tissue samples obtained from January 2000 to December 2022 after being diagnosed with CM and undergoing enucleation surgery in our hospital,as well as choroidal tissue samples obtained from eye content removal surgery due to non tumor diseases such as trauma or eye atrophy.Use immunoblotting experiments and immunohistochemical techniques to detect the expression of E2F2 protein in CM tissue and normal choroidal tissue.After silencing and overexpression of E2F2,in vitro cell experiments were conducted to investigate the changes in migration and invasion ability of CM cells.Immunoblotting experiments were used to detect the expression of EMT related proteins in CM cells after silencing and overexpression of E2F2.In vivo experiments were conducted to investigate the changes in the metastatic ability of CM cells in nude mice after overexpression of E2F2.3.The bioinformatics website GEPIA2.0 analyzes and predicts the correlation between IGF2BP2 and E2F2 transcript expression levels.After silencing IGF2BP2 by si RNA transfection in CM cells,q RT PCR was used to detect changes in E2F2 m RNA transcription levels,and immunoblotting was used to detect changes in E2F2 protein expression levels.CM cells were treated with transcriptional inhibitor actinomycin D at the same time gradient and the change in E2F2 m RNA degradation rate was detected through q RT PCR assay.By using the bioinformatics website Starbase2.0 to predict the binding of IGF2BP2 and E2F2 m RNA,and using RNA immunoprecipitation(RIP)technology to detect the binding of IGF2BP2 protein to E2F2 m RNA.By overexpressing IGF2BP2 on CM cells and then overexpressing E2F2,functional recovery experiments were conducted.Immunoblotting was used to detect changes in E2F2 protein expression during the recovery experiment,while Transwell experiments were used to detect changes in CM cell migration and invasion ability during the recovery experiment.4.By searching the bioinformatics database,the potential downstream target gene HSPA8 of E2F2 was identified.After silencing and overexpressing E2F2 in CM cell lines,the protein content of the downstream target gene HSPA8 was detected using immunoblotting experiments.The transcription binding sites of E2F2 and HSPA8 gene promoter regions were predicted using the online database Ch IPBase V3.0,Application of chromatin immunoprecipitation(Ch IP)technology to verify the specific binding of E2F2 protein to DNA fragments in the HSPA8 promoter region.Detect the protein expression of HSPA8 in CM and normal choroidal tissue through immunoblotting experiments,and predict the differences in survival prognosis of patients with different levels of HSPA8 transcript expression through bioinformatics website analysis.Silencing HSPA8 in CM cells by transfecting si RNA and detecting changes in migration and invasion ability of CM cells using Transwell assay.By bioinformatics analysis and immunoblotting experiments,the correlation between E2F2 and HSPA8 with MAPK(RAF/MEK/ERK)pathway marker proteins was detected.And use functional recovery experiments to verify that E2F2 activates the RAF/MEK/ERK pathway by regulating HSPA8 transcription.Results:1.The expression level of IGF2BP2 in the CM tissues we collected was significantly lower than that in normal choroidal tissue.Elevated expression levels of IGF2BP2 can inhibit the migration and invasion of CM cells,while decreased expression levels of IGF2BP2 can promote the migration and invasion of CM cells.The metastasis rate of CM cells stably overexpressing IGF2BP2 decreased in nude mice compared to the negative control group.2.The expression of E2F2 protein was significantly upregulated in the CM tissue collected.Elevated expression of E2F2 can promote the migration and invasion of CM cells,while decreased expression of E2F2 can inhibit the migration and invasion of CM cells.Compared with the negative control group,CM cells with stable overexpression of E2F2 showed an increased metastasis rate in nude mice.3.The bioinformatics website GEPIA2.0 suggests a negative correlation between IGF2BP2 and E2F2 at the RNA level in CM.The bioinformatics website Starbase 2.0predicts a specific binding between IGF2BP2 protein and E2F2 m RNA in the 3’UTR region.After knocking down IGF2BP2 in CM,the expression level of E2F2 increased at both RNA and protein levels.RNA immunoprecipitation experiments confirmed the direct binding between IGF2BP2 protein and E2F2 m RNA.The stability test of m RNA using actinomycin D method confirmed that after knocking down IGF2BP2 in CM cells,the degradation efficiency of E2F2 m RNA was significantly lower than that of the non knockdown group.Conversely,overexpression of IGF2BP2 significantly increased the degradation efficiency of E2F2 m RNA.Experimental results have shown that overexpression of E2F2 in CM cells can partially restore the decreased migration and invasion ability of tumor cells caused by overexpression of IGF2BP2.4.HSPA8 was predicted to be a downstream gene of E2F2 through bioinformatics databases.There is a specific binding site for E2F2 in the HSPA8 DNA promoter region.The TCGA public database was used to detect a significant positive correlation between the two at the RNA level.Immunoblotting experiments confirmed that silencing and overexpression of E2F2 can cause a decrease and increase in the expression level of HSPA8 protein.The Ch IP experiment results showed that E2F2 binds to the HSPA8 DNA promoter region and can transcriptionally regulate the expression of HSPA8.GEPIA2.0 bioinformatics website analysis shows that patients with high HSPA8 expression have a shorter survival period.The protein expression of HSPA8 in CM tissue was detected to be higher than that in normal choroidal tissue by immunoblotting.In vitro cell experiments,silencing the expression of HSPA8 protein can reduce the migration and invasion ability of CM cells.Through analysis on the GEPIA2.0 website,it was found that E2F2 and HSPA8 are highly correlated with MAPK signaling pathway marker protein genes RAF1,MEK1,and ERK2.Immunoblotting experiments confirmed that E2F2 expression is increased,and the expression levels of p MEK1/2,MEK1/2,p MEK1,p ERK1/2,ERK1/2,and ERK2 proteins are also increased.STRING Bioinformatics website predicts direct binding between HSPA8 protein and RAF1 protein.Conclusions:1.IGF2BP2 shows low expression in CM,and the decrease in IGF2BP2 expression is closely related to poor prognosis in patients.IGF2BP2 can inhibit the metastasis of CM.2.E2F2 is highly expressed in CM and is associated with its occurrence,progression,and poor prognosis in patients.E2F2 can promote the migration and invasion of CM.3.In CM,IGF2BP2 protein acts as an RNA binding protein and binds to E2F2 m RNA,promoting E2F2 m RNA degradation and reducing its stability.4.E2F2 can regulate the expression of HSPA8 through transcriptional regulation.HSPA8 activates the RAF/MEK/ERK pathway by binding to RAF1 protein,thereby promoting the migration and invasion of CM.The expression of HSPA8 is elevated in CM tissue,which is associated with poor prognosis in patients. |
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