【作者】 李军；

【导师】 宁宏；

【作者基本信息】 中国医科大学 ， 眼科学， 2023， 博士

【摘要】 目的:脉络膜黑色素瘤（Choroidal melanoma,CM）是成年人最常见的原发性眼内恶性肿瘤,极易发生转移,常累及肝脏、肺、骨骼等多处器官,即使经过早期积极治疗和严密随访,仍然有大约50%的CM患者死于肿瘤转移^[1],不仅导致患者视功能的丧失,更严重威胁患者的生命。至今CM的转移机制尚不清楚,因此,深入研究CM转移过程中的相关分子机制对于疾病的理解以及发掘新的治疗靶点具有重要意义。N6-甲基腺嘌呤（N6-methyladenosine,m6A）是真核生物中最普遍的RNA修饰形式之一,该修饰过程在甲基转移酶、去甲基化酶和阅读器蛋白三者共同参与下保持相对稳定。m6A甲基化修饰过程的紊乱参与多种癌症的发生发展,在肿瘤的机制研究中备受关注^[2]。科学家发现m6A甲基化修饰在CM中发挥着复杂且重要的作用。IGF2BP2作为经典的m6A阅读器蛋白,可以通过结合m6A修饰甲基对目的RNA进行翻译、编辑、稳定性等方面的调节^[3]。研究发现IGF2BP2在众多肿瘤中表达上调且对肿瘤的发生发展起到重要作用。但其在CM中的表达和功能尚无报道。E2F家族包括E2F1-E2F8,是知名的转录因子家族,其对于维持基因组完整性、调控细胞增殖、分化和凋亡等多种生理过程具有重要意义。其中E2F2不仅影响细胞分裂、增殖,还对于血管生成、细胞外基质重塑以及肿瘤细胞迁移、侵袭发挥作用。近年来E2F2在肿瘤研究领域备受关注,但在CM中的表达与功能尚无报道。经过生物信息学分析预测,E2F2在CM中表达量高于正常脉络膜组织,且高表达的E2F2在CM中与更差的生存预后相关。通过检索公共数据库及生物信息网站,发现E2F2作为转录因子与HSPA8基因启动子区存在特异性结合位点,可以促进HSPA8转录,二者在CM中呈可靠正相关,HSPA8作为E2F2可能有意义的下游因子,同时也是热休克蛋白家族的一员,在多种肿瘤中表达升高,并参与MAPK（RAF/MEK/ERK）等多种信号通路的激活。由此可见,E2F2在CM中发挥着复杂且重要的作用,有望成为CM新的治疗靶点。本课题组的前期研究中对CM细胞所进行的RNA转录组测序中发现E2F2 m RNA受到m6A甲基化修饰,该结果提示我们E2F2功能的实现与阅读器蛋白的调控相关。在m6A甲基化修饰的背景下探究E2F2、IGF2BP2在CM中的表达、功能以及二者的调控关系,并深入了解E2F2对CM转移的作用机制,在补充CM中m6A甲基化修饰研究的同时,旨在为该肿瘤探寻新的治疗靶点。方法:1、检索TCGA公共数据库并结合生物信息学分析预测IGF2BP2在CM不同临床分期及组织病理类型中的表达情况以及在IGF2BP2不同表达量下,患者生存预后的差异性。收集自2000年1月至2022年12月之间于我院确诊为CM并行眼球摘除手术而获得的患眼组织标本及因眼外伤或眼球萎缩等非肿瘤性疾病而行眼内容去除术的眼脉络膜组织标本,分别通过免疫印迹实验、免疫组织化学技术检测IGF2BP2蛋白在CM组织及正常脉络膜组织中的表达情况。对CM细胞分别成功进行沉默和过表达IGF2BP2后,进行体外细胞实验探究CM细胞迁移、侵袭能力的变化,同时通过免疫印迹实验分别检测沉默和过表达IGF2BP2后CM细胞中上皮间质转化（Epithelial-mesenchymal transition,EMT）相关蛋白质表达水平的变化。将成功稳定过表达IGF2BP2的CM细胞通过尾静脉注射在裸鼠体内建立肺转移模型观察肿瘤细胞在裸鼠体内转移能力的改变。2、检索TCGA公共数据库及UALCAN生物信息网站,预测E2F2在CM中不同临床分期和组织病理类型的表达情况,以及不同E2F2 m RNA转录水平下,患者生存预后的差异性。收集自2000年1月至2022年12月之间于我院确诊为CM并行眼球摘除术而取得的患眼组织标本及因外伤或眼球萎缩等非肿瘤性疾病而行眼内容去除术而获得的眼脉络膜组织标本,利用免疫印迹实验及免疫组织化学技术检测E2F2蛋白质在CM组织及正常脉络膜组织中的表达情况。分别进行沉默和过表达E2F2后,体外细胞实验探究CM细胞迁移、侵袭能力的变化,通过免疫印迹实验检测沉默及过表达E2F2后CM细胞中EMT相关蛋白质的表达情况。体内实验探究过表达E2F2后CM细胞在裸鼠体内转移能力的改变。3、生物信息网站GEPIA2.0分析预测IGF2BP2与E2F2转录本表达水平的相关性。在CM细胞中通过转染si RNA沉默IGF2BP2后,使用q RT-PCR检测E2F2m RNA转录水平的变化,利用免疫印迹实验检测E2F2蛋白水平表达量的变化。使用转录抑制剂放线菌素D按相同时间梯度处理CM细胞并通过q RT-PCR实验检测E2F2m RNA降解速率的变化。通过利用生物信息网站Starbase2.0对I GF2BP2与E2F2m RNA进行结合预测,使用RNA免疫共沉淀技术（RNA Imm unoprecipitation,RIP）检测IGF2BP2蛋白质与E2F2m RNA结合情况。通过对C M细胞先行过表达IGF2BP2,而后进行E2F2的过表达,进行功能回复实验,使用免疫印迹实验检测回复实验中E2F2蛋白表达的变化,同时通过Transwell实验检测回复实验中CM细胞迁移、侵袭能力的变化。4、通过检索生物信息数据库,寻找到E2F2可能下游靶基因HSPA8,在CM细胞系中分别沉默和过表达E2F2后,利用免疫印迹实验检测其下游靶基因HSP A8蛋白含量的变化,利用在线数据库Ch IPBase V3.0预测E2F2与HSPA8基因启动子区转录结合位点,应用染色质免疫共沉淀（Chromatin-Immunoprecipitatio n,Ch IP）技术验证E2F2蛋白与HSPA8启动子区DNA片段特异性结合。通过免疫印记实验检测HSPA8在CM和正常脉络膜组织的蛋白表达情况,并通过生物信息网站分析预测不同HSPA8转录本表达水平的患者生存预后的差异性。在CM细胞中通过转染si RNA沉默HSPA8,并使用Transwell实验检测CM细胞迁移和侵袭能力的变化。通过生物信息学分析及免疫印迹实验,分别检测E2F2和HSPA8与MAPK（RAF/MEK/ERK）通路标志蛋白的相关性。并利用功能回复实验,验证E2F2通过调控HSPA8转录而激活RAF/MEK/ERK通路。结果:1、在我们收集的CM组织中IGF2BP2表达量明显低于正常脉络膜组织。IGF2BP2表达水平升高可以抑制CM细胞的迁移与侵袭,而IGF2BP2表达水平下降可以促进CM细胞的迁移与侵袭。稳定过表达IGF2BP2的CM细胞与阴性对照组相比在裸鼠体内转移率下降。2、E2F2蛋白在我们所收集的CM组织中表达明显上调。E2F2表达水升高可以促进CM细胞的迁移与侵袭,而E2F2表达水平降低可以抑制CM细胞的迁移与侵袭。稳定过表达E2F2的CM细胞与阴性对照组相比在裸鼠体内转移率升高。3、生物信息网站GEPIA2.0提示CM中IGF2BP2与E2F2在RNA水平呈负相关。生物信息网站Starbase2.0预测IGF2BP2蛋白与E2F2m RNA在3’UTR区存在特异性结合。在CM中敲减IGF2BP2后,E2F2在RNA和蛋白质水平表达量均升高。RNA免疫共沉淀实验验证IGF2BP2蛋白与E2F2 m RNA存在直接结合。放线菌素D法检测m RNA稳定性实验证实,在CM细胞中敲减IGF2BP2后,E2F2m RNA降解效率明显低于未敲减组,反之,过表达IGF2BP2后,E2F2m RNA降解效率明显升高。回复实验证明,在CM细胞中过表达E2F2可部分回复过表达IGF2BP2引起的肿瘤细胞迁移和侵袭能力下降。4、通过生物信息数据库预测到HSPA8是E2F2的下游基因。在HSPA8 DNA启动子区存在E2F2的特异性结合位点,利用TCGA公共数据库检测二者在RNA水平存在明显正相关,免疫印迹实验验证沉默与过表达E2F2可以引起HSPA8蛋白水平表达量的下降与升高。Ch IP实验结果显示E2F2与HSPA8 DNA启动子区结合,可转录调控HSPA8的表达。GEPIA2.0生物信息网站分析显示,HSPA8表达量高者患者生存期更短。通过免疫印迹法检测HSPA8在CM组织中的蛋白表达量高于正常脉络膜组织。在体外细胞实验中,沉默HSPA8的蛋白表达可以使CM细胞迁移、侵袭能力下降。通过GEPIA2.0网站分析提示E2F2和HSPA8均与MAPK信号通路标志蛋白基因RAF1、MEK1及ERK2高度相关,免疫印迹实验验证E2F2表达升高,p MEK1/2、MEK1/2、p MEK1、p ERK1/2、ERK1/2及ERK2蛋白表达量升高。STRING生物信息网站预测HSPA8蛋白与RAF1蛋白存在直接结合。结论:1、IGF2BP2在CM中呈现低表达,IGF2BP2表达量的下降与患者不良预后密切相关,IGF2BP2可以抑制CM的转移。2、E2F2在CM中高表达并且与其发生、进展以及患者的不良预后相关。E2F2可以促进CM细胞的迁移和侵袭。3、在CM中IGF2BP2蛋白作为RNA结合蛋白与E2F2m RNA相互结合并且促进E2F2m RNA降解,降低其稳定性。4、E2F2可以通过转录调控调节HSPA8的表达,HSPA8通过与RAF1蛋白结合而激活RAF/MEK/ERK通路,从而促进CM的迁移和侵袭。而HSPA8在CM组织中表达升高,与患者不良预后相关。更多还原

【Abstract】 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.更多还原