摘要
目的探讨ALKBH5在卵巢子宫内膜异位囊肿中的作用及其机制。方法收集2022-01-01-10-10青岛市中心医院5例行卵巢子宫内膜异位囊肿剥除术患者的囊肿组织和正常子宫内膜组织,通过蛋白质印迹实验和实时荧光定量聚合酶链反应(qRT-PCR)实验检测组织中ALKBH5蛋白和mRNA的表达,并在卵巢子宫内膜异位囊肿组织中分离培养子宫内膜间质细胞(EESCs)。应用转染小干扰RNA敲低ALKBH5的表达,通过细胞计数盒8(CCK8)、克隆形成实验和Transwell实验验证ALKBH5对EESCs增殖、迁移和侵袭能力的影响,通过脉管形成实验验证ALKBH5对血管生成能力的影响。通过检测谷胱甘肽(GSH)和铁离子含量验证ALKBH5对EESCs铁死亡的影响,通过透射电镜观察ALKBH5对细胞的亚细胞结构的影响,通过qRT-PCR实验和蛋白质印迹实验验证ALKBH5对BECN1表达的影响,通过RIP和MeRIP实验明确ALKBH5是否通过m6A去甲基化修饰调控BECN1的表达。采用Student t检验进行统计学分析。结果蛋白质印迹实验结果显示,ALKBH5蛋白在卵巢子宫内膜异位囊肿组织中的表达明显高于正常子宫内膜组织。qRT-PCR结果显示,ALKBH5 mRNA在正常子宫内膜组织中的相对表达量为1.000±0.058,在卵巢子宫内膜异位囊肿组织中的相对表达量为2.270±0.044,t=17.56,P<0.001。CCK8实验结果显示,在72 h时siNC组和siALKBH5组的光密度值分别为1.575±0.000和0.957±0.000,t=23.05,P<0.001。克隆形成实验结果显示,siNC组和siALKBH5组形成的克隆数分别为75.330±3.712和22.670±1.764,t=12.82,P<0.001。Transwell实验结果显示,siNC组和siALKBH5组迁移细胞数分别为196.700±8.819和77.000±5.859(t=11.30,P<0.001),侵袭细胞数分别为62.000±2.309和18.330±2.028(t=14.21,P<0.001)。脉管形成实验结果显示,siNC组和siALKBH5组形成的脉管数分别为73.330±4.667和19.330±1.764,t=10.82,P<0.001。GSH含量检测结果显示,siNC组和siALKBH5组GSH相对含量分别为1.067±0.088和0.430±0.025,t=6.942,P<0.001。铁离子含量检测结果显示,siNC组和siALKBH5组铁离子相对含量分别为1.000±0.058和2.773±0.059,t=21.43,P<0.001。透射电镜观察到敲低ALKBH5后细胞的线粒体和内质网结构破坏和溶解。蛋白质印迹实验结果显示,敲低ALKBH5显著促进BECN1蛋白的表达。qRT-PCR结果显示,siNC组和siALKBH5组BECN1 mRNA相对表达量分别为1.033±0.067和3.277±0.043,t=28.21,P<0.001。RNA免疫沉淀实验结果显示,IgG组和ALKBH5组BECN1相对富集量分别为1.073±0.101和79.980±3.695,t=21.35,P<0.001。甲基化RNA免疫沉淀实验结果显示,siNC组和siALKBH5组m6A+BECN1相对富集量分别为1.007±0.052和2.453±0.098,t=13.01,P<0.001。结论ALKBH5通过介导m6A去甲基化抑制BECN1的表达而抑制EESCs铁死亡,促进卵巢子宫内膜异位囊肿的进展。
Objective To explore the role and mechanism of ALKBH5 on ovarian endometriosis cysts.Methods The cyst tissue and normal endometrial tissue of 5 patients undergoing ovarian endometriosis cyst exfoliation from January 1,2022,to October 10,2022 at Qingdao Central Hospital were collected.The expression of ALKBH5 in mRNA and protein were detected by western blot and quantitative real-time polymerase chain reaction(qRT-PCR)assay.The ectopic endometriosis stromal cells(EESCs)were isolated and cultured.After knocking down ALKBH5 expression by transfecting small interfering RNA,the effects of ALKBH5 on the ability of proliferation,migration and invasion were verified by cell counting kit 8(CCK8),colony formation and transwell assays in EESCs.The effects of ALKBH5 on the ability of angiogenesis was verified by tube formation assay.Glutathione(GSH)and iron content were detected to verify the effect of ALKBH5 on ferroptosis in EESCs.The effect of ALKBH5 on subcellular structure of EESCs was observed by transmission electron microscopy.The effect of ALKBH5 on BECN1 expression was verified by qRT-PCR and western blot.RIP and MeRIP assays were used to verify whether ALKBH5 regulated BECN1 expression through m6A demethylation modification.The Student t test was used for statistical analysis.Results The results of western blot showed that the expression of ALKBH5 protein in ovarian endometriosis cysts tissue was significantly higher than that in normal endometrial tissue.The results of qRT-PCR showed that the relative expression level of ALKBH5 mRNA in normal endometrial tissue was 1.000±0.058,and that in ovarian endometriosis cysts tissue was 2.270±0.044,t=17.56,P<0.001.The results of CCK8 assay showed that the D(450 nm)value of siNC group and siALKBH5 group was 1.575±0.000 and 0.957±0.000,t=23.05,P<0.001.The results of colony formation assay showed that the number of colony formed in siNC group and siALKBH5 group were 75.330±3.712 and 22.670±1.764,t=12.82,P<0.001.The results of transwell assay showed that the number of migrated cells in siNC group and siALKBH5 group were 196.700±8.819 and 77.000±5.859(t=11.30,P<0.001).The number of invaded cells in siNC group and siALKBH5 group were 62.000±2.309 and 18.330±2.028(t=14.21,P<0.001).The results of tube formation assay showed that the number of tubes formed in siNC group and siALKBH5 group were 73.330±4.667 and 19.330±1.764,t=10.82,P<0.001.Additionally,the results of GSH content detection showed that the relative content of GSH in siNC group and siALKBH5 group were 1.067±0.088 and 0.430±0.025,t=6.942,P<0.001.The results of iron content detection showed that the relative content of iron in siNC group and siALKBH5 group were 1.000±0.058 and 2.773±0.059,t=21.43,P<0.001.The results of transmission electron microscopy showed that the mitochondrial and endoplasmic reticulum structures of siALKBH5 group were damaged and dissolved.The results of western blot showed that ALKBH5 knockdown promoted the expression of BECN1 protein.The results of qRT-PCR showed that the relative expression level of BECN1 mRNA in siNC group and siALKBH5 group were 1.033±0.067 and 3.277±0.043,t=28.21,P<0.001.The results of RIP assay showed that the relative enrichment of BECN1 in IgG group and ALKBH5 group were 1.073±0.101 and 79.980±3.695,t=21.35,P<0.001.The results of MeRIP assay showed that the relative enrichment of m6A+BECN1 in siNC group and siALKBH5 group were 1.007±0.052 and 2.453±0.098,t=13.01,P<0.001.Conclusion ALKBH5 inhibits the ferroptosis of EESCs by mediating m6A demethylation to inhibit BECN1 expression,and promotes the progression of ovarian endometriosis cysts.
作者
吕红梅
程亚玉
惠雅梦
程海燕
刘榕娟
马会清
田甜
LYU Hongmei;CHENG Yayu;HUI Yameng;CHENG Haiyan;LIU Rongjuan;MA Huiqing;TIAN Tian(Medical College,Qingdao University,Qingdzo,Shzndong 266071,Chinz;Department of Gynecology,Qingdao Central Hospital,University of Health and Rehabilitation Sciences(Qingdao Central Hospital),Qingdzo,Shzndong 266042,Chinz;Department of Gynecology,Affiliated Hospital of Qingdao University,Qingdzo,Shzndong 266061,Chinz)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2024年第15期926-932,共7页
Chinese Journal of Cancer Prevention and Treatment
基金
青岛市医药卫生科研指导项目(2022-WJZD188)
青岛市医药卫生科研计划(2021-WJZD089)
吴阶平医学基金会临床科研专项资助基金(320.6750.2021-10-21)。
作者简介
第一作者:吕红梅,女,山东青岛人,主要从事妇科的临床和基础研究工作。E-mail:339792781@qq.com;通信作者:田甜,女,山东青岛人,博士,硕士生导师,主要从事妇科肿瘤的临床和基础研究工作。E-mail:tiantian@qdu.edu.cn。
