circRNA-NDUFA10抑制膀胱癌血管新生的机制研究  

circRNA-NDUFA10inhibits angiogenesis of bladder cancer

作　　者：艾乐 冯梓贤 阴艺娜 苏民 李志花[1] AI Le;FENG Zi-xian;YIN Yi-na;SU Min;LI Zhi-hua(Department of Medical Oncology,Sun Yat-sen Memorial Hospital,Guangzhou510120,China;Department of Hematology&Oncology,Lianjiang People's Hospital,Lianjiang524400,China)

机构地区：[1]中山大学孙逸仙纪念医院肿瘤科,广东广州510120 [2]廉江市人民医院肿瘤血液科,广东廉江524400

出　　处：《中华肿瘤防治杂志》2022年第23期1668-1675,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省自然科学基金(2021A1515010215)。

摘　　要：目的揭示circNDUFA10(has_circ_0001118)在膀胱癌组织中的表达情况,并进一步探究其是否通过调控第10号染色体同源缺失性磷酸酶-张力蛋白基因(PTEN)/蛋白激酶B(Akt)/人血管内皮生长因子A(VEGF-A)信号通路抑制膀胱癌血管新生。方法收集2016-08-02-2018-07-26于中山大学孙逸仙纪念医院行根治性膀胱切除术的膀胱癌患者癌组织和配对癌旁正常组织标本62例,qRT-PCR检测circNDUFA10在临床组织标本及人正常膀胱上皮永生化细胞(SV-HUC-1)和人膀胱癌细胞系(T24、UM-UC-3和5637)的表达。选取T24和UM-UC-3细胞进行siRNA转染以沉默circNDUFA10的表达,分别获得si-NC组、si-circNDUFA10#1组和si-circNDUFA10#2组。成管实验和Transwell实验分别检测各组细胞成管长度及细胞迁移情况。RNA pull-down实验检测miR-20b-5p与circNDUFA10的结合情况。选取T24和UM-UC-3细胞进行inhibitor和siRNA转染,分别获得NC inhibitor组、miR-20b-5p inhibitor组和miR-20b-5p inhibitor+si-circNDUFA10组。qRT-PCR检测miR-20b-5p对PTEN表达的影响。蛋白质印迹法检测circNDUFA10沉默后PTEN及其下游相关蛋白(Akt、p-Akt)的表达。qRT-PCR和ELISA实验检测circNDUFA10沉默后VEGF-A的表达。结果circNDUFA10在膀胱癌组织中的相对表达量(-2.450±0.192)低于癌旁正常组织(0±0.212),差异具有统计学意义,t=8.576,P<0.001。circNDUFA10在膀胱癌细胞株T24(0.332±0.017)、UM-UC-3(0.359±0.021)和5637(0.422±0.024)中的相对表达量低于正常膀胱细胞SV-HUC-1(1.000±0.060),差异具有统计学意义,F=80.605,P<0.001。circNDUFA10沉默后,与T24si-NC组(1.000±0.026)和UM-UC-3si-NC组(1.000±0.023)相比,T24 si-circNDUFA10#1组(3.265±0.100),si-circNDUFA10#2组(3.227±0.068)及UM-UC-3si-circNDUFA10#1组(2.812±0.089)和si-circNDUFA10#2组(2.760±0.056)膀胱癌细胞诱导HUVECs的相对成管长度均升高,差异具有统计学意义,F值分别为326.820和275.671,均P<0.001。与此一致,Transwell实验表明,circNDUFA10沉默后膀胱癌�Objective To identify the expression of circNDUFA10(has_circ_0001118)in bladder cancer(BCa),and explore the underlying mechanisms in inhibiting angiogenesis of BCa by regulating phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/vascular endothelial growth factor A(VEGF-A)signaling pathway.Methods Totally 62pairs of tumor tissues and normal adjacent tissues(NAT)were collected from BCa patients who underwent surgery at Sun Yat-sen Memorial Hospital from 2016-8-2to 2018-7-26.The expression of circNDUFA10in tissue samples and SV-HUC-1,T24,UM-UC-3,5637cells were analyzed by qRT-PCR.T24and UM-UC-3cells were transfected with siRNA to silence siRNA,and grouped as followed:si-NC,si-circNDUFA10#1,si-circNDUFA10#2,respectively.The effect of circNDUFA10on angiogenesis were performed by tuber formation assays and Transwell assays.The relationship of circNDUFA10and miR-20b-5p were detected by RNA pull-down assays.T24and UM-UC-3cells were transfected with inhibitor and siRNA,and grouped as followed:NC inhibitor,miR-20b-5p inhibitor and miR-20b-5p inhibitor+si-circNDUFA10,respectively.The effect of miR-20b-5p on the expression of PTEN were identified by qRT-PCR.The protein expression of PTEN,Akt and p-Akt were detected by Western blot.qRT-PCR and ELISA assays were employed to detected the expression of VEGF-A induced by circNDUFA10silencing.Results The relative expression level of circ-NDUFA10in BCa tissues(-2.450±0.192)was lower than that in paired NAT(0±0.212),t=8.576,P<0.001.The relative expression level of circNDUFA10in SV-HUC-1(1.000±0.060)was higher than that in T24(0.332±0.017),UM-UC-3(0.359±0.021)and 5637(0.422±0.024),F=80.605,P<0.001.Compared with T24si-NC group(1.000±0.026)and UM-UC-3si-NC group(1.000±0.023),the relative tube formation length of HUVECs induced by BCa cells in T24si-circNDUFA10#1group(3.265±0.100),si-circNDUFA10#2group(3.227±0.068)and UM-UC-3si-circ-NDUFA10#1group(2.812±0.089),si-circNDUFA10#2group(2.760±0.056)were higher after silencing circNDUFA10,Fvalues

关 键 词：膀胱肿瘤 环状RNA 血管新生 PTEN基因 血管内皮生长因子A 

分 类 号：R737.14[医药卫生—肿瘤]