转录通读环状RNA rt-circ-HS促进低氧诱导因子1α表达和肾癌细胞增殖与侵袭  

Read-through circular RNA rt-circ-HS promotes hypoxia inducible factor 1αexpression and renal carcinoma cell proliferation,migration and invasiveness

作　　者：许云屹 苏征征[1] 郑林茂 张孟尼 谭珺娅 杨亚蓝[1] 张梦鑫 徐苗 陈铌[1,2] 陈雪芹[1,2] 周桥[1,2] XU Yun-yi;SU Zheng-zheng;ZHENG Lin-mao;ZHANG Meng-ni;TAN Jun-ya;YANG Ya-lan;ZHANG Meng-xin;XU Miao;CHEN Ni;CHEN Xue-qin;ZHOU Qiao(Department of Pathology,West China Hospital,Sichuan University,Chengdu 610041,China;Research Laboratory of Pathology,West China Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西医院病理科,成都610041 [2]四川大学华西医院病理研究室,成都610041 [3]四川大学华西公共卫生学院卫生毒理与病理系

出　　处：《北京大学学报（医学版）》2023年第2期217-227,共11页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81872108、82273073、81872107、82273047);四川大学专职博士后研发基金(2022SCU12022)。

摘　　要：目的:鉴定肾癌细胞中由染色体14q23上相邻基因低氧诱导因子1α(hypoxia inducible factor 1α,HIF1α)和小核RNA激活复合多肽(small nuclear RNA activating complex polypeptide 1,SNAPC1)形成的转录通读RNA及转录通读环状RNA(read-through circular RNA HIF1α-SNAPC1,rt-circ-HS),研究rt-circ-HS在肾癌细胞及组织样本中的表达、对肾癌细胞生物学行为的影响以及对其亲本分子HIF1α的调控机制。方法:逆转录PCR(reverse transcription-polymerase chain reaction,RT-PCR)和Sanger测序检测不同肿瘤细胞中由HIF1α-SNAPC1形成的转录通读RNA和rt-circ-HS的表达。构建不同类型的肾细胞癌(renal cell carcinoma,RCC)组织芯片共437例,用原位杂交检测rt-circ-HS表达。采用小干扰RNA(small interference RNA,si-RNA)和人工过表达质粒干预rt-circ-HS,用细胞计数实验(cell counting kit 8,CCK8)、EdU掺入实验、Transwell细胞迁移和细胞侵袭实验分别检测rt-circ-HS对肾癌细胞增殖、迁移和侵袭的影响。用RT-PCR和Western blot验证干预rt-circ-HS对亲本分子HIF1α和SNAPC1表达的影响。构建包含rt-circ-HS、HIF1α3′端非翻译区(3′untranslated region,3′UTR)与微小RNA 539(microRNA 539,miR-539)结合序列的野生型和突变型质粒,用双荧光素酶报告基因系统检测rt-circ-HS、HIF1α3′UTR与miR-539的结合。结果:发现一个新的rt-circ-HS,由HIF1α外显子(exon)6-SNAPC1 exon 2转录通读本产生,在肾癌细胞786-O中高表达。Sanger测序证实rt-circ-HS全长1144 nt,包括HIF1αexon 2-exon 6和SNAPC1 exon 2-exon 4,是一个新的转录通读环状RNA。原位杂交结果显示,rt-circ-HS在RCC中阳性表达率为67.5%(295/437),在不同类型RCC中表达率不同。发现miR-539是HIF1α的转录后负调控分子;rt-circ-HS作为分子海绵与miR-539结合,竞争性抑制miR-539与HIF1α3′UTR的结合,解除其对HIF1α的转录后负调控作用,促进亲本分子HIF1α表达及肾癌细胞增殖、迁移和侵袭。结论:rt-circ-HS作为分子Objective:To identify and characterize read-through RNAs and read-through circular RNAs(rt-circ-HS)derived from transcriptional read-through hypoxia inducible factor 1α(HIF1α)and small nuclear RNA activating complex polypeptide 1(SNAPC1)the two adjacent genes located on chromosome 14q23,in renal carcinoma cells and renal carcinoma tissues,and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α.Methods:Reverse transcription-polymerase chain reaction(RT-PCR)and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells.Tissue microarrays of 437 different types of renal cell carcinoma(RCC)were constructed,and chromogenic in situ hybridization(ISH)was used to investigate expression of rt-circ-HS in different RCC types.Small interference RNA(siRNA)and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation,migration and invasiveness by cell counting kit 8(CCK8),EdU incorporation and Transwell cell migration and invasion assays.RT-PCR and Western blot were used to exa-mine expression of HIF1αand SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA,respectively.The binding of rt-circ-HS with microRNA 539(miR-539),and miR-539 with HIF1α3′untranslated region(3′UTR),and the effects of these interactions were investigated by dual luciferase reporter gene assays.Results:We discovered a novel 1144 nt rt-circ-HS,which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1αexon 2-6 and SNAPC1 exon 2-4.Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells.ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5%(295/437),and the expression was different in different types of RCCs.Mechanistically,rt-circ-HS promoted renal carcinoma cell proliferation,migration and invasiveness by functioning as a competitive endogen

关 键 词：转录通读环状RNA HIF1α-SNAPC1 肾细胞癌 低氧诱导因子1Α 小核RNA激活复合多肽1 微小RNA 539 

分 类 号：R365[医药卫生—病理学]