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

doi:10.19723/j.issn.1671-167X.2023.02.004

摘要/Abstract

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、HIF1α 3′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、HIF1α 3′ 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1 144 nt，包括HIF1α exon 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HIF1α 3′ 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作为分子海绵结合miR-539，抑制其对HIF1α的负调控作用，促进亲本分子HIF1α表达及肾癌细胞增殖、迁移和侵袭。

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

Abstract:

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 HIF1α and 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 HIF1α 3′ untranslated region (3′ UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays. Results: We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 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 endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α. Conclusion: The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.

Key words: Read-through circular RNA HIF1α-SNAPC1, Renal cell carcinoma, Hypoxia inducible factor 1α, Small nuclear RNA activating complex polypeptide 1, microRNA 539

中图分类号:

R365

许云屹,苏征征,郑林茂,张孟尼,谭珺娅,杨亚蓝,张梦鑫,徐苗,陈铌,陈雪芹,周桥. 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[J]. 北京大学学报（医学版）, 2023, 55(2): 217-227.

Yun-yi XU,Zheng-zheng SU,Lin-mao ZHENG,Meng-ni ZHANG,Jun-ya TAN,Ya-lan YANG,Meng-xin ZHANG,Miao XU,Ni CHEN,Xue-qin CHEN,Qiao ZHOU. Read-through circular RNA rt-circ-HS promotes hypoxia inducible factor 1α expression and renal carcinoma cell proliferation, migration and invasiveness[J]. Journal of Peking University (Health Sciences), 2023, 55(2): 217-227.

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mRNA name	Primer sequence(5′-3′)	Length/bp
rt-circ-HS (divergent primer)	FP：TCACTTTACAGCAATGCCCA RP：TCCTCACACGCAAATAGCTGA	306
rt-circ-HS (convergent primer)	FP：GTGAACCCATTCCTCACCCA RP：AGCACCAACTCTGATCTGGA	209
rt-circ-HS full length (divergent primer)	FP：CACTTTACAGCAATGCCCA RP：TCAGCACCAAGCAGGTCATAGG	761
rt-circ-HS full length (convergent primer)	FP：GGTATAAGAAACCACCTATGAC RP：CACACGATCACTTGGGTCC	518
HIF1α	FP：GATGTAATGCTCCCCTCACC RP：ATTCATTGACCATATCACTATCCAC	295
SNAPC1	FP：CAGGAGACGGACAGTGTACG RP：TCGCCAAGCCAAAGCTAAAGC	138
BNIP3	FP：ACCAACAGGGCTTCTGAAAC RP：GAGGGTGGCCGTGCGC	202
VEGF	FP：AGGAGGGCAGAATCATCACG RP：GCACACAGGATGGCTTGAAGA	140
β-actin	FP：CTGGCACCACACCTTCTACAATG RP：CCTCGTAGATGGGCACAGTGTG	248
U6	FP: TGGAACGATACAGAGAAGATTAGCA RP：AACGCTTCACGAATTTGCGT TaqMan probe：FAM-CCCCTGCGCAAGGA-MGB	75
miR-539	FP: CGGCGGGAGAAATTATCCT RP：GTGCAGGGTCCGAGGT TaqMan probe：FAM-ACTGGATACGACACTCCACACCA-MGB	74

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