摘要
目的研究表明环状RNA在许多生物学过程及疾病中发挥了重要的功能。文中旨在研究由抗氧化基因SOD2转录而来的环状RNA hsacirc0004662的基本特征和功能预测。方法通过PCR扩增和一代测序验证hsacirc0004662反向剪接位点,根据核糖核酸酶R(RNase R)处理分为RNase R组和对照组(ddH2O),细胞质细胞核RNA分离和定量PCR及蛋白翻译的潜能分析研究hsacirc0004662的基本特征。利用生物信息学方法预测hsacirc0004662上微小RNA(microRNA,miRNA)的结合位点、AGO2结合位点以及miRNA的靶基因,并构建circRNA-miRNA-mRNA网络,随后对miRNA的靶基因进行功能富集分析以及信号通路富集分析。结果序列分析显示,hsacirc0004662箭头左端为SOD2基因4号外显子末端序列,右侧为2号外显子的起始序列。环状RNA CDR1as、hsacirc0004662在细胞质中的表达量显著高于细胞核。RNase R组的环状RNA hsacirc0004662、CDR1as的表达与对照组比较差异无统计学意义(P>0.05),而GAPDH基因的线性mRNA的表达则明显降低(P<0.05)。hsacirc0004662开放阅读框编码的多肽含149个氨基酸,起始密码子始于第456位核苷酸,终止密码子位于第443位核苷酸。hsacirc0004662含10个AGO2结合位点。GO分析结果显示,hsa-miR-224和hsa-miR-532的靶基因分别在12个生物学过程条目和13个分子功能条目中显著富集;其中最显著富集的生物学过程条目包括神经冲动传递、行为、蛋白氨基酸磷酸化;最显著的分子功能条目包括蛋白激酶活性、转录因子结合、蛋白丝氨酸/苏氨酸激酶活性。KEGG通路分析结果显示,该靶基因合集在Wnt信号通路和泛素介导的蛋白降解信号通路显著富集。结论抗氧化基因SOD2的新型环状RNA hsacirc0004662的反向剪切位点、RNA酶的抵抗性和主要分布在细胞质等基本特征,从蛋白翻译角度和circRNA-miRNA-mRNA角度为hsacirc0004662的功能研究提供了新的方向。
Objective The feature and function prediction of circular RNA hsa_circ_0004662 transcribed from antioxidant gene SOD2 were investigated in this study.Methods We have studied the feature of hsa_circ_0004662 through PCR amplification,Sanger sequencing,RNase R treatment,isolation and quantification of the cytoplasmic and nuclear RNA and analysis of the proteincoding ability.Additionally,bioinformatic methods were used to predict the microRNA(miRNA)binding sites,the AGO2 binding sites and the miRNA target genes of hsa_circ_0004662.The circRNAmiRNA-mRNA regulatory network was constructed.Subsequently,Gene Oncology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis on the miRNA target genes were performed.Results Hsa_circ_0004662 was generated through backsplicing of exon 4 to exon 2 of SOD2 gene.Hsa_circ_0004662 was resistant to RNase R treatment,and was predominantly localized in the cytoplasm.A putative open reading frame(ORF)was found in hsa_circ_0004662 that encoded a 149 aa protein.Hsa_circ_0004662 functioned as hsa-miR-224 or hsa-miR-532 sponge.Target genes of hsa-mir-224 and hsa-mir-532 were significantly enriched in neuromuscular biological processes,Wnt signaling pathway and ubiquitin mediated proteplysis signaling pathway.Conclusion Our study successfully characterize the novel circRNA hsa_circ_0004662 derived from antioxidant gene SOD2,and provides a novel insight into functional research of hsa_circ_0004662 from protein-coding and circRNAmiRNA-mRNA view.Moreover,this study expands a novel research field of SOD2 gene.
作者
董志杰
周新科
潘劲辉
林铭珍
林海
梁敏
姚文霞
DONG Zhi-jie;ZHOU Xin-ke;PAN Jin-hui;LIN Ming-zhen;LIN Hai;LIANG Min;YAO Wen-xia(Department of Centeral Laboratory,the Fifth Affiliated Hospital of Guangzhou Medical University,Guangzhou 510700,Guangdong,China)
出处
《医学研究生学报》
CAS
北大核心
2020年第10期1014-1020,共7页
Journal of Medical Postgraduates
基金
国家自然科学基金(31500142,81673206)。
