摘要
目的:研究胃癌中环状RNA hsa_circ_0003071的基本特征、功能预测及生存预后分析。方法:通过PCR扩增验证胃癌与癌旁组织中hsa_circ_0003071的表达量,利用PCR扩增及一代测序验证hsa_circ_0003071反向剪接位点,利用核糖核酸酶R(RNase R)处理,细胞质细胞核RNA分离和定量PCR以及蛋白质翻译的潜能分析研究hsa_circ_0003071的基本特征。利用生信方法预测hsa_circ_0003071上微RNA(miRNA)的结合位点以及miRNA的靶基因,并构建circRNA-miRNA-mRNA网络,随后利用基因本体论(GO)及京都基因和基因组百科全书(KEGG)对miRNA的下游靶基因合集进行功能注释。结合生信方法获得hsa_circ_0003071调控的下游关键靶基因在胃癌的生存预后分析。结果:PCR实验证实胃癌中hsa_circ_0003071的表达量显著低于癌旁组织。CircBase注释显示hsa_circ_0003071是由基因SPATS2L转录本NM_001100422中第6号外显子经反向剪接构成,并且PCR扩增和一代测序共同验证了hsa_circ_0003071的反向剪接位点。hsa_circ_0003071可以抵抗RNase R处理,并且主要分布于细胞质。蛋白质翻译分析显示,hsa_circ_0003071具有翻译蛋白的潜能,其开放阅读框可能编码含124个氨基酸的蛋白质。生信预测显示hsa_circ_0003071具有多个miRNA结合位点,取数据库交集的两个miRNA(has-miR-604、has-miR-1205)预测其下游靶基因共148个,随之构建circRNA-miRNA-mRNA调控网络。GO分析结果显示,上述靶基因合集分别在12个生物学过程条目和2个分子功能条目中显著富集;KEGG通路分析结果显示,上述靶基因合集在mRNA监测通路和Hippo信号通路显著富集。结论:本研究分析了胃癌中新型环状RNA hsa_circ_0003071的表达量、反向剪接位点、RNase R酶的抵抗性和主要分布在细胞质等基本特征,从蛋白质翻译、circRNA-miRNA-mRNA调控网络和环状RNA下游调控关键基因在胃癌中的预后生存分析角度为hsa_circ_0003071的功能研究提供了新的方向。
Objective:To investigate the basic characteristics,predicted functions and prognostic role of the circular RNA hsa_circ_0003071 in gastric cancer.Methods:The expression levels of hsa_circ_0003071 in gastric cancer and the adjacent tissues were measured by PCR amplification.The hsa_circ_0003071 back splicing site was verified by PCR amplification and first-generation Sanger sequencing.Ribonuclease R(RNase R)digestion,cytoplasmic nuclear RNA isolation,quantitative PCR,and analysis of protein translational potential were performed for basic characterization of hsa_circ_0003071.Bioinformatics approach was used to predict the binding sites of microRNAs(miRNAs)on hsa_circ_0003071 and the target genes of these miRNAs.A circRNA-miRNA-mRNA network was then generated.Functional annotation of the downstream target gene set of these miRNAs was performed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).The survival and prognostic role of the key downstream target genes regulated by hsa_circ_0003071 in gastric cancer was determined bioinformatically.Results:PCR confirmed that the hsa_circ_0003071 expression level was significantly lower in gastric cancer than that in the adjacent tissues.CircBase annotation showed that hsa_circ_0003071 was formed by back-splicing of the exon 6 of NM_001100422,a SPATS2L transcript.The back-splicing site of hsa_circ_0003071 was confirmed by PCR amplification and the first-generation Sanger sequencing.hsa_circ_0003071 showed resistance to RNase R digestion and was mainly localized in cytoplasm.Protein translation analysis showed that hsa_circ_0003071 has protein translation potential,harboring an open reading frame that may encode a protein with 124 amino acids in length.Bioinformatics prediction showed that hsa_circ_0003071 has multiple miRNA binding sites.Based on two miRNAs from the intersection of databases(has-miR-604 and has-miR-1205),a total of 148 downstream target genes of hsa_circ_0003071 were predicted.A gene regulatory network of circRNA-miRNA-mRNA was generated accordingly.The GO analysis showed that these target genes were significantly enriched in 12 biological processes and 2 molecular functions.KEGG pathway analysis showed that these target genes were significantly enriched in mRNA surveillance pathway and Hippo signaling pathway.Conclusion:This study shed light on the basic characteristics of the novel circRNA hsa_circ_0003071 in gastric cancer,regarding the expression level,back splicing site,RNase R resistance,and its localization in cytoplasm.Our analyses from perspectives of protein translational protential,circRNA-miRNA-mRNA gene regulatory network and the survival and prognostic role of the key downstream target genes in gastric cancer offer new ideation for functional study of hsa_circ_0003071.
作者
黄伟
周新科
林铭珍
肖瑶
梁敏
姚文霞
Huang Wei;Zhou Xinke;Lin Mingzhen;Xiao Yao;Liang Min;Yao Wenxia(Department of Oncology,Fifth Affiliated Hospital of Guangzhou Medical University,Guangzhou Key Laboratory of Enhanced Recovery After Surgery in Abdominal Surgery,Guangzhou 510700,China)
出处
《广州医科大学学报》
2022年第3期21-31,共11页
Academic Journal of Guangzhou Medical University
基金
广州市加速康复腹部外科重点实验室项目(201905010004)。
