肝癌中环状RNA的表达模式及其编码潜能的研究

Research on the expression pattern and coding potential of circRNAs in hepatocellular carcinoma

目的:环状RNA作为一类特殊的非编码RNA,近些年多有报道其可以通过编码多肽的方式参与多种恶性肿瘤的演变和进展,然而其在肝癌中的研究是有限的。本研究基于RNA-seq结合在线数据库,探讨肝癌中环状RNA的表达模式并评估其编码潜能。方法:应用Novaseq 6000 PE150高通量测序仪对3对肝癌与癌旁组织行circRNA测序。生物信息学分析寻找差异性表达的circRNA并行序列分析,应用GO、KEGG和Reactome分别进行功能、通路富集分析;联合在线数据库Transcirc和Ribocirc筛选可翻译的circRNA;应用IRES finder识别IRES元件;ORFfinder识别ORF序列;m6A SRAMP识别m6A位点序列;CPAT法评估编码潜能。qPCR检测上述寻找到的差异性表达circRNA表达情况。结果:本研究共发现了416个丰度相对可观的circRNA,包括35个上调和31个下调的circRNA(均P<0.05),序列分析提示这些基因多以小分子量(长度<1 000 nt)、外显子-外显子类型及(2-5)外显子个数构成为主。GO分析显示这些表达差异性的circRNA主要富集在GTP酶活性的调节(P<0.001)、小GTP酶介导的信号转导的调节(P<0.001)、GTP酶活性阳性调节(P<0.001)(生物学过程),核散斑(P=0.016)(细胞成分),GTP酶调节因子活性(P<0.001)、GTP酶激活因子活性(P<0.001)(分子功能)等;KEGG分析显示这些表达差异的circRNA主要富集在补体系统(P=0.002)、mRNA监测通路(P=0.003)和血小板激活(P=0.005)等;Reactome分析显示主要富集在RHO GTP酶周期(P=0.008)、RHOA GTP酶周期(P<0.001)和CDC42 GTP酶周期(P=0.001)等。进一步联合Transcirc和Ribocirc数据库交集获得17个circRNA,其中以hsa_circ_0000231、hsa_circ_0000417、 hsa_circ_0000745、 hsa_circ_0005455、 hsa_circ_0000847、 hsa_circ_0005552、 hsa_circ_0060849、hsa_circ_0008234、hsa_circ_0075796、hsa_circ_0001742及hsa_circ_0001686共11个基因编码潜能评分最显著且均大于0.9分。同时在10例肝癌和癌旁组织中qPCR验证提示:hsa_circ_0000231、hsa_circ_0000745、hsa_circ_0005552、和hsa_circ_0000847在肝癌组织中高表达,hsa_circ_0060849和hsa_circ_0008234呈低表达,差异具有意义(P<0.05)。结论:综合分析了肝癌中cicRNA的表达模式,发现了6个circRNA存在差异性表达且具有高的翻译潜能评分,值得进一步研究。

Objective:As a special class of non-coding RNAs,circular RNAs(circRNAs)have been frequently reported to participate in the evolution and progress of malignant tumors by encoding polypeptides.However,its functions in hepatocellular carcinoma(HCC)are rarely addressed in previous research.Based on the RNA-seq and online data,our study investigated the ex-pression pattern and coding potential of circRNAs in HCC.Methods:The circRNA sequencing procedure was performed on three pairs of HCC and adjacent tissues using a Novaseq 6000 PE150 high-throughput sequencer.Bioinformatics analysis was conducted to identify circRNAs that expressed differentially and reveal the sequence characteristics of these circRNAs.GO,KEGG,and Re-actome studies were performed to analyze the functions and pathway enrichment of the identified circRNAs.Translatable cir-cRNAs were screened through online databases such as Transcirc and Ribocirc.The IRES finder,ORF finder,and m6A SRAMP were used to predict IRES elements,ORF sequences,and m6A sites,respectively.The coding potential was evaluated following the CPAT method.The expression of differentially expressed circRNA was detected by qPCR.Results:A total of 416 circRNAs were found to be relatively abundant,of which 35 were up-regulated and 31 were down-regulated circRNAs(P<0.05 for all the circRNAs).Sequence analysis suggested that the majority of these circRNAs are of a small molecular weight(length<1000 nt)and the exon-exon type that contains 2-5 exons.GO analysis showed that differentially expressed circRNAs were enriched in the regulation of GTPase activity(P<0.001),the regulation of small GTPase mediated signal transduction(P<0.001),and the pos-itive regulation of GTPase activity(P<0.001)in the biological process;the nuclear speck(P=0.016)in the cellular component;the GTPase regulator activity(P<0.001),and the GTPase activator activity(P<0.001)in the molecular function,respectively.KEGG analysis indicated that the circRNAs were mainly enriched in the complement and coagulation cascades(P=0.002),the mRNA surveillance pathway(P=0.003),and the platelet activation(P=0.005),while Reactome analysis revealed that the RHO GTPase cycle(P=0.008),the RAC1 GTPase cycle(P<0.001),and the CDC42 GTPase cycle(P=0.001)were the main pathway columns where enrichment of the studied circRNAs appeared.Furthermore,a total of 17 circRNAs were obtained from the intersection of Transcirc and Ribocirc databases,of which 11 genes(hsa_circ_0000231,hsa_circ_0000417,hsa_circ_0000745,hsa_circ_0005455,hsa_circ_0000847,hsa_circ_0005552,hsa_circ_0060849,hsa_circ_0008234,hsa_circ_0075796,hsa_circ_0001742,and hsa_circ_0001686)had the most significant coding potential scores greater than 0.9.At the same time,we used qPCR to verify 10 cases of liver cancer and adjacent tissues,suggesting:hsa_circ_0000231,hsa_circ_0000745,hsa_circ_0005552,and hsa_circ_0000847 are highly expressed in liver cancer tissue,while hsa_circ_0060849 and hsa_circ_0008234 are low expressed,the difference was significant(P<0.05).Conclusion:We comprehensively analyzed the expression patterns of cicRNA in liver cancer,and found that 6 Circrnas with differential expression and high translation potential score were worthy of further study.