深度挖掘肝细胞癌中遗传变异对可选择性多聚腺苷酸化的影响

In-depth Mining of the Effect of Genetic Variation on Alternative Polyadenylation in Liver Hepatocellular Carcinoma

目的通过整合组学和临床信息,深度挖掘肝细胞癌中重要的可选择性多聚腺苷酸化(alternative polyadenylation,APA)数量性状基因位点(alternative polyadenylation quantitative trait locus,apaQTL),并研究这些重要位点相关的下游靶基因及上游RNA结合蛋白(RNA-binding protein,RBP)。方法从SNP2APA数据库下载泛癌顺式和反式apaQTL数据,用R语言分析肝细胞癌apaQTL的分布特征。根据位置提取apaQTL上下游50 bp范围内的序列,使用STREME进行特征基序富集,进一步结合肝细胞癌临床信息,筛选改变基序和临床预后相关的apaQTL。利用Tomtom挖掘潜在调控APA的上游RBP,并通过ENCODE以及TCGA预后数据分别验证RBP在APA调控和肝细胞癌发生发展中的重要性。将apaQTL和表达数量性状位点(expression quantitative trait locus,eQTL)进行共定位,并结合差异表达和预后分析,筛选影响表达和预后的apaQTL和下游靶基因。结果通过对肝细胞癌apaQTL进行特征分析,发现肝细胞癌顺式apaQTL在其他癌症中广泛出现,而反式apaQTL在肝细胞癌中具有特异性。通过对apaQTL进行基序分析,筛选出20个可以改变多聚腺苷酸化基序的apaQTL。其中rs16742可以生成新的多聚腺苷酸化基序,从而导致赖氨酰-tRNA合成酶(leucyl-tRNA synthetase,LARS)基因的转录本增长。进一步分析表明,rs16742与肝细胞癌的预后相关。通过预测新的apaQTL基序和上游靶基因,并结合外部数据验证,发现RBP聚(RC)结合蛋白2[poly(RC)binding protein 2,PCBP2]参与肝细胞癌APA事件的调控,并与预后相关。进一步分析表明,预后显著的apaQTL rs115865883和rs150628203可能影响PCBP2与转录本的结合。通过apaQTL和eQTL共定位分析,筛选出30个潜在的影响表达和肝细胞癌发生发展的apaQTL和下游靶基因。结论研究发现了一系列功能性肝细胞癌apaQTL及下游靶基因,基于apaQTL相关分析,发现RBP PCBP2可调控肝细胞癌的APA事件及预后。

Objective By integrating omics and clinical information,we explored the important quantitative trait loci(apaQTL)of alternative polyadenylation(APA)in liver hepatocellular carcinoma(LIHC),and investigated the downstream target genes and upstream RNA-binding protein(RBP)associated with these important loci.Methods The pan-cancer cis and trans-apaQTL data were downloaded from the SNP2APA database,and the distribution characteristics of LIHC apaQTL were analyzed by R language.Sequences in the upstream and downstream of apaQTL with a range of 50 bp were extracted according to the position,and characteristic motifs in these sequences were enriched by STREME.Combined with clinical information of LIHC,apaQTL that could not only change the motif but also be related to clinical prognosis was screened.Tomtom was used to explore upstream RBP potentially regulating APA,and the importance of RBP in the regulation of APA and the development of LIHC was verified by ENCODE and TCGA prognostic data,respectively.Finally,by integrating the data of apaQTL,expression quantitative trait locus(eQTL),expression and clinical survival times,we identified important apaQTLs which could not only affect APA,but also affect expression and prognosis.Results Through analyzing the characteristics of LIHC apaQTLs,we found that cis-apaQTLs of LIHC were widely found in other cancers,while most trans-apaQTLs showed specificity in LIHC.Through motif analysis of apaQTLs,20 apaQTLs that could change the motif of polyadenylation were screened out.Among them,rs16742 can generate new alternative polyadenylation motifs,which leads to the increase of leucyl-tRNA synthase(LARS)gene transcript.Further analysis showed that rs16742 was associated with the prognosis of LIHC.By predicting the novel apaQTL motif and upstream target genes,and combining with external data validation,we found that the RBP poly(RC)binding protein 2(PCBP2)was involved in the regulation of APA events in LIHC,and was associated with the prognosis of LIHC.Further analysis showed that the apaQTL rs115865883 and rs150628203 with significant prognosis may affect the binding of PCBP2 to the transcript.Finally,we screened 30 apaQTLs which could also affect expression and development of LIHC by apaQTL and eQTL co-location analysis.Conclusion A series of potential functional LIHC apaQTLs and downstream target genes have been identified.Based on apaQTL correlation analysis,RBP PCBP2 was found to regulate APA events and the prognosis of LIHC.