CDR1as在前列腺癌中表达及作用机制探索

Expression and mechanism of CDR1as in prostate cancer

目的探索CDR1as在前列腺癌中的作用及机制。方法选取34例前列腺癌患者术后标本的癌及癌旁配对组织样本进行高通量测序筛选出的差异表达circRNA-CDR1as作为研究对象,另选取17例前列腺癌患者术后标本的癌及癌旁配对组织样本和3种前列腺癌相关细胞系进行RT-qPCR验证CDR1as表达量,利用3种CDR1as的siRNA干扰处理PC3细胞并进行高通量测序,选取与组织测序结果中差异表达mRNA变化趋势一致的mRNA作为被CDR1as调控的下游靶基因,采用Pearson相关性分析组织测序结果中与CDR1as表达变化存在相关性的基因,利用癌症基因组图谱(TCGA)数据库中的临床数据信息,分析差异表达的基因对前列腺癌预后的影响,采用miRanda软件预测CDR1as与miRNA的结合位点,通过circinteractome网站预测CDR1as是否与蛋白质存在结合位点,利用ORF Finder网站及IRES网站对CDR1as是否具有翻译蛋白质潜能进行分析。结果CDR1as在癌组织中的表达量低于癌旁组织,差异具有统计学意义(P<0.05)。相比于前列腺正常上皮细胞系RWPE1,CDR1as在前列腺癌细胞系PC3中高表达,在LNCap中低表达(P<0.05);共筛选出7个显著上调的mRNA和37个显著下调的mRNA作为在前列腺癌发生、发展中被CDR1as调控的下游靶基因;共有19个基因在前列腺癌患者肿瘤组织中与CDR1as表达变化存在相关性,其中12个基因在癌与癌旁组织中表达差异变化与CDR1as表达差异变化存在相关性,低表达的GSTM5与前列腺癌不良预后有关;经相关数据库预测,CDR1as与68个miRNA存在结合位点,CDR1as反向剪接位点与IGF2BP2存在结合位点,其侧翼序列与FUS、IGF2BP2、IGF2BP3存在结合位点,CDR1as具有14个开放阅读框及8个内部核糖体进入位点。结论低表达的CDR1as可能促进前列腺癌的发生及发展,其主要机制可能是通过吸附miRNA下调具有抑制肿瘤进展的GSTM5等下游靶基因,也可能通过减弱对IGF2BP3等靶蛋白的作用或减少翻译蛋白而发挥功能,其具体机制有待进一步研究。

Objective To explore the role and mechanism of CDR1as in prostate cancer.Methods CDR1as was selected as the target by high-throughput sequencing from 34 pairs of prostate cancer tissues and corresponding paracancerous tissues.Another 17 pairs of prostate cancer tissues and corresponding paracancerous tissues and three prostate cancer-related cell lines were selected to verify the expression of CDR1as by RT-qPCR,differentially expressed mRNAs and CDR1as were detected in PC3 cells treated with 3 differerent siRNAs by high-throughput sequencing.MRNAs with consistent expression trends were selected as the downstream target genes of CDR1as.Pearson correlation analysis was used to analyze the relationship between CDR1as and differentially expressed mRNAs from the sequencing results,and prognosis analysis was used to predict if these above differentially expressed mRNAs were related to poor prognosis of prostate cancer using the TCGA database.MiRanda software was used to predict the binding sites of CDR1as and miRNA.The circinteractome website was used to predict whether CDR1as had binding sites with proteins,and the ORF Finder website and IRES website were used to predict whether CDR1as had the potential to be translated into proteins.Results The expression of CDR1as in cancer tissues was lower than that in paracancerous tissues,which was statistically significant(P<0.05).Compared with the normal prostate epithelial cell line RWPE1,CDR1as was highly expressed in PC3 cell line,and lower expressed in LNCap cell(P<0.05).A total of 7 significantly up-regulated mRNA and 37 significantly down-regulated mRNA were screened as candidate downstream target gene for CDR1as in the occurrence and development of prostate cancer.A total of 19 genes were associated with CDR1as expression in the cancer tissues of prostate cancer patients,and expression changes from cancer to paracancerous tissues were related between 12 genes and CDR1as.The low expression of GSTM5 meant poor prognosis of prostate cancer.CDR1as had binding sites with 68 miRNAs.The reverse splicing site of CDR1as had binding sites with IGF2BP2,and its flanking sequence had binding sites with FUS,IGF2BP2 and IGF2BP3.CDR1as had 14 ORFs and 8 IRES.Conclusions The low expression of CDR1as promotes the occurrence and development of the prostate cancer.The main mechanism may be through down-regulation of downstream target genes such as GSTM5 that inhibit cancer progression by miRNA sponse,or alleviating binding with target proteins such as IGF2BP3 or attenuating being translated into proteins.The specific mechanism remains to be further researched.