基于高通量测序技术的胰腺癌环状RNA差异表达谱分析

Analysis of differential expression profile of circRNA in pancreatic cancer based on high-throughput sequencing technology

目的筛查胰腺癌组织环状RNA(circRNA)差异表达谱并初步分析其潜在功能。方法随机选取4对胰腺癌组织和对应癌旁胰腺组织标本进行高通量测序,按表达量差异倍数>2且P<0.05为筛选标准,筛选差异表达的circRNA。qRT-PCR检测随机选取的5个差异表达的circRNA在20对胰腺癌组织标本中的表达,对测序结果进行验证。对差异表达的circRNA进行GO和KEGG富集分析,并构建top50 circRNA-microRNA互作网络图。进一步分析hsa_circ_0046523与胰腺癌临床病理特征的关系,并通过敲减试验观察其对胰腺癌细胞增殖、迁移和侵袭能力的影响。结果高通量测序共预测出17182个circRNA,在胰腺癌组织中差异表达的circRNA有302个,其中137个表达上调,165个表达下调。qRT-PCR结果显示,与癌旁组织相比,胰腺癌组织中has_circ_0046523、hsa_circ_0004220和hsa_circ_0000690表达水平上调(P<0.05),hsa_circ_0008676和hsa_circ_0004416表达水平下调(P<0.05),与测序结果一致。GO分析提示差异表达的circRNA主要参与底物依赖性细胞迁移、蛋白激酶复合物和细胞骨架蛋白结合等过程。KEGG通路富集分析显示差异表达的circRNA主要参与蛋白消化和吸收通路、ABC转运蛋白通路、癌症中心碳代谢通路以及甘氨酸、丝氨酸和苏氨酸代谢通路等。hsa_circ_0046523表达水平与胰腺癌肿瘤分化程度(P=0.002)、T分期(P=0.006)、淋巴结转移(P=0.011)及TNM分期(P=0.001)密切相关。与HPDE6-C7细胞相比,hsa_circ_0046523在胰腺癌细胞SW1990、Mia PaCa-2、BxPC-3和Capan-2中的表达水平升高(P<0.05)。下调hsa_circ_0046523的表达可以抑制胰腺癌细胞(Mia PaCa-2、BxPC-3)的增殖、迁移及侵袭能力(P<0.05)。结论胰腺癌组织中存在特征性差异表达的circRNA,这些差异表达的circRNA可能参与胰腺癌的发生发展。

Objective To screen the differential expression profile of circular RNA(circRNA)in pancreatic cancer and analyze its potential function.Methods Four pairs of pancreatic cancer tissues and corresponding adjacent pancreatic tissues were selected for high-throughput sequencing,and the differentially expressed circRNA was screened according to fold change>2 and P<0.05.qRT-PCR was used to detect the expression of 5 randomly selected differentially expressed circRNA in 20 pairs of pancreatic cancer tissue samples.GO and KEGG enrichment analysis of differentially expressed circRNA was performed,and the top 50 circRNA-microRNA interaction network was constructed.The relationship between hsa_circ_0046523 and the clinicopathological features of pancreatic cancer was further analyzed,and the effects of hsa_circ_0046523 on the proliferation,migration and invasion of pancreatic cancer cells were observed by functional experiments.Results A total of 17182 circRNA were predicted by high-throughput sequencing,of which 302 circRNA were differentially expressed including 137 circRNA were upregulated and 165 circRNA were downregulated in pancreatic cancer tissues.The qRT-PCR results showed that the expression levels of hsa_circ_0046523,hsa_circ_0004220 and hsa_circ_0000690 in pancreatic cancer tissues were significantly upregulated(P<0.05),while the expression levels of hsa_circ_0008676 and hsa_circ_0004416 were significantly downregulated(P<0.05),which was consistent with the sequencing results.GO analysis indicated that differentially expressed circRNA were mainly involved in substrate-dependent cell migration,protein kinase complex and cytoskeletal protein binding.KEGG pathway enrichment analysis showed that differentially expressed circRNA were mainly involved in protein digestion and absorption,ABC transporters,central carbon metabolism in cancer,and glycine,serine and threonine metabolism pathways.The expression level of hsa_circ_0046523 was closely correlated with tumor differentiation(P=0.002),T stage(P=0.006),lymph node metastasis(P=0.011)and TNM stage(P=0.001).Compared with HPDE6-C7 cells,the expression of hsa_circ_0046523 significantly increased in pancreatic cancer SW1990,Mia PaCa-2,BxPC-3 and Capan-2 cells(P<0.05).hsa_circ_0046523 knockdown significantly inhibited the proliferation,migration and invasion of pancreatic cancer Mia PaCa-2 BxPC-3 cells(P<0.05).Conclusion There are characteristic differentially expressed circRNAs in pancreatic cancer tissues,and these differentially expressed circRNAs may be involved in the occurrence and development of pancreatic cancer.