结直肠癌中miR-31的表达及预测靶基因的生物信息学分析

Micro RNA-31 Expression in Colorectal Cancer and Bioinformatic Analysis of Its Predicted Target Genes

目的检测micro RNA-31(mi R-31)在结直肠癌中的表达及功能,预测其靶基因并进行生物信息学分析,为研究其作用和调控机制奠定基础。方法采用实时荧光定量反转录聚合酶链反应(q RT-PCR)检测8种结肠癌细胞株、40例结直肠癌(colorectal cancer,CRC)患者癌组织及匹配的正常黏膜组织及33例结直肠腺瘤组织中mi R-31的表达情况,并分析癌组织中mi R-31表达与患者临床特征的关系。MTS法观察转染mi R-31模拟物(mimics)组、抑制剂(inhibitor)组和对照组(mi RControl)及空白细胞组细胞生长的差异,Western blot检测空白细胞组、mi R-31 mimics和inhibitor三组细胞PCNA蛋白的表达。Target Scan、DIANA-micro T、mi Randa等软件预测mi R-31的靶基因,并进行KEGG功能和信号通路富集分析。结果 mi R-31在8种结肠癌细胞株中高表达,同时CRC患者癌组织中的表达高于腺瘤及正常黏膜组织(P<0.05)。其中癌组织和匹配的正常黏膜相比,mi R-31表达明显上调(P=0.035),但腺瘤和正常黏膜相比,差异无统计学意义(t=0.122,P=0.904)。mi R-31的表达与临床病理特征间未见明显关系(P均>0.05)。转染mi R-31 mimics后,mi R-31的表达明显上调且和mi R-Control组及空白细胞组相比,mi R-31 mimics组细胞生长加快;而转染mi R-31 inhibitor组mi R-31表达显著降低,细胞生长活力明显受抑制。同时转染mi R-31 inhibitor组PCNA蛋白表达较mi R-31 mimics组和空白细胞组显著降低。生物信息学分析mi R-31靶基因功能集中于转录后及翻译水平的调节、细胞连接、迁移及细胞运动等生物学过程。信号通路主要富集于内吞作用、轴突导向、T细胞受体信号通路、Wnt及MAPK信号通路。结论 mi R-31在结直肠癌中高表达,且mi R-31可以促进细胞生长和增殖,其靶基因可能通过调节多种生物学过程发挥作用。

Objective To detect the expression and the function of micro RNA-31(mi R-31) in colorectal cancer(CRC) tissues, and to predict its target genes and conduct bioinformatic analysis, so as to investigate its role and the regulation mechanism. Methods mi R-31 expression were examined in 8 CRC cell lines, 40 CRC tissues and their matched normal tissues, and 33 adenoma tissues by quantitative reverse transcription PCR(q RT-PCR). The relationship between mi R-31 expression and clinicopathological information were analyzed. MTS method was used to detect the cell growth of HCT116 in different groups, including mi RControl, mi R-31 mimics, mi R-31 inhibitor and the blank groups. Western blot was used to detect the expression of proliferation cell nuclear antigen(PCNA) in mi R-31 mimic and mi R-31 inhibitor groups. Target genes of mi R-31 were predicted by the software including Target Scan, DIANA-micro T, mi Randa, et al, and further to analyze the information by KEGG, GO(gene ontology) and signaling pathway enrichment. ResultsThe relative expression levels of mi R-31 in 8 CRC cell lines and tumor tissues were much higher than those in adenoma and normal tissues(P<0.05), however, there was no significant difference between adenoma and the normal tissues(t=0.122, P=0.904). No correlation was found between mi R-31 expression and the clinicopathological variables of CRC(all P>0.05). After transfection with mi R-31 mimics, the expression level of mi R-31 was remarkably up-regulated while down-regulated in mi R-31 inhibitor group, compared with transfection with the mi R-Control. Consequently, the cell grew faster in mi R-31 mimics group, while the cell growth was inhibited in mi R-31 inhibitor group, compared with the blank group and the mi R-Control group(P<0.05). PCNA protein level in mi R-31 inhibitor group was much lower than those in mi R-31 mimics group and the blank group. The gene ontology analysis showed the function of predicted target genes of mi R-31 concentrated on the regulation of post transcription and translation regulation, cell junction, migration and motility, etc.. And the KEGG pathway analysis showed the target genes mainly involved in endocytosis, axon guidence, T cell receptor signaling pathway, Wnt and MAPK signaling pathway. Conclusion The expression level of mi R-31 is up-regulated in CRC tissues, and mi R-31 could promote cell growth and proliferation. The predicted target genes of mi R-31 provide some clues for CRC via regulating multiple biological processes.