摘要目的 探讨核内不均一核糖核蛋白(heterogeneous nuclear ribonucleoprotein,HnRNPL)在肾母细胞瘤中的表达及其与肿瘤细胞增殖、凋亡的关系.方法 对3例肾母细胞瘤组织进行全转录组测序了解肿瘤差异表达基因,对差异基因进行GO分析和KEGG分析,并用qPCR检测验证HnRNPL在肾母细胞瘤中的表达情况.采用siRNA干扰肿瘤细胞HnRNPL的表达,设置转染组(转染siRNA)、空白组(不转染siRNA)及阴性对照组(转染NC-siRNA),用qPCR及Western Blot检测干扰后HnRNPL、P53和BCL2基因及蛋白的表达情况,用MTT法和流式细胞术检测干扰后肿瘤细胞的增殖及凋亡情况.采用RNA免疫共沉淀检测HnRNPL蛋白与P53 mRNA的相互作用关系.结果 全转录组测序显示肾母细胞瘤组织中有748个基因差异表达.通过GO功能显著性富集分析发现:差异基因参与的生物进程主要是生化活动调节、细胞增殖、系统分化、RNA多聚酶Ⅱ转录调节等.通过KEGG显著性富集分析发现:差异基因参与的与肿瘤进程相关的信号通路主要是白细胞跨内皮迁移信号通路、p53信号通路、肿瘤转录调控异常等.qPCR检测证实HnRNPL在肾母细胞瘤组织中表达上调,与芯片测序结果一致.HnRNPL-siRNA干扰肿瘤细胞后,qPCR检测显示转染siRNA组的HnRNPL、p53和Bcl2mRNA相对表达量分别为0.348 5±0.019 7、0.248 5±0.035 5和0.372 2±0.014 2,阴性对照组分别为1.143 1±0.071 6、0.976 1±0.118 6和0.9060±0.032 3,空白对照组分别为1.1980±0.126 3、1.008 3±0.112 3和1.013 1±0.106 7,转染组与阴性对照组和空白对照组的组间差异有统计学意义(P＜0.05及P＜0.01).Western Blot检测显示siRNA转染组中HnRNPL、p53和Bcl2蛋白相对表达量分别为0.131 5±0.005 7、0.466 2±0.043 3和0.185 1±0.0231,阴性对照组分别为0.370 2±0.006 2、0.744 4±0.0206和0.8463±0.0020,空白对照组分别为0.399 9±0.0234、0.819 3±0.0201和0.993 5±0.058 7,转染组与阴性对照组及空白对照组比较,差异有统计学意义(P均＜0.05).MTT法检测显示siRNA转染组肿瘤细胞的生长曲线24、48和72 h的吸光度分别为0.457 5±0.061 2、0.552 7±0.033 2和0.662 5±0.005 7,阴性对照组分别为0.653 3±0.011 2、0.797 4±0.002 1和0.901 1±0.014 7,空白对照组分别为0.687 9±0.013 2、0.813 4±0.0231和0.913 1±0.071 2,转染组各个时间段与阴性对照组和空白对照组之间差异均有统计学意义(P均＜0.05).流式细胞术检测结果显示,siRNA转染组肿瘤细胞凋亡率为(37.57±2.30)％,远高于空白对照组(2.01±0.33)％及阴性对照组(3.46±0.41)％,组间比较,差异有统计学意义(P＜0.05).RIP检测显示,HnRNPL蛋白能与p53 mRNA相互作用.结论 HnRNPL可能通过与p53 mRNA特异性的结合调控P53的表达,并通过p53相关信号通路参与了肿瘤细胞的增殖与抗凋亡过程.

abstractsObjective To explore the mechanism of HnRNPL regulating cell proliferation and apoptosis in Wilms' tumor.Methods Next generation sequencing was employed for detecting differentially expressed genes between Wilms' tumor and adjacent non-tumor tissues.After RNA sequencing,gene ontology (GO) analysis was performed for detecting the biological functions of differentially expressed genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for examining the related signal pathways.The expression of HnRNPL was identified by quantitative polymerase chain reaction (qPCR).The cells were divided into transfection group with hnRNPL siRNA transfection,negative control group with NC siRNA transfection and blank control group without transfection.After silencing,the expressions of HnRNPL,p53 and bcl-2 in G401 cell line were detected by qPCR and Western blot.After silencing HnRNPL,cell proliferation and apoptosis were measured by thiazolyl blue tetrazolium bromide (MTT) assay and flow cytometry.And RNA-binding protein immuno-precipitation was employed for confirming a direct interaction of HnRNPL with p53 mRNA.Results There were a total of 748 differentially expressed genes in 3 paired samples,including 328 consistently up-regulated differentially expressed genes and 420 consistently down-regulated differentially expressed genes.GO analysis revealed that differentially expressed genes were involved in different biological processes,such as biochemical regulation,cell proliferation,system differentiation and RNA polymerase Ⅱ transcriptional regulation.And KEGG enrichment analysis indicated that differentially expressed genes were involved in different signaling pathways,such as leukocyte migration signaling pathways,p53 signaling pathways and tumor transcriptional regulation.Markedly down-regulated HnRNPL,p53 and Bcl2 mRNA were detected in transfection group (HnRNPL mRNA:0.348 5 ± 0.019 7;p53 mRNA:0.248 5 ± 0.035 5;Bcl2 mRNA:0.372 2 ± 0.014 2) as compared with negative control group (HnRNPL mRNA:1.143 1 ± 0.071 6;p53 mRNA:0.976 1 ± 0.118 6;Bcl2mRNA:0.906 0 ± 0.032 3,P＜0.05) and blank control group (HnRNPL mRNA:1.198 0 ± 0.126 3;p53 mRNA:1.008 3 ± 0.112 3;Bcl2 mRNA:1.013 1 ± 0.106 7,P＜0.01).HnRNPL,p53 and Bcl2 protein were obviously weaker in transfection group (HnRNPL:0.131 5± 0.005 7;p53:0.466 2 ± 0.043 3;Bcl2:0.185 1 ± 0.023 1) than those in negative control group (HnRNPL:0.370 2 ± 0.006 2;p53:0.744 4 ± 0.020 6;Bcl2:0.846 3 ± 0.002 0,P＜0.05) and blank control group (HnRNPL:0.399 9 ± 0.023 4;p53:0.819 3 ± 0.020 1;Bcl2:0.993 5 ± 0.058 7,P＜0.05).Silencing HnRNPL could inhibit cell growth in transfection group (24 h:0.457 5 ± 0.061 2;48 h:0.552 7 ± 0.033 2;72 h:0.662 5 ± 0.005 7) as compared with negative control group (24 h:0.653 3 ± 0.011 2;48 h:0.797 4 ± 0.002 1;72 h:0.901 1 ± 0.014 7,P＜0.05) and blank control group (24 h:0.687 9 ± 0.013 2;48 h:0.813 4 ± 0.023 1;72 h:0.913 1 ± 0.071 2,P ＜ 0.05).And silencing HnRNPL could accelerate cell apoptosis in transfection group (37.57％ ±2.39％ vs 2.01％ ±0.33％ and 3.46％ ±0.41％,P＜0.05).In RIP products,HnRNPL directly conjugated with p53 mRNA.Conclusions Acting as a p53 mRNA-binding protein,HnRNPL plays important roles in the proliferation and apoptosis of Wilms' tumor through p53 signaling pathway.