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  • 王璋,张丹,张朝霞,王钊颖,谭小军,卢虹旭,沈炼桔,龙春兰,魏光辉,何大维.Piwi互作RNA的筛选及其在儿童肾母细胞瘤组织中的表达[J].第二军医大学学报,2020,41(2):167-175    [点击复制]
  • WANG Zhang,ZHANG Dan,ZHANG Zhao-xia,WANG Zhao-ying,TAN Xiao-jun,LU Hong-xu,SHEN Lian-ju,LONG Chun-lan,WEI Guang-hui,HE Da-wei.Screening of Piwi-interacting RNAs and their expression in childhood nephroblastoma[J].Acad J Sec Mil Med Univ,2020,41(2):167-175   [点击复制]
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Piwi互作RNA的筛选及其在儿童肾母细胞瘤组织中的表达
王璋1,2,张丹1,2,张朝霞1,2,王钊颖1,2,谭小军1,3,卢虹旭1,3,沈炼桔1,2,4,龙春兰1,2,4,魏光辉3,4,5,6,何大维3,4,5,6*
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(1. 重庆医科大学附属儿童医院儿童泌尿生殖发育与组织工程重庆市重点实验室, 重庆 400014;
2. 重庆医科大学附属儿童医院儿科学重庆市重点实验室, 重庆 400014;
3. 重庆医科大学附属儿童医院泌尿外科, 重庆 400014;
4. 重庆医科大学附属儿童医院儿童发育疾病研究教育部重点实验室, 重庆 400014;
5. 重庆医科大学附属儿童医院儿童发育重大疾病国家国际科技合作基地, 重庆 400014;
6. 重庆医科大学附属儿童医院国家儿童健康与疾病临床医学研究中心, 重庆 400014
*通信作者)
摘要:
目的 探讨Piwi互作RNA(piRNA)在儿童肾母细胞瘤中的表达及临床意义。方法 对Piwi样蛋白2(Piwil2)基因重编程人成纤维细胞所形成的肿瘤样干细胞进行高通量测序,筛选出差异表达piRNA,并利用miRanda软件预测其靶基因,对靶基因进行基因本体(GO)功能分析。采用qRT-PCR检测差异表达piRNA及其靶基因在34例肾母细胞瘤患儿肿瘤组织及癌旁正常肾组织标本中的表达,并分析差异表达piRNA及其靶基因与肾母细胞瘤临床病理特征的关系。结果 通过高通量测序筛选出230个差异表达piRNA,利用miRanda软件预测出43个靶基因,经GO分析发现其参与的生物学过程主要为调节细胞钙离子浓度,分子功能主要为参与ATP酶的活动及多聚A尾RNA结合。选取5个未知的差异表达piRNA及其靶基因检测其在肾母细胞瘤肿瘤组织及癌旁正常组织中的表达情况,结果显示第13位未知上调piRNA(NU13)表达量在肿瘤组织中下调(P<0.01),其靶基因NOP56核糖核蛋白(NOP56)表达在肿瘤组织和癌旁正常组织中差异表达无统计学意义(P=0.58);第9位未知上调piRNA(NU9)表达量在肿瘤组织中下调(P<0.01),其靶基因40S核糖体蛋白S8(RPS8)基因在肿瘤组织和癌旁正常组织中差异表达无统计学意义(P=0.29);第5、7、9位未知下调piRNA(ND5、ND7、ND9)及其共同靶基因MT-RNR2样蛋白1(MTRNR2L1)基因的表达量在肿瘤组织中均下调(P均<0.01)。以上在儿童肾母细胞瘤组织中出现差异表达的piRNA及其靶基因与患儿年龄、性别、肿瘤临床分期、病理分型无明显相关性(P均>0.05)。结论 piRNA NU9、NU13、ND5、ND7、ND9及靶基因MTRNR2L1在儿童肾母细胞瘤组织中出现差异表达,有望成为鉴别肾母细胞瘤与正常肾组织的标志物。
关键词:  高通量测序  肿瘤干细胞  Piwi互作RNA  肾母细胞瘤
DOI:10.16781/j.0258-879x.2020.02.0167
投稿时间:2019-11-23修订日期:2020-02-10
基金项目:重庆市科学技术委员会项目(7000025).
Screening of Piwi-interacting RNAs and their expression in childhood nephroblastoma
WANG Zhang1,2,ZHANG Dan1,2,ZHANG Zhao-xia1,2,WANG Zhao-ying1,2,TAN Xiao-jun1,3,LU Hong-xu1,3,SHEN Lian-ju1,2,4,LONG Chun-lan1,2,4,WEI Guang-hui3,4,5,6,HE Da-wei3,4,5,6*
(1. Chongqing Key Laboratory of Child Urogenital Development and Tissue Engineering, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
2. Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
3. Department of Urology, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
4. Key Laboratory of Child Development and Disorders of Ministry of Education, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
5. China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, Chongqing 400014, China;
6. National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Chongqing 400014, China
*Corresponding author)
Abstract:
Objective To investigate the expression and clinical significance of P-element-induced wimpy testis (Piwi)-interacting RNAs (piRNAs) in childhood nephroblastoma. Methods The differential piRNAs of Piwil2-iCSC (P-element-induced wimpy testis like 2[Piwil2] reprogramming human fibroblast) were screened by high-throughput sequencing. Target genes were predicted by miRanda software and performed by gene ontology (GO) function analysis. Differential piRNAs and their target genes were detected by qRT-PCR in tumor tissues and normal kidney tissues of 34 children with nephroblastoma. The correlation of differential piRNAs and their target genes with clinicopathological characteristics of nephroblastoma was analyzed. Results A total of 230 differential piRNAs were screened out through high-throughput sequencing, and 43 target genes were predicted by miRanda. The GO analysis showed that the biological process of target genes was mainly involved in regulating the cytosolic calcium ion concentration and its molecular function was mainly involved in the activity of ATPase and poly (A) RNA binding. The expression of five unknown differentially expressed piRNAs and their target genes were detected in nephroblastoma and normal kidney tissues. The expression of NU13 (13th unknown upregulated piRNA) and NU9 (9th unknown upregulated piRNA) were significantly downregulated in tumor tissues (both P<0.01), while the expression of their target genes NOP56 ribonucleoprotein (NOP56, P=0.58) and 40S ribosomal protein S8 (RPS8, P=0.29) had no significant difference between tumor tissues and normal kidney tissues. The expression of ND5, ND7, ND9 (5th, 7th, 9th unknown downregulated piRNA) and their target gene MT-RNR2 like protein 1 (MTRNR2L1) were significantly downregulated in tumor tissues (all P<0.01). The above-mentioned piRNAs and their target genes had no significant correlation with age, gender, tumor stage, or pathological type of the children (all P>0.05). Conclusion piRNA NU9, NU13, ND5, ND7, ND9 and the target gene MTRNR2L1 are differentially expressed in children with nephroblastoma. They are expected to be markers to distinguish nephroblastoma from normal kidney tissues.
Key words:  high-throughput sequencing  cancer stem cells  Piwi-interacting RNA  nephroblastoma