Yang Guanghua, Zeng Chen, Liu Yang, Li Dongliang, Cui Juanjuan
First Department of Oncology, Zhumadian Central Hospital Zhumadian, Henan, China.
Department of Pediatric Oncology, Zhumadian Central Hospital Zhumadian, Henan, China.
Am J Transl Res. 2021 Jun 15;13(6):5866-5879. eCollection 2021.
ZNRD1-AS1 plays an important role in liver cancer, endometrial cancer and other diseases. However, the relationship between ZNRD1-AS1 and retinoblastoma has not been studied in detail. This study aimed to determine the role of ZNRD1-AS1 in retinoblastoma.
Differentially expressed genes in retinoblastoma downloaded from GEO database were identified by Limma package, and the expression and cell location of ZNRD1-AS1 were detected by real-time quantitative PCR (RT-qPCR). The relationships between miR-128-3p and two genes (ZNRD1-AS1 and BMI1) were analyzed by bioinformatics and dual-luciferase assay. After manipulating the expressions of ZNRD1-AS1, miR-128-3p and BMI1, cell viability, tube length, migration, invasion and the protein expressions (PCNA, E-Cadherin, N-Cadherin) of retinoblastoma cells were determined by cell counting kit-8 (CCK-8), tube formation, transwell and Western blot assays, respectively. Subcutaneous transplantation tumor assay, immunohistochemistry, and RT-qPCR were applied to verify the functions of the target gene .
ZNRD1-AS1 was up-regulated in the cytoplasm of retinoblastoma and regulated BMI1 via sponging miR-128-3p. ZNRD1-AS1 knockdown alleviated the malignant phenotype (viability, tube length, migration and invasion) of retinoblastoma cells, reduced tumor volume and weight, and inhibited BMI1 and CD34 expressions. Different from miR-128-3p mimic, miR-128-3p inhibitor promoted malignant phenotype of retinoblastoma cells, and partially reversed the inhibitory effect of siZNRD1-AS1. MiR-128-3p mimic down-regulated BMI1, PNCA, N-Cadherin expressions, and up-regulated p16 and E-Cadherin expressions. The regulatory effect of miR-128-3p was partially reversed by BMI1.
ZNRD1-AS1, acting as a "sponge" of miR-128-3p, up-regulates BMI1, thereby promoting the progression of retinoblastoma.
ZNRD1-AS1在肝癌、子宫内膜癌等疾病中发挥重要作用。然而，ZNRD1-AS1与视网膜母细胞瘤之间的关系尚未得到详细研究。本研究旨在确定ZNRD1-AS1在视网膜母细胞瘤中的作用。
使用Limma软件包从GEO数据库中识别视网膜母细胞瘤中差异表达的基因，并通过实时定量PCR（RT-qPCR）检测ZNRD1-AS1的表达和细胞定位。通过生物信息学和双荧光素酶报告基因检测分析miR-128-3p与两个基因（ZNRD1-AS1和BMI1）之间的关系。在调控ZNRD1-AS1、miR-128-3p和BMI1的表达后，分别通过细胞计数试剂盒-8（CCK-8）、管腔形成、Transwell和蛋白质免疫印迹法检测视网膜母细胞瘤细胞的活力、管腔长度、迁移、侵袭以及蛋白质表达（PCNA、E-钙黏蛋白、N-钙黏蛋白）。采用皮下移植瘤实验、免疫组织化学和RT-qPCR验证靶基因的功能。
ZNRD1-AS1在视网膜母细胞瘤细胞的细胞质中上调，并通过吸附miR-128-3p来调控BMI1。敲低ZNRD1-AS1可减轻视网膜母细胞瘤细胞的恶性表型（活力、管腔长度、迁移和侵袭），减小肿瘤体积和重量，并抑制BMI1和CD34的表达。与miR-128-3p模拟物不同，miR-128-3p抑制剂可促进视网膜母细胞瘤细胞的恶性表型，并部分逆转siZNRD1-AS1的抑制作用。miR-128-3p模拟物可下调BMI1、PNCA以及N-钙黏蛋白的表达，并上调p16和E-钙黏蛋白的表达。BMI1可部分逆转miR-128-3p的调控作用。
ZNRD1-AS1作为miR-128-3p的“海绵”，上调BMI1，从而促进视网膜母细胞瘤的进展。
