lncRNA ADPGK-AS1对视网膜母细胞瘤细胞生物学行为的抑制作用及其调控机制

Inhibitory effects of lncRNA ADPGK-AS1 on the biological behaviours of human retinoblastoma cells and its regulating mechanism

目的探讨长链非编码RNA二磷酸腺苷依赖的葡萄糖激酶反义RNA1(lncRNA ADPGK-AS1)对人视网膜母细胞瘤(RB)细胞增生、迁移和侵袭的影响及其调控机制。方法收集2017年2月至2018年11月在驻马店市中心医院和郑州大学第一附属医院接受RB手术治疗的RB患者39例39眼的术中瘤旁组织和瘤体组织标本,采用实时荧光定量PCR法检测ADPGK-AS1和miR-623在标本组织中的相对表达量。体外培养人RB细胞Y-79,将培养细胞分为小干扰RNA正常对照组(siRNA-NC组)、siRNA-ADPGK-AS1组、微小RNA正常对照组(miR-NC组)、miR-623组、siRNA-ADPGK-AS1+anti-miR-NC组和siRNA-ADPGK-AS1+anti-miR-623组,采用MTT法检测各组细胞增生率;采用Transwell小室实验检测各组细胞迁移及侵袭数;采用双荧光素酶报告实验检测Y-79细胞中ADPGK-AS1和miR-623的靶向关系;采用Western blot法检测不同干预组细胞中Ki-67、基质金属蛋白酶(MMP)-2和MMP-9蛋白表达量。结果与瘤旁组织比较,RB组织中ADPGK-AS1相对表达量升高,miR-623相对表达量明显降低,差异均有统计学意义(t=40.522、48.497,均P<0.01);与siRNA-NC组比较,siRNA-ADPGK-AS1组细胞中Ki-67蛋白相对表达量明显下降,Y-79细胞增生A值显著降低,差异均有统计学意义(t=26.833、18.522,均P<0.01);siRNA-ADPGK-AS1组细胞中MMP-2和MMP-9蛋白相对表达量明显低于siRNA-NC组,差异均有统计学意义(t=22.123、26.183,均P<0.01);siRNA-ADPGK-AS1组迁移细胞数和侵袭细胞数均明显少于siRNA-NC组,差异均有统计学意义(t=12.385、19.201,均P<0.01);双荧光素酶报告实验证实ADPGK-AS1可靶向结合miR-623;miR-623组细胞中Ki-67、MMP-2、MMP-9蛋白相对表达量明显低于miR-NC组,差异均有统计学意义(t=22.137、22.200、21.094,均P<0.01);与miR-NC组比较,miR-623组Y-79细胞增生A值显著降低且迁移细胞数和侵袭细胞数均明显减少,差异均有统计学意义(t=16.398、11.400、17.846,均P<0.01);siRNA-ADPGK-AS1+anti-miR-623组细胞中Ki-67、MMP-2和MMP-9蛋白相对表达量均明显高于siRNA-ADPGK-AS1+anti-miR-NC组,差异均有统计学意义(t=20.795、17.493、23.479,均P<0.01);与siRNA-ADPGK-AS1+anti-miR-NC组比较,siRNA-ADPGK-AS1+anti-miR-623组增生A值明显升高且迁移细胞数及侵袭细胞数明显增多,差异均有统计学意义(t=15.600、14.495、17.855,均P<0.01)。结论敲低ADPGK-AS1基因可抑制RB细胞增生、迁移和侵袭,其作用机制与miR-623的表达上调有关。

Objective To explore the effects of long noncoding RNA adenosine diphosphate-dependent glucokinase antisense RNA 1(ADPGK-AS1)on the proliferation,migration and invasion of human retinoblastoma(RB)Y-79 cells and its regulatory effect on microRNA-623(miR-623).Methods The peritumoral tissue and RB specimens were collected from 39 eyes of 39 patients with RB during surgery in The First Affiliated Hospital of Zhengzhou University and Zhumadian Central Hospital from February 2017 to November 2018.Real-time fluorescence quantitative PCR was employed to detect the expression of ADPGK-AS1 and miR-623 in the specimens.Human RB line Y-79 cells were cultured in vitro and divided into small interfering RNA-normal control(siRNA-NC)group,siRNA-ADPGK-AS1 group,microRNA(miR)-NC group,miR-623 group,siRNA-ADPGK-AS1+anti-miR-NC group and siRNA-ADPGK-AS1+anti-miR-623 group.The cell proliferation rate was detected by MTT method.Transwell cell experiment was performed to detect the number of migrating and invading cells.The dual luciferase reporter experiment was used to evaluate the targeting relationship between ADPGK-AS1 and miR-623.The expression of Ki-67,matrix metalloproteinases(MMP)-2,and MMP-9 in the cells was detected by Western blot assay.Written informed consent was obtained from each patient prior to any medical examination and treatment.This study protocol adhered to the Declaration of Helsinki.The use of the human specimens was approved by an Ethics Committee of The First Affiliated Hospital of Zhengzhou University(No.2017-KY-73).Results Compared with the peritumoral tissue,the relative expression level of ADPGK-AS1 in the RB tissue was significantly increased,and the relative expression level of miR-623 was significantly reduced(t=40.522,48.497;both at P<0.01).Compared with the siRNA-NC group,both the relative expression level of Ki-67 protein and the proliferation A value of RB Y-79 cells were significantly reduced in the siRNA-ADPGK-AS1 group(t=26.833,18.522;both at P<0.01).The relative expression levels of MMP-2 and MMP-9 proteins in the siRNA-ADPGK-AS1 group were significantly lower than those in the siRNA-NC group(t=22.123,26.183;both at P<0.01).The number of migrating and invading cells in the siRNA-ADPGK-AS1 group was significantly less than that in the siRNA-NC group(t=12.385,19.201;both at P<0.01).The dual luciferase report experiment confirmed that ADPGK-AS1 targeted miR-623.The protein expression levels of the Ki-67,MMP-2 and MMP-9 in the miR-623 group were significantly lower than those in the miR-NC group(t=22.137,22.200,21.094;all at P<0.01).Compared with the miR-NC group,the proliferation A value of Y-79 cells in the miR-623 group was significantly lower,and the number of migrating and invadoing cells was significantly less(t=16.398,11.400,17.846;all at P<0.01).The relative expressions levels of Ki-67,MMP-2 and MMP-9 proteins in the siRNA-ADPGK-AS1+anti-miR-623 group were significantly higher than those in the siRNA-ADPGK-AS1+anti-miR-NC group(t=20.795,17.493,23.479;all at P<0.01).Compared with the siRNA-ADPGK-AS1+anti-miR-NC group,the proliferation A value of Y-79 cells in the siRNA-ADPGK-AS1+anti-miR-623 group was significantly increased(t=15.600,P<0.01),and the number of migrating and invading cells was obviously elevated(t=14.495,17.855;both at P<0.01).Conclusions Knockdown of ADPGK-AS1 gene can inhibit the proliferation,migration and invasion of Y-79 cells by up-regulating the expression of miR-623.