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

Authors: Zhang Jun,  Liu Cailin,  Bu Zhanyun
DOI: 10.3760/cma.j.cn115989-20200819-00599
Published 2021-03-10
Cite as Chin J Exp Ophthalmol, 2021, 39(3): 207-215.

Abstract

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.

Key words:

Long noncoding RNA; Adenosine diphosphate-dependent glucokinase antisense RNA 1; MicroRNA; Retinoblastoma

Contributor Information

Zhang Jun
Department of Laboratory, Zhumadian Central Hospital, Zhumadian 463000, China
Liu Cailin
Department of Laboratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Bu Zhanyun
Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
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Updated: March 16, 2021 — 8:02 am