MicroRNA-101-3p inhibits proliferation in retinoblastoma cells by targeting EZH2 and HDAC9

Abstract

Retinoblastoma is the most frequent intraocular malignant tumor type to occur in childhood. MicroRNA (miR)-101-3p has been reported to function as a tumor suppressor in various types of cancer. However, the biological function and underlying mechanisms of miR-101-3p in retinoblastoma are largely unknown. In the present study, it was identified that miR-101-3p was downregulated in retinoblastoma. MTT and flow cytometry assays demonstrated that ectopic overexpression of miR-101-3p significantly inhibited cell viability and cell cycle progression in WERI-Rb-1 and Y79 cells. In vivo mouse experiments further confirmed the anti-proliferative role of miR-101-3p in retinoblastoma. Additionally, predictions with TargetScan software indicated that the 3'-untranslated regions of enhancer of zeste homolog 2 (EZH2) and histone deacetylase (HDAC9) mRNAs are targeted by miR-101-3p. Accordingly, a dual luciferase reporter gene assay demonstrated that miR-101-3p directly targeted EZH2 and HDAC9 to suppress the proliferation of retinoblastoma cells. Meanwhile, the restoration of EZH2 or HDAC9 expression countered the anti-proliferative effect of miR-101-3p on WERI-Rb-1 and Y79 cells. Collectively, these data highlight the role of miR-101-3p in the tumorigenesis of retinoblastoma, and indicate its suitability as a novel therapeutic target.

Keywords: enhancer of zeste homolog 2; histone deacetylase 9; microRNA-101-3p; proliferation; retinoblastoma.

Figure 1.

miR-101-3p is downregulated in retinoblastoma. Reverse transcription-quantitative polymerase chain reaction assays were performed in 12 retinoblastoma and three normal retina specimens. The expression level of miR-101-3p in retinoblastoma samples was significantly downregulated compared with normal retinas. *P<0.05. miR, microRNA.

Figure 2.

miR-101-3p suppresses proliferation in retinoblastoma cells. (A) miR-101-3p agomir transfection significantly upregulated the expression of miR-101-3p in WERI-RB-1 and Y79 cells. (B) WERI-RB-1 and Y79 cells were transfected with an miR-control and miR-101-3p agomir, and cell viability at the indicated times was assessed by MTT assays. (C and D) Effects of miR-101-3p on WERI-RB-1 and Y79 cell cycle were analyzed by flow cytometry. Overexpression of miR-101-3p resulted in G₁ arrest in WERI-RB-1 and Y79 cells. *P<0.05. miR, microRNA.

Figure 3.

miR-101-3p inhibits tumor growth in nude mice. (A) Tumor volume was assessed every 4 days. The results demonstrated that the growth of tumors in the miR-101-3p agomir group was significantly slower compared with the miR-control group. (B) Images of tumors extracted from mice inoculated with Y79 cells and treated with miR-101-3p agomir or miR-control. (C) Mean tumor weight of the indicated groups. *P<0.05. miR, microRNA.

Figure 4.

miR-101-3p directly targets EZH2 and HDAC9. (A) EZH2 and HDAC9 were identified as candidate targets for miR-101-3p. The wild-type or mutant putative miR-101-3p binding sites in the 3′UTRs of EZH2 and HDAC9 mRNAs are shown. (B) Luciferase activity assays demonstrated that miR-101-3p overexpression significantly decreased the luciferase activity in WERI-RB-1 and Y79 cells transfected with the wild-type 3′UTR of EZH2 or HDAC9. No effects on the mutant 3′UTRs for EZH2 or HDAC9 were observed. *P<0.05. (C) Overexpression of miR-101-3p suppressed the levels of EZH2 and HDAC9 protein. (D) Overexpression of miR-101-3p reduced the mRNA levels of EZH2 and HDAC9. *P<0.05 vs. relative control. miR, microRNA; EZH2, enhancer of zeste homolog 2; HDAC9, histone deacetylase 9; 3′UTR, 3′-untranslated region; WT, wild-type; Mut, mutant; ORF, open reading frame.

Figure 5.

Restoration of EZH2 or HDAC9 expression reverses the effect of miR-101-3p on WERI-RB-1 and Y79 cells. (A) Overexpression of EZH2 or HDAC9 in indicated cells was confirmed by performing western blotting. (B) WERI-RB-1 and Y79 cells were transfected with miR-control or miR-101-3p, and pEnter or pEnter-EZH2. Cell viability was determined by an MTT assay. (C) WERI-RB-1 and Y79 cells were transfected with miR-control or miR-101-3p, and pEnter or pEnter-HDAC9. Cell viability was determined by an MTT assay. *P<0.05. EZH2, enhancer of zeste homolog 2; HDAC9, histone deacetylase 9; miR, microRNA.