L Yao1, L Yang, H Song, T-G Liu, H Yan. 1. Department of Ophthalmology, Tianjin Medical University General Hospital, Tianjin, China. hanzxu@163.com.
Abstract
OBJECTIVE: Retinoblastoma (RB) is the most prevalent intraocular malignancy in childhood. Long non-coding RNAs (lncRNAs) have been found as critical oncogenic drivers and tumor suppressor in RB. The aim of the present work was to investigate the impact and mechanism of XIST on RB cell autophagy and vincristine (VCR) sensitivity. MATERIALS AND METHODS: The levels of XIST and miR-204-5p were assessed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Western blot analysis was used for the determination of related protein levels. Cell proliferation and IC50 value of VCR were detected using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Flow cytometry was performed to evaluate cell apoptosis. The activities of caspase-3 and caspase-9 were identified using a corresponding assay kit. The direct interaction between XIST and miR-204-5p was confirmed using Dual-Luciferase reporter assay. Xenograft model was established to observe the effect of XIST on RB in vivo. RESULTS: Our data indicated that XIST was highly expressed in RB tissues and cell lines. XIST knockdown weakened the proliferation and autophagy and enhanced VCR sensitivity in RB cells. XIST acted as a molecular sponge of miR-204-5p. Moreover, the regulatory effects of XIST silencing on RB cell proliferation, autophagy and VCR sensitivity were mediated by miR-204-5p. Additionally, XIST silencing weakened tumor growth and enhanced VCR sensitivity in vivo through up-regulating miR-204-5p. CONCLUSIONS: Our current study suggested that XIST silencing suppressed RB progression and promoted VCR sensitivity in vitro and in vivo at least partially by acting as a miR-204-5p sponge, highlighting a powerful therapeutic strategy for RB treatment.
OBJECTIVE:Retinoblastoma (RB) is the most prevalent intraocular malignancy in childhood. Long non-coding RNAs (lncRNAs) have been found as critical oncogenic drivers and tumor suppressor in RB. The aim of the present work was to investigate the impact and mechanism of XIST on RB cell autophagy and vincristine (VCR) sensitivity. MATERIALS AND METHODS: The levels of XIST and miR-204-5p were assessed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Western blot analysis was used for the determination of related protein levels. Cell proliferation and IC50 value of VCR were detected using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Flow cytometry was performed to evaluate cell apoptosis. The activities of caspase-3 and caspase-9 were identified using a corresponding assay kit. The direct interaction between XIST and miR-204-5p was confirmed using Dual-Luciferase reporter assay. Xenograft model was established to observe the effect of XIST on RB in vivo. RESULTS: Our data indicated that XIST was highly expressed in RB tissues and cell lines. XIST knockdown weakened the proliferation and autophagy and enhanced VCR sensitivity in RB cells. XIST acted as a molecular sponge of miR-204-5p. Moreover, the regulatory effects of XIST silencing on RB cell proliferation, autophagy and VCR sensitivity were mediated by miR-204-5p. Additionally, XIST silencing weakened tumor growth and enhanced VCR sensitivity in vivo through up-regulating miR-204-5p. CONCLUSIONS: Our current study suggested that XIST silencing suppressed RB progression and promoted VCR sensitivity in vitro and in vivo at least partially by acting as a miR-204-5p sponge, highlighting a powerful therapeutic strategy for RB treatment.