Up-regulation of miR-101 inhibits proliferation and invasion of retinoblastoma cells / 眼科新进展

ABSTRACT

Objective To investigate the effects of miR-101 expression on retinoblastoma cell proliferation and invasion.Methods A total of 31 cases of retinoblastoma tissues and 7 cases of normal retinal tissues were collected.Human normal retinal vascular endothehal cell line ACBRI-181 and retinoblastoma cell line HXO-Rb44 were cultured.HXO-Rb44 cells were transfected with Lipofectamine 2000 and grouped as followsnegative control (NC) group 1,in which cells were transfected with miRNA-101 negative controls,miR-101 expression group,cells transfected with miRNA-101 mimics;NC group 2,in which cells were transfected with histone-lysine N-methyltransferase (EZH2) negative controls,siRNA-EZH2 group,cells transfected with EZH2 siRNA,siRNA-EZH2 + mimics group,cells transfected with EZH2 siRNA and miRNA-101 mimics,and EZH2 + mimics group,cells transfected with EZH2 expression vector and miRNA-101 mimics.Normal HXO-Rb44 cells were served as blank group,miR-101 and EZH2 mRNA expression were detected by qRT-PCR,and EZH2 protein expression was measured by Western blot.Cell proliferation and invasion ability were determined by MTT and Transwell assays,respectively.Luciferase reporter assay was used to assess the targeting relationship between miR-101 and EZH2.Xenograft in nude mice was performed to detect cell proliferation ability in vivo.Results Compared with normal retinal tissues and ACBRI-181 cells,the relative expression of miR-101 in retinoblastoma tissues and HXO-Rb44 cells was significantly down-regulated (both P ＜ 0.05).The relative expression of EZH2 mRNA and protein in the miR-101 expression group was significantly lower than that in the blank group and NC group 1 (all P ＜ 0.05).At 72-96 h,the A values of the miR-101 expression group was significantly lower than those of the blank group and NC group 1 (both P ＜0.01).The number of invasive cells in the miR-101-expressing group (51 ± 6) was significantly lower than that of the blank group (97 ± 11)and NC group 1 (92 ± 8) (both P ＜ 0.01).EZH2 was the target gene of miR-101.At 48-96 h,the A values of the siRNA-EZH2 group and siRNA-EZH2 + mimics group were significantly lower than those of the blank group and NC group 2 (all P ＜0.01).At 72-96 h,the A values of the siRNA-EZH2 + mimics group were obviously lower than those of the siRNA-EZH2 group (both P ＜ 0.05).At 24-96 h,the A values of EZH2 + mimics group were not statistically different from the blank group or NC group 2 (all P ＞ 0.05).The number of invasive cells in the siRNA-EZH2 group (48 ± 4) and siRNA-EZH2 +mimics group (38 ±3) was significantly lower than that in the blank group (95 ± 10) and NC group 2 (90 ±6) (all P ＜0.01),and siRNA-EZH2 + mimics group was significantly lower than siRNA-EZH2 group (P ＜0.05).The number of invading cells of the EZH2 +mimics group (101 ± 11) was not statistically different from the blank group and NC group 2 (both P ＞ 0.05).At 5-7 weeks,the tumor volumes of siRNA-EZH2 group and siRNA-EZH2 + mimics group were significantly lower than those of the blank group and NC group 2 (all P ＜ 0.01),and siRNA-EZH2 + mimics group was significantly lower than the siRNA-EZH2 group (P ＜ 0.05).There was no significant difference in the tumor volume between the EZH2 + mimics group and the blank group or the NC group 2 (all P ＞ 0.05).Conclusion Up-regulation of miR-101 expression can inhibit the proliferation and invasion of HXO-Rb44 cells,which might be achieved by inhibiting the expression of EZH2.