Résumé
Objective:To investigate the effects of long noncoding RNA (LncRNA) lung cancer associated transcript 1 (LUCAT1) targeting microRNA (miR)-502-5p on the proliferation, migration and invasion of human retinoblastoma (RB) cells.Methods:RB tissue samples were collected from 27 RB patients who underwent eyeball enucleation in Henan Eye Hospital from May 2019 to January 2021.Another 27 normal retinal tissue specimens were collected from 12 patients with eyeball rupture, 7 with eyeball atrophy, and 8 with eyeball penetrating injury combined with pigment film incarceration who underwent the eyeball enucleation in Henan Eye Hospital during the same period.The expressions of LncRNA LUCAT1 and miR-502-5p in RB tissues, cell lines (Y-79, WERI-Rb-1, HXo-RB44) and human retinal epithelial cells (ARPE-19) were detected by real-time quantitative PCR.Y-79 RB cell was divided into control group, small interfering RNA (si)-LncRNA LUCAT1 group, si-control (con) group, pcDNA group, pcDNA-LncRNA LUCAT1 group, miR-con group, miR-502-5p group, si-LncRNA LUCAT1+ anti-miR-con group and si-LncRNA LUCAT1+ anti-miR-502-5p group, and cells in different groups were transfected with corresponding reagents.The expressions of MMP2 and MMP9 proteins were detected by Western blot.Cell proliferation activity was assayed by cell counting kit 8.Cell proliferation capability was detected by colony formation assay.Cell migration and invasion ability were determined by Transwell assay.The targeting regulation of LncRNA LUCAT1 against miR-502-5p was confirmed by dual luciferase reporter assay and real-time quantitative PCR.The study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2021[32]). Written informed consent was obtained from guardians of subjects.Results:LncRNA LUCAT1 expression level in RB tissue was 2.73±0.34, which was significantly higher than 1.00±0.15 in normal retinal tissue ( t=24.190, P<0.001). The miR-502-5p expression level in RB tissues was 0.42±0.06, which was significantly lower than 1.00±0.13 in normal retinal tissue ( t=21.049, P<0.001). LncRNA LUCAT1 expression level was significantly higher and the miR-502-5p expression level was significantly lower in human RB cell lines Y-79, WERI-Rb-1 and HXO-RB44 than those in ARPE-19 cells, with statistically significant differences (all at P<0.05). The LncRNA LUCAT1 expression, the relative expressions of MMP2 and MMP9 proteins, the absorbance ( A) value, and the number of proliferated, migrating and invading Y-79 cells in si-LncRNA LUCAT1 group were significantly reduced in comparison with control group, and the differences were statistically significant (all at P<0.05). The miR-502-5p expression level was higher, and the relative expression levels of MMP2 and MMP9, A value, as well as the number of proliferated, migrating and invading Y-79 cells were lower in miR-502-5p group than in miR-con group, showing statistically significant differences ( t=20.274, 14.884, 14.181, 12.692, 17.749, 20.889, 21.913; all at P<0.001). The miR-502-5p expression level was lower and the relative expression levels of MMP2 and MMP9, A value as well as the number of proliferated, migrating and invading Y-79 cells were higher in si-LncRNA LUCAT1+ anti-miR-502-5p group than in si-LncRNA LUCAT1+ anti-miR-con group, showing statistically significant differences ( t=14.097, 15.839, 15.757, 11.860, 16.235, 16.565, 16.487; all at P<0.001). When co-transfected with LncRNA LUCAT1-wild type, the relative luciferase activity of miR-502-5p group was lower than that of miR-con group, and the difference was statistically significant ( t=16.379, P<0.001). The LncRNA LUCAT1 expression level was higher and the miR-502-5p expression level was lower in pcDNA-LncRNA LUCAT1 group than in pcDNA group, and the differences were statistically significant (both at P<0.05). The LncRNA LUCAT1 expression level was lower and the miR-502-5p expression level was higher in si-LncRNA LUCAT1 group than in si-con group, and the differences were statistically significant (both at P<0.05). Conclusions:Inhibition of LncRNA LUCAT1 can attenuate the proliferation, migration and invasion ability of human RB cells by the targeting up-regulation of miR-502-5p.
Résumé
@#AIM: To investigate whether circular RNA(circRNA)circ_0000144 targets microRNA(miRNA)-502-5p to regulate the proliferation, apoptosis, migration and invasion of human retinoblastoma Y79 cells. <p>METHODS: Y79 cells were divided into si-NC group(transfected with si-NC), si-circ_0000144 group(transfected with si-circ_0000144), miR-NC group(transfected with miR-NC), miR-502-5p group(transfected with miR-502-5p mimic), pcDNA group(transfected with pcDNA), pcDNA-circ_0000144 group(transfected with pcDNA-circ_0000144), si-circ_0000144+anti-miR-NC group(transfected with si-circ_0000144+anti-miR-NC), si-circ_0000144+anti-miR-502-5p group(transfected with si-circ_0000144+anti-miR-502-5p). Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of circ_0000144 and miR-502-5p in retinoblastoma tissues and cells, thiazole blue tetrazolium bromide(MTT)detected cell proliferation, and western blot was employed to determine the expression of nuclear associated antigen Ki67(Ki-67), B cell lymphoma/lewkmia-2(Bcl-2), Bcl-2 associated X protein(Bax), matrix metalloprotease(MMP)-2, MMP-9 protein. Flow cytometry detected cell apoptosis, and Transwell measured cell migration and invasion. Bioinformatics prediction and dual luciferase report experiment analyzed whether circ_0000144 targets miR-502-5p. <p>RESULTS: The expression of circ_0000144 in 31 cases of retinoblastoma tissue was higher than that of adjacent tissues, and the expression of miR-502-5p was lower than that of adjacent tissues(<i>P</i><0.05). Compared with the si-NC group, the circ_0000144 expression, OD value, expression of Ki-67, Bcl-2, MMP-2, MMP-9 protein, number of migration and invasion cells of the Y79 cells in the si-circ_0000144 group decreased, and the expression of Bax protein and apoptosis rate increased(<i>P</i><0.05). circ_0000144 targets and negatively regulates the expression of miR-502-5p. Compared with miR-NC group, miR-502-5p group increased cell apoptosis rate and expression of Bax protein of the Y79 cells, while decreased OD value, number of migration and invasion cells, and the expression of Ki-67, Bcl-2, MMP-2 and MMP-9 protein(<i>P</i><0.05). Compared with the si-circ_0000144+anti-miR-NC group, cell apoptosis rate and Bax protein expression of the Y79 cells in the si-circ_0000144+anti-miR-502-5p group decreased, but the OD value, number of migration and invasion cells, the protein expression of Ki-67, Bcl-2, MMP-2 and MMP-9 increased. <p>CONCLUSION: circ_0000144 was highly expressed in retinoblastoma tissue, and inhibiting circ_0000144 can reduce the proliferation, migration and invasion of retinoblastoma Y79 cells, and promote apoptosis through negative regulation of miR-502-5p.