DADS上调miR-200b抑制视网膜母细胞瘤细胞的生长与侵袭

目的 探索miR-200b和二烯丙基二硫(DADS)相关的抑瘤机制,为揭示DADS抑制视网膜母细胞瘤生长及侵袭的内在分子机制提供理论依据.方法 采用qRT-PCR分别检测不同浓度的DADS对Y79细胞miR-200b表达的影响,DADS分别设置六种浓度:0 μmol/L、25 μmol/L、50 μmol/L、100 μmol/L、200 μmol/L和400 μmol/L.MTT和Transwell侵袭实验分别检测DADS和miR-200b对Y79细胞生长及侵袭能力的影响,将实验设置成五个组,即miRNA阴性对照组(瞬时转染scramble 40μmol/L);miR-200b组(瞬时转染miR-200b-mimics 40μmol/L);DADS阴性对照组(DMSO 10μmol/L,即mock组);DADS组(DADS 200μmol/L)和DADS+miR-200b组(瞬时共转染miR-200b-mimics 40μmol/L)和DADS (200μmol/L).结果 DADS可上调Y79细胞中miR-200b的表达,且其呈剂量依赖性(P<0.05);在MTT实验中,miR-200b组的OD值(0.549±0.057)较miRNA阴性对照组(0.737±0.135)明显降低;DADS组的OD值(0.508±0.064)较DADS阴性对照组(0.722±0.145)明显降低;而DADS+miR-200b组的OD值(0.362±0.081)较miRNA阴性对照组和DADS阴性对照组降低最为显著(P<0.05),即DADS可以通过上调miR-200b抑制Y79细胞的增殖能力.在Transwell侵袭实验中,外源性的高表达miR-200b组(105±13)较miRNA阴性对照组(162±12)能够显著抑制Y79细胞的穿膜细胞数,DADS组(102±13)较DADS阴性对照组(154±8)能够显著降低Y79的穿膜细胞数,且DADS+miR-200b组(77±8),细胞穿膜细胞数较miRNA阴性对照组和DADS阴性对照组减少最显著(P<0.05),即DADS通过上调miR-200b抑制Y79细胞侵袭.结论 DADS通过上调miR-200b的表达抑制视网膜母细胞瘤细胞的生长及侵袭.

Suppression of diallyl disulfide on the proliferation and invasion of retinoblastoma cells by up-regulating miR-200b

Objective To explore the internal molecular mechanisms related to miR-200b for the suppressing ef-fect of diallyl disulfide (DADS) on the proliferation and invasion of retinoblastoma cells. Methods The expres-sion of miR-200b regulated by different dose of DADS (0 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L and 400 μmol/L) in Y79 cells was detected by quantitative real time polymerase chain reaction (qRT-PCR). The pro-liferation and invasion ability of Y79 cells in vitro regulated by miR-200b and DADS were assessed by MTT and Transwell invasion assays, with five groups set up: miRNA-negative control group (transient transfection with scramble 40μmol/L);miR-200b group (transient transfection with miR-200b-mimics 40μmol/L);DADS-negative control group (mock group, DMSO 40μmol/L);DADS group (DADS 200μmol/L) and DADS+miR-200b group (tran-sient transfection with both miR-200b-mimics 40μmol/L and DADS 200μmol/L). Results miR-200b expression was increased with the increase of the doses of DADS, in a dose-dependent manner (P<0.05). For MTT, the OD value was sig-nificantly lower in miR-200b group than miRNA-negative control group (0.549 ± 0.057) vs (0.737 ± 0.135)], in DADS group than DADS-negative control group (0.508±0.064) vs (0.722±0.145)], and the OD value in DADS+miR-200b group was the lowest (0.362±0.081)]. The differences were all statistically significant (P<0.05). The results indicate that DADS could inhibit the proliferation capacity of Y79 cells by increasing the expression of miR-200b. In Transwell invasion as-says, the number of cells penetrating membrane was significantly less in miR-200b group than miRNA-negative control group (105 ± 13) vs (162 ± 12)], in DADS group than DADS-negative control group (102 ± 13) vs (154 ± 8)], and the number in DADS+miR-200b group was the lowest (77±8)]. The differences were all statistically significant (P<0.05). The results indicate that DADS could inhibit the invasion capacity of Y79 cells by increasing the ex-pression of miR-200b. Conclusion DADS can suppress the proliferation and invasion of human retinoblastoma cells by up-regulation of miR-200b.