Ma Y G, Zhang Z S, Yu Y, Xu X F, Yuan L
Department of Gastroenterology, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Zhonghua Zhong Liu Za Zhi. 2020 Jan 23;42(1):30-36. doi: 10.3760/cma.j.issn.0253-3766.2020.01.004.
To investigate the effects of miR-513a-3p on proliferation, migration and invasion of gastric cancer cells and its mechanism. The miR-NC (miR-negative control mimics), miR-513a-3p (miR-513a-3p mimics), anti-miR-NC, anti-miR-513a-3p, si-NC, si-MDM2 (murine double minute 2), miR-513a-3p+ pcDNA3.1 (co-transfected with miR-513a-3p and pcDNA3.1), miR-513a-3p+ pcDNA3.1-MDM2 (co-transfected with miR-513a-3p and pcDNA3.1-MDM2) were transfected into BGC-823 cells, respectively. The expression of miR-513a-3p was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the protein expressions of cyclin D1, MMP-2, p21, E-cadherin, MDM2 were detected by western blot. The viability of BGC-823 cells of each group was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The migration and invasion of each group were detected by Transwell, the targeting relationship between miR-513a-3p and MDM2 was detected by double luciferase reporter gene assay. The expression of miR-513a-3p in gastric epithelial cells GES-1 was 0.76±0.08, significantly higher than 0.21±0.02 in gastric cancer cells BGC-823 and 0.34±0.03 in MGC-803, respectively (<0.05). The cell viabilities of the miR-NC group at 24 h, 48 h and 72 h were 0.57±0.05, 1.03±0.10, 1.43±0.14, respectively, while those of the miR-513a-3p group were 0.36±0.03, 0.48±0.05, and 0.63±0.06, respectively. The migration and invasion numbers of miR-NC group were 130±11.80 and 117±10.60, respectively, those of miR-513a-3p group were 58±5.64 and 50±5.13, respectively, and the differences were statistically significant (<0.05). The cell viabilities of the si-NC group at 24 h, 48 h and 72 h were 0.53±0.05, 0.95±0.10, 1.36±0.14, respectively. Those of the si-MDM2 group were 0.39±0.04, 0.57±0.06, and 0.80±0.08, respectively. The cell migration and invasion of the si-NC group were 141±12.02 and 109±10.60, respectively, while those of the MDM2 group were 66±6.67 and 61±6.18, respectively, and the differences were statistically significant (<0.05). The cell viabilities of the miR-513a-3p+ pcDNA3.1 group at 24 h, 48 h and 72 h were 0.34±0.03, 0.46±0.05, and 0.61±0.06, respectively. Those of miR-513a-3p+ pcDNA3.1-MDM2 group were 0.48±0.05, 0.82±0.08, 1.17±0.12, respectively. The migration and invasion of miR-513a-3p+ pcDNA3.1 group were 56±5.71 and 51±5.16, respectively, while those of miR-513a-3p+ pcDNA3.1-MDM2 group were 113±10.28 and 104±10.02, respectively, and the differences were statistically significant (<0.05). miR-513a-3p may inhibit the proliferation, migration and invasion of gastric cancer cells through targeting regulation of MDM2, which will provide new targets for the prevention and treatment of gastric cancer.
探讨miR-513a-3p对胃癌细胞增殖、迁移和侵袭的影响及其机制。分别将miR-NC（miR阴性对照模拟物）、miR-513a-3p（miR-513a-3p模拟物）、抗miR-NC、抗miR-513a-3p、si-NC、si-MDM2（鼠双微体2）、miR-513a-3p+pcDNA3.1（与miR-513a-3p和pcDNA3.1共转染）、miR-513a-3p+pcDNA3.1-MDM2（与miR-513a-3p和pcDNA3.1-MDM2共转染）转染至BGC-823细胞。采用实时定量逆转录聚合酶链反应（qRT-PCR）检测miR-513a-3p的表达，采用蛋白质免疫印迹法检测细胞周期蛋白D1、基质金属蛋白酶-2、p21、E-钙黏蛋白、MDM2的蛋白表达。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-溴化四氮唑（MTT）法检测各组BGC-823细胞的活力。采用Transwell检测各组细胞的迁移和侵袭能力，采用双荧光素酶报告基因检测法检测miR-513a-3p与MDM2的靶向关系。胃上皮细胞GES-1中miR-513a-3p的表达为0.76±0.08，显著高于胃癌细胞BGC-823中的0.21±0.02和MGC-803中的0.34±0.03（<0.05）。miR-NC组在24 h、48 h和72 h的细胞活力分别为0.57±0.05、1.03±0.10、1.43±0.14，而miR-513a-3p组分别为0.36±0.03、0.48±0.05、0.63±0.06。miR-NC组的迁移和侵袭细胞数分别为130±11.80和117±10.60，miR-513a-3p组分别为58±5.64和50±5.13，差异具有统计学意义（<0.05）。si-NC组在24 h、48 h和72 h的细胞活力分别为0.53±0.05、0.95±0.10、1.36±0.14。si-MDM2组分别为0.39±0.04、0.57±0.06、0.80±0.08。si-NC组的细胞迁移和侵袭能力分别为141±12.02和109±10.60，而MDM2组分别为66±6.67和61±6.18，差异具有统计学意义（<0.05）。miR-513a-3p+pcDNA3.1组在24 h、48 h和72 h的细胞活力分别为0.34±0.03、0.46±0.05、0.61±0.06。miR-513a-3p+pcDNA3.1-MDM2组分别为0.48±0.05、0.82±0.08、1.17±0.12。miR-513a-3p+pcDNA3.1组的迁移和侵袭细胞数分别为56±5.71和51±5.16，而miR-513a-3p+pcDNA3.1-MDM2组分别为113±10.28和104±10.02，差异具有统计学意义（<0.05）。miR-513a-3p可能通过靶向调控MDM2抑制胃癌细胞的增殖、迁移和侵袭，为胃癌的防治提供新靶点。
