超声微泡介导的膀胱癌抑制因子miR-490-5p调控转录因子ZEB1对膀胱癌细胞的影响研究

Research on effects of ultrasound microbubble-mediated bladder cancer suppressor miR-490-5p regulating transcription factor ZEB1 on bladder cancer cells

目的:探究微泡介导的膀胱癌抑制因子微小核糖核酸(miR)-490-5p调控锌指E-盒结合同源盒蛋白1(ZEB1)对膀胱癌细胞增殖、侵袭和迁移的影响。方法:检测人膀胱上皮细胞.人膀胱癌细胞VM-CUB-3、639V和5637、HT-1197细胞中mi R-490-5p和ZEB1 mRNA与蛋白的表达情况。选择人膀胱癌639V细胞株进行实验,对639V细胞给予1%、5%、10%、20%浓度水平的微泡以及超声强度为0.5 W/cm2、0.75 W/cm2的超声照射30 s处理,筛选超声强度为0.5 W/cm2、微泡浓度为10%;将常规培养639V细胞纳入对照组,将培养及微泡制备后的639V细胞分别纳入超声微泡组、超声微泡转染miR-490-5p组、脂质体转染miR-490-5p组及超声微泡转染miR-490-5p+ZEB1组,分别检测5组639V细胞增殖抑制率、侵袭细胞数及划痕愈合率,并用Westernblot法检测5组639V细胞ZEB1、增殖细胞核抗原(PCNA)、钙黏蛋白E(E-cad)和钙黏蛋白N(N-cad)水平。结果:膀胱癌VM-CUB-3、639V、5637和HT-1197细胞与人膀胱上皮细胞相比,miR-490-5p水平降低,ZEB1mRNA和ZEB1蛋白水平均升高,其中639V细胞差异最明显。在超声0.5 W/cm2、微泡浓度10%的处理条件下,超声微泡组细胞增殖抑制率和E-cad蛋白均高于对照组,差异有统计学意义(t=22.282,t=4.517;P<0.05),PCNA蛋白水平、侵袭细胞数和N-cad蛋白和划痕愈合率降低,差异有统计学意义(t=2.441,t=2.325,t=4.572,t=4.417;P<0.05);超声微泡转染miR-490-5p组、脂质体转染miR-490-5p组细胞增殖抑制率、E-cad蛋白高于超声微泡组,差异有统计学意义(t细胞增殖抑制率=16.902,t=12.426;tE-cad=5.586,t=2.765;P<0.05);PCNA蛋白水平、侵袭细胞数、N-cad蛋白和划痕愈合率低于超声微泡组,差异有统计学意义(tPCNA蛋白水平=7.530,t=4.164;t侵袭细胞数=9.007,t=5.682;tN-cad蛋白=8.089,t=4.297;t划痕愈合率=11.366,t=6.665,P<0.05)。超声微泡转染miR-490-5p+ZEB1组细胞增殖抑制率、E-cad蛋白低于超声微泡转染miR-490-5p组,差异有统计学意义(t=2.903,t=6.114,P<0.05),PCNA蛋白水平、侵袭细胞数、N-cad蛋白和划痕愈合率高于超声微泡转染miR-490-5p组,差异有统计学意义(t=6.586,t=4.487,t=5.695,t=4.603,P<0.05);双荧光素酶报告基因显示miR-490-5p与ZEB1存在靶向关系。结论:超声微泡介导的miR-490-5p可能通过负向调控ZEB1,抑制人膀胱癌细胞639V增殖、侵袭及迁移,超声微泡介导是一种行之有效的转染方式。

Objective: To explore the effect of microbubble mediated regulation of zinc finger E-box binding homeobox protein 1(ZEB1) by bladder cancer suppressor microribonucleic acid(MIR)-490-5p on the proliferation, invasion and migration of bladder cancer cells. Methods: The mir-490-5p, ZEB1 mRNA and protein expression in human bladder epithelial cells, human bladder cancer cells VM-CUB-3, 639V and 5637, HT-1197 cells were detected, and 639V cell lines were selected for experiments;639V cells were treated with 1%, 5%, 10%, 20% microbubbles and 0.5 W/cm2,0.75 W/cm2 ultrasound for 30 seconds, the ultrasound intensity was 0.5 W/cm~2 and the microbubble concentration was 10%. The routinely cultured 639V cells were included in the control group. The cultured and microbubble prepared 639V cells were respectively included in the ultrasound microbubble group, the ultrasound microbubble transfection miR-490-5p group, the liposome transfection miR-490-5p group, and the ultrasound microbubble transfection miR-490-5p+ZEB1 group. The proliferation inhibition rate, the number of invasive cells, and the scratch healing rate of 639V cells in five groups were detected respectively. The protein levels of ZEB1, proliferating cell nuclear antigen(PCNA),cadherin E(E-cad) Calmodulin N(N-cad) in 639V cells in the five groups were detected by Western blot method.Results: Compared with human bladder epithelial cells, the levels of mir-490-5p in vm-cub-3, 639V, 5637 and ht-1197cells of bladder cancer decreased, and the levels of ZEB1 mRNA and ZEB1 protein increased, the difference was most significant in 639V cells. Under the treatment conditions of ultrasonic 0.5 W/cm~2 and microbubble concentration 10%,the cell proliferation inhibition rate and E-cad protein in the ultrasound microbubble group were higher than those in the control group, the difference was statistically significant(t=22.282, t=4.517;P<0.05), the PCNA protein level,number of invasive cells, N-cad protein and scratch healing rate were lower, the difference was statistically significant(t=2.441, t=2.325, t=4.572, t=4.417;P<0.05). The cell proliferation inhibition rate and E-cad protein in the ultrasonic microbubble transfection miR-490-5p group and the liposome transfection miR-490-5p group were higher than those in the ultrasonic microbubble group, the difference was statistically significant(tCell Proliferation Inhibition Rate=16.902,t=12.426, tE-cad=5.586, t=2.765;P<0.05). PCNA protein level, number of invasive cells, N-cad protein, and scratch healing rate were lower than those in the ultrasonic microbubble group, the difference was statistically significant(tPCNA protein level=7.530, t=4.164, tnumber of invasive cells=9.007, t=5.682, tN-cad protein=8.089, t=4.297, tscratch healing rate=11.366,t=6.665;P<0.05). The cell proliferation inhibition rate and E-cad protein in the ultrasound microbubble transfection miR-490-5p+ZEB1 group were lower than those in the ultrasound microbubble transfection miR-490-5p group, the difference was statistically significant(t=2.903, t=6.114;P<0.05);and the PCNA protein level, number of invasive cells,N-cad protein and scratch healing rate were higher than those in the ultrasonic microbubble transfection miR-490-5p group,the difference was statistically significant(t=6.586, t=4.487, t=5.695, t=4.603;P<0.05). The double luciferase reporter gene showed that there was a targeting relationship between miR-490-5p and ZEB1. Conclusion: Ultrasonic microbubblemediated miR-490-5p may inhibit the proliferation, invasion and migration of Human bladder cancer cells 639V cells by negatively regulating ZEB1. Ultrasonic microbubble-mediated transfection is an effective transfection method.