微小 RNA-494-3p靶向配子生成素结合蛋白 2促进结直肠癌细胞侵袭、迁移及上皮间质转化的实验研究

目的 研究微小RNA-494-3p(miR-494-3p)影响结直肠癌细胞迁移、侵袭及上皮间质转化(EMT)的分子机制.方法 于2019年1—12月体外培养购自美国ATCC的结直肠上皮细胞NCM460和结直肠癌细胞系T84、LS1034和HCT116,实时定量聚合酶链式反应(RT-qPCR)检测细胞中miR-494-3p和配子生成素结合蛋白2(GGNBP2)mRNA的表达量,蛋白质印迹法(Western blot)检测细胞中GGNBP2蛋白表达.将LS1034细胞分为对照(NC)组、miR-494-3p抑制剂(anti-miR-494-3p)组、抑制剂阴性对照(anti-miR-con)组、GGNBP2过表达载体(pcDNA-GGNBP2)组、空载体(pcDNA-con)组、anti-miR-494-3p+GGNBP2小干扰RNA(si-GGNBP2)组和anti-miR-494-3p+小干扰RNA阴性对照(si-con)组,甲基噻唑基四唑(MTT)法测定LS1034细胞存活率,Transwell实验检测细胞迁移和侵袭,Western blot检测相关蛋白细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶-2(MMP-2)、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)的表达.双荧光素酶报告系统验证miR-494-3p和GGNBP2的调控关系.结果 与NCM460细胞相比,结直肠癌细胞T84、LS1034和HCT116中miR-494-3p表达量均显著升高(1.91±0.11)、(2.23±0.14)、(2.08±0.12)比(1.00±0.04),P<0.001],GGNBP2 mRNA表达量均显著下降(0.51±0.08)、(0.38±0.06)、(0.45±0.07)比(1.00±0.11),P<0.001],GGNBP2蛋白表达量均显著下降(P<0.001).与anti-miR-con组相比,anti-miR-494-3p组LS1034细胞存活率、迁移数和侵袭数均显著下降(41.29±3.89)％比(84.36±7.28)％,(132.56±7.56)个比(246.3±10.64)个,(55.64±5.64)个比(145.62±9.56)个,均P<0.001],细胞中Cyclin D1、MMP-2、Vimentin蛋白表达量均显著下降,均P<0.001,E-cadherin蛋白表达升高、蛋白表达降低(P<0.001).与pcDNA-con组相比,pcDNA-GGNBP2组LS1034细胞存活率、迁移数和侵袭数均显著下降,P<0.05,细胞中Cyclin D1、MMP-2、Vimentin蛋白表达均下降,均P<0.001,E-cadherin蛋白表达升高,蛋白表达降低,P<0.001.miR-494-3p靶向负调控GGNBP2的表达.与anti-miR-494-3p+si-con组相比,anti-miR-494-3p+si-GGNBP2组LS1034细胞存活率、迁移数和侵袭数均显著上升,细胞中Cyclin D1、MMP-2和Vimentin蛋白表达均升高,E-cadherin蛋白表达下降,均P<0.001.结论 miR-494-3p通过靶向抑制GGNBP2促进结直肠癌LS1034细胞迁移、侵袭及EMT.

by targeting gametopoietin binding protein 2

Objective To study the molecular mechanism of microRNA-494-3p (miR-494-3p) on migration, invasion and epithelialmesenchymal transition (EMT) of colorectal cancer cells.Methods From January 2019 to December 2019, colorectal epithelial cellsNCM460 and colorectal cancer cell lines T84, LS1034 and HCT116 were purchased from ATCC in the United States and cultured in vitro, and then the expression of miR-494-3p and gametopoietin binding protein 2 (GGNBP2) mRNA in cells were detected by qRT-PCR,the expression of GGNBP2 protein in the cells was detected by Western blot. LS1034 cells were assigned into control (NC) group, miR494-3p inhibitor (anti-miR-494-3p) group, inhibitor negative control (anti-miR-con) group, GGNBP2 overexpression vector (pcDNAGGNBP2 ) group, empty vector (pcDNA-con) group, anti-miR-494-3p+GGNBP2 small interfering RNA (si-GGNBP2) group and anti-miR-494-3p+small interfering RNA negative control (si-con) group. And then methyl thiazolyl tetrazolium (MTT) method was used tomeasure the survival rate of LS1034 cells. Transwell experiment was used to detect cell migration and invasion. Western blot was usedto detect the protein expression of Cyclin D1, MMP-2 and E-cadherin and Vimentin. The dual luciferase reporter system verified the regulatory relationship between miR-494-3p and GGNBP2.Results Compared with the NCM460 cells, the expression of miR-494-3p in colorectal cancer cell lines T84, LS1034 and HCT116 were significantly increased (1.91±0.11), (2.23±0.14), (2.08±0.12) vs. (1.00± 0.04), all P<0.001], but the expression of GGNBP2 mRNA were all significantly decreased (0.51±0.08), (0.38±0.06), (0.45±0.07) vs. (1.00±0.11), all P<0.001], and the expression of GGNBP2 protein expression levels were all significantly decreased,all P<0.001. Compared with the anti-miR-con group, the survival rate of LS1034 cells, the number of migration and invasion in the anti-miR-494-3p group were significantly reduced (41.29±3.89)% vs. (84.36±7.28)%, (132.56±7.56) vs. (246.3±10.64), (55.64±5.64) vs. (145.62±9.56), all P< 0.001], and the protein expression of Cyclin D1, MMP-2 and Vimentin in cells were all significantly decreased, all P<0.001, but the protein expression of E-cadherin was increased . Compared with the pcDNA-con group, the survival rate of LS1034 cells, the number of migration and invasion in the pcDNA-GGNBP2 group were significantly reduced , all P<0.05, and the protein expression of Cyclin D1, MMP-2, and Vimentin in the cells were all significantly decreased, all P<0.001, but the protein expression of E-cadherin were significantly increased. miR-494-3p could target and negatively regulate the expression of GGNBP2. Compared with the anti-miR-494-3p+si-con group, the survival rate of LS1034 cells, the number of migration and invasion in the anti-miR-494-3p+si-GGNBP2 group were significantly increased, and the protein expression of Cyclin D1, MMP-2 and Vimentin in cells were all increased, but the protein expression of E-cadherin was decreased, P<0.001.Conclusion MiR-494-3p promotes the migration, invasion and EMT of colorectal cancer cells LS1034 by targeting to inhibit the expression of GGNBP2.