| 刘敏,吴运.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[J].安徽医药,2021,25(10):1998-2003. |
| 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 |
| by targeting gametopoietin binding protein 2 |
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| DOI：10.3969/j.issn.1009-6469.2021.10.021 |
| 中文关键词: 结直肠肿瘤 微小 RNA-494-3p 配子生成素结合蛋白(GGNBP2) 侵袭 迁移 上皮间质转化 |
| 英文关键词: Colorectal neoplasms MiR-494-3p GGNBP2 Invasion Migration EMT |
| 基金项目: |
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| 中文摘要: |
| 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 antimiR-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， Westernblot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］GGNBP2mRNA表达量均显著下降［(0.51±0.08)(0.38±0.06)(0.45±0.07)(1.00±0.11)，P<0.0、01］，GGNBP2蛋白表达量均，显著下降，(P<0.001)。与 anti-miRcon组相比，anti-miR-494、-3p组LS1034、细胞存活率、迁比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< |
| 英文摘要: |
| 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. |
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