miR-325-3p靶向PRELID1基因介导EMT通路对结肠癌细胞侵袭和迁移的影响及其机制

doi:10.13481/j.1671-587X.20250504

摘要/Abstract

摘要：

目的探讨微小RNA-325-3p（miR-325-3p）过表达对结肠癌细胞侵袭和迁移的影响，阐明其作用机制。方法收集25例明确诊断为结肠癌患者的肿瘤组织及对应的癌旁正常组织。采用实时荧光定量PCR（RT-qPCR）法检测癌旁正常组织和结肠癌组织中miR-325-3p和进化和淋巴兴趣相关结构域蛋白1（PRELID1）mRNA表达水平。采用RT-qPCR法和Western blotting法检测人正常结肠细胞NCM460及结肠癌细胞SW480、HCT116和HT-29中miR-325-3p及PRELID1 mRNA表达水平和PRELID1蛋白表达水平。将SW480细胞分为对照组、NC-mimics组、miR-325-3p mimics组、miR-325-3p mimics+oe-NC组和miR-325-3p mimics+oe-PRELID1组。采用Transwell小室实验和细胞划痕实验分别检测各组细胞侵袭及迁移能力，Western blotting法检测各组细胞中上皮-间质转化（EMT）中E-钙黏蛋白（E-Cadherin）、N-钙黏蛋白（N-Cadherin）和波形蛋白（Vimentin）表达水平。使用Targetscan生物信息学网站预测miR-325-3p下游靶基因，采用双荧光素酶实验验证miR-325-3p与PRELID1的靶向调控关系。结果与癌旁正常组织比较，结肠癌组织中miR-325-3p表达水平明显降低（P<0.05），PRELID1 mRNA表达水平明显升高（P<0.05），且结肠癌组织中miR-325-3p和PRELID1 mRNA表达水平呈负相关关系（r<0，R²=0.392， P<0.001）。与NCM460细胞比较， SW480、 HCT116和HT-29细胞中miR-325-3p表达水平均明显降低（P<0.05），PRELID1 mRNA表达水平明显升高（P<0.05）。与对照组和NC-mimics组比较，miR-325-3p mimics组SW480细胞侵袭细胞数明显减少（P<0.05），划痕愈合率降低（P<0.05），E-Cadherin蛋白表达水平升高（P<0.05），N-Cadherin和Vimentin蛋白表达水平降低（P<0.05）。双荧光素酶实验证实PRELID1是miR-325-3p的直接靶标。与miR-325-3p mimics+oe-NC组比较，miR-325-3p mimics+oe-PRELID1组SW480细胞侵袭细胞数明显增加（P<0.05），划痕愈合率升高（P<0.05），E-Cadherin蛋白表达水平降低（P<0.05），N-Cadherin和Vimentin蛋白表达水平均升高（P<0.05）。结论过表达miR-325-3p可靶向下调PRELID1表达，抑制EMT进程，进而抑制SW480细胞侵袭和迁移。

关键词: 微小RNA-325-3p, 进化和淋巴兴趣相关结构域蛋白1, 结肠肿瘤, 细胞侵袭, 细胞迁移, 上皮-间质转化

Abstract:

Objective To investigate the effects of microRNA-325-3p （miR-325-3p） over-expression on the invasion and migration of colon cancer cells， and to clarify their mechanisms. Methods The tumor tissue and the corresponding adjacent normal tissue of 25 patients clearly diagnosed with colon cancer were collected. The expression levels of miR-325-3p and relevant evolutionary and lymphoid interest domain containing protein 1（PRELID1） mRNA were detected by real-time fluorescence quantitative PCR （RT-qPCR） method. The expression levels of miR-325-3p and PRELID1 mRNA and the expression levels of PRELID1 protein in human normal colon cells NCM460 and colon cancer cells SW480， HCT116 and HT-29 were detected by RT-qPCR and Western blotting methods. The SW480 cells were divided into control group， NC-mimics group， miR-325-3p mimics group， miR-325-3p mimics+oe-NC group and miR-325-3p mimics+oe-PRELID1 group. The invasion and migration abilities of cells in various groups were detected by Transwell chamber assay and scratch healing assay， respectively. The expression levels of E-Cadherin， N-Cadherin and Vimentin in the epithelial-mesenchymal transition （EMT） of cells in various groups were detected by Western blotting method.The downstream target genes of miR-325-3p were predicted using the Targetscan bioinformatics website， and the targeted regulatory relationship between miR-325-3p and PRELID1 was verified by the dual-luciferase assay. Results Compared with adjacent normal tissue， the expression level of miR-325-3p in cancer tissue was significantly decreased （P<0.05）， while the expression level of PRELID1 mRNA was significantly increased （P<0.05）. The expression levels of miR-325-3p and PRELID1 mRNA was negatively correlated in colon cancer tissue （r<0， R²=0.392， P<0.001）. Compared with NCM460 cells， the expression levels of miR-325-3p in SW480， HCT116 and HT-29 cells were significantly decreased （P<0.05）， and the expression levels of PRELID1 mRNA were significantly increased （P<0.05）. Compared with control group and NC-mimics group， the number of invasive SW480 cells in miR-325-3p mimics group was significantly reduced （P<0.05）， the rate of scratch healing was decreased （P<0.05）， and the expression level of E-Cadherin protein was increased， while the expression levels of N-Cadherin and Vimentin proteins were decreased （P<0.05）. The dual-luciferase assay confirmed that PRELID1 was the direct target of miR-325-3p. Compared with miR-325-3p mimics+oe-NC group， the number of invasive SW480 cells in miR-325-3p mimics+oe-PRELID1 group was significantly increased （P<0.05）， the rate of scratch healing was increased （P<0.05）， the expression level of E-Cadherin protein was decreased， and the expression levels of N-Cadherin and Vimentin proteins were increased （P<0.05）. Conclusion Over-expression of miR-325-3p can inhibit EMT process by down-regulating PRELID1 expression， thereby inhibiting SW480 cell invasion and migration.

Key words: MicroRNA-325-3p, Relevant evolutionary and lymphoid interest domain containing protein 1, Colon neoplasm, Cell invasion, Cell migration, Epithelial-mesenchymal transition

中图分类号:

R735.35

朱平胜,葛思堂,左芦根,陈德利,张洋洋. miR-325-3p靶向PRELID1基因介导EMT通路对结肠癌细胞侵袭和迁移的影响及其机制[J]. 吉林大学学报(医学版), 2025, 51(5): 1185-1193.

Pingsheng ZHU,Sitang GE,Lugen ZUO,Deli CHEN,Yangyang ZHANG. Effect of miR-325-3p targeting PRELID1 gene in regulation of EMT pathway on invasion and migration of colon cancer cells and their mechanisms[J]. Journal of Jilin University(Medicine Edition), 2025, 51(5): 1185-1193.

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Group	Number of invasion cells	Rate of scratch healing（η/%）
Control	213.33±7.51	76.38±4.65
NC-mimics	212.33±4.16	74.51±5.36
MiR-325-3p mimics	69.00±3.61^*△	20.99±2.35^*△