微小RNA-155-5p靶向尾型同源盒基因1抑制人胰腺癌细胞迁移、侵袭和上皮-间充质转化

MicroRNA-155-5p targets caudal-type homeobox 1 to inhibit the migration, invasion and epithelial-mesenchymal transition of human pancreatic cancer cells

目的观察尾型同源盒基因1(CDX1)对胰腺癌(PC)细胞迁移、侵袭和上皮-间充质转化(EMT)的作用,探讨微小RNA-155-5p(miR-155-5p)/CDX1轴调控PC细胞迁移、侵袭和EMT的分子机制。方法选取2020年1月至2023年1月在临沂市人民医院肝胆外科行PC切除术的30例PC患者的癌组织为研究对象,采用免疫组织化学染色、免疫印迹和实时荧光定量聚合酶链反应(RT-qPCR)检测PC癌和癌旁组织CDX1的表达水平。通过细胞计数试剂(CCK-8)、划痕实验、Matrigel侵袭实验检测细胞迁移、侵袭能力;采用免疫印迹和RT-qPCR检测EMT相关基因E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)的表达水平,采用荧光素酶报告基因实验检测miR-155-5p与CDX1启动子区的结合情况。组间差异采用t检验或方差分析(ANOVA)。结果PC组织中CDX1蛋白和RNA表达水平(7.15±1.72、9.17±2.83)明显高于癌旁组织(1.00±0.28、1.00±0.38),差异有统计学意义(t=11.290、13.912,P<0.05)。过表达CDX1促进PC细胞的迁移侵袭能力,划痕实验显示,过表达CDX1(Ad-CDX1)组PC细胞的迁移细胞数明显高于病毒对照(Ad-GFP)组(81.92±5.08比53.10±4.19,t=10.031,P<0.01),过表达miR-155-5p抑制PC细胞的迁移侵袭能力,miR-155-5p类似物(mimics)组PC细胞的迁移细胞数明显低于对照(NC mimics)组(38.80±5.76比53.48±6.19,t=7.861,P<0.01)。RT-qPCR结果显示,miR-155-5p抑制剂(inhibitor)+小干扰RNA(si-CDX1)组EMT相关基因E-cadherin的表达水平高于miR-155-5p inhibitor+小干扰RNA对照(si-NC)组(1.73±0.36比1.00±0.12,t=6.001,P<0.05),N-cadherin和Vimentin的表达水平低于miR-155-5p inhibitor+si-NC(N-cadherin:1.16±0.21比3.18±0.67,t=4.337,P<0.05;Vimentin:1.23±0.41比2.76±0.54,t=6.230,P<0.05)。结论CDX1在PC中表达显著增加,miR-155-5p通过直接靶向CDX1抑制人PC细胞迁移、侵袭和EMT。

Objective To investigate the role of caudal-type homeobox 1(CDX1)in the migration,invasion and epithelial-mesenchymal transition(EMT)of pancreatic cancer(PC)cells,and the molecular mechanism of microRNA-155-5p(miR-155-5p)/CDX1 axis in regulating the migration,invasion and EMT of PC cells.Methods Cancer tissues from 30 patients with PC who underwent PC resection in Linyi People’s Hospital from January 2020 to January 2023 were selected as the research objects.Immunohistochemical staining,Western blotting and real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)were used to detect the expression level of CDX1 in PC tissues.Cell counting kit-8(CCK-8)assay,wound healing assay and Matrigel invasion assay were used to detect cell migration and invasion ability.Western blotting and RT-qPCR were used to detect the expression levels of EMT-related genes E-cadherin,N-cadherin and Vimentin.Luciferase reporter gene assay was used to detect the binding of miR-155-5p to CDX1 promoter region.The differences between groups were analyzed by t-test or analysis of variance(ANOVA).Results The expression levels of CDX1 protein and RNA in PC tissues(7.15±1.72,9.17±2.83)was significantly higher than that in adjacent tissues(1.00±0.28,1.00±0.38)(t=11.290,13.912,P<0.05).Overexpression of CDX1 promoted the migration and invasion of PC cells.Scratch test showed that the number of migrating cells in overexpression(Ad-CDX1)group was significantly higher than that in control(Ad-GFP)group(81.92±5.08 vs.53.10±4.19,t=10.031,P<0.01).Overexpression of miR-155-5p inhibited the migration and invasion of PC cells,and the number of migrating cells in miR-155-5p mimics group was significantly less than that in negative control(NC)mimics group(38.80±5.76 vs.53.48±6.19,t=7.861,P<0.01).The results of RT-qPCR showed that the expression level of EMT-related gene E-cadherin in miR-155-5p inhibitor+small interfering RNA(si)-CDX1 group was higher than that in miR-155-5p inhibitor+si-NC group(1.73±0.36 vs.1.00±0.12,t=6.001,P<0.05),and the expression levels of N-cadherin and Vimentin were lower than those of miR-155-5p inhibitor+si-NC(N-cadherin:1.16±0.21 vs.3.18±0.67,t=4.337,P<0.05;Vimentin:1.23±0.41 vs.2.76±0.54,t=6.230,P<0.05).The results of Western blotting and luciferase reporter gene assay indicated that miR-155-5p directly targeted CDX1 gene to exert the above effects.Conclusion The expression of CDX1 is significantly increased in PC cells,and miR-155-5p inhibits the migration,invasion and EMT of human PC cells by directly targeting CDX1.