miR-23b-3p在结直肠癌中的表达及其靶向KLF3抑制结直肠癌细胞侵袭和迁移能力的机制研究

目的:研究miR-23b-3p在结直肠癌中的表达情况及对结直肠癌SW620细胞侵袭和迁移的影响机制。方法:收集2018年01月至2020年12月我院胃肠外科手术切除的58例结直肠癌组织及癌旁组织标本，以及结直肠癌细胞株SW480、SW620、HCT116、HT-29、Lovo和人正常结直肠黏膜细胞FHC，采用qPCR法检测miR-23b-3p和KLF3相对表达水平。采用脂质体转染技术将miR-23b-3p mimics、mimics-NC转染至SW620细胞，采用Transwell实验和划痕实验检测其侵袭和迁移能力的改变。利用生物信息学软件预测并通过双荧光素酶报告基因实验验证miR-23b-3p和KLF3的结合位点。将KLF3过表达质粒（pcDNA3.1-KLF3）或空载质粒（Vector）单独或联合miR-23b-3p mimics转染至SW620细胞，采用Transwell实验和划痕实验检测其侵袭和迁移能力的改变，采用qPCR和Western Blot实验检测SW620细胞中KLF3 mRNA及蛋白的表达。结果:miR-23b-3p在结直肠癌组织中的表达水平显著低于癌旁组织（P＜0.05），并与患者TNM分期和远处转移有关（均P＜0.05）;miR-23b-3p在结肠癌细胞系中的表达水平均显著低于FHC细胞，上调miR-23b-3p表达能显著抑制SW620细胞的侵袭和迁移能力。KLF3在结直肠癌组织和细胞中高表达，与miR-23b-3p在结直肠癌组织中的表达呈负相关（r=-0.326，P=0.013），双荧光素酶报告基因实验证实miR-23b-3p直接靶向调节KLF3的表达， KLF3过表达能促进SW620细胞的侵袭和迁移，同时转染miR-23b-3p mimics可下调KLF3蛋白和mRNA表达，逆转KLF3过表达对SW620细胞侵袭和迁移能力的促进作用。结论:miR-23b-3p在结直肠癌中低表达并与肿瘤患者TNM分期及远处转移相关，miR-23b-3p靶向调控KLF3表达抑制结直肠癌SW620细胞的侵袭和迁移能力。

The expression of miR-23b-3p in colorectal cancer and the mechanism of targeting KLF3 to inhibit the invasion and migration of colorectal cancer cells

Objective:To investigate the expression of miR-23b-3p in colorectal cancer (CRC) and its effect on the invasion and migration of SW620 cells.Methods:58 cases of CRC tissue and para-cancerous tissue specimens were collected from January 2018 to December 2020 in gastrointestinal surgery in our hospital,in addition,CRC cell lines (SW480,SW620,HT-29 and Lovo) and human normal colorectal mucosal cells (FHC) were also chosen for this study.The relative expression level of miR-23b-3p and KLF3 was detected by quantitative polymerase chain reaction (qPCR).Transwell assay and wound healing assay were used to observe the invasion and migration of SW620 cells which was transfected with miR-23b-3p mimics or mimics-NC.The binding sites of miR-23b-3p and KLF3 were predicted by bioinformatics software and verified by double luciferase reporter gene assay.pcDNA3.1-KLF3 or vector was transfected into SW620 cells alone or in combination with miR-23b-3p mimics,transwell assay and wound healing assay were used to detected the invasion and migration of SW620 cells,the expression of KLF3 protein and mRNA in SW620 cells was detected by Western Blot and qPCR,respectively.Results:The expression of miR-23b-3p in CRC tissues was significantly lower than that in adjacent tissues (P＜0.05),which was related to the TNM stage and distant metastasis of CRC patients (all P＜0.05).The expression of miR-23b-3p in CRC cell lines was significantly lower than that in FHC cells,and up-regulation of miR-23b-3p expression could significantly inhibit the invasion and migration of SW620 cells.KLF3 was highly expressed in CRC tissues and cells,which was negatively correlated with miR-23b-3p expression in CRC tissues (r=-0.326,P=0.013),dual luciferase reporter gene assay confirmed that miR-23b-3p directly regulates KLF3 expression.Up-regulation of KLF3 can promote the invasion and migration of SW620 cells,meanwhile,transfection with miR-23b-3p mimics can down-regulate the expression of KLF3 protein and mRNA,reverse the promoting effection of pcDNA3.1-KLF3 on invasion and migration of SW620 cells.Conclusion:miR-23b-3p is low expressed in colorectal cancer and is associated with TNM stage and distant metastasis.miR-23b-3p inhibits the invasion and migration of SW620 cells by targeting KLF3.