miRNA-5089-5p体外通过调节组织蛋白酶B表达抑制食管癌细胞增殖和迁移

miRNA-5089-5p inhibits proliferation and migration of esophageal cancer cells by regulating the expression of cathepsin B in vitro

目的探讨miRNA-5089-5p(miR-5089-5p)体外对食管癌细胞增殖和迁移能力的影响及其与组织蛋白酶B(CTSB)基因表达的关系。方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测2017年3月至2019年12月鄂东医疗集团黄石市中心医院食管癌手术切除癌组织标本及相应癌旁组织标本31例,以及食管癌TE-13、EC9706、Eca109、KYSE30细胞株和正常食管黏膜上皮HET-1A细胞miR-5089-5p的表达水平。选择miR-5089-5p表达量最低的食管癌细胞,分为两组,分别转染miR-5089-5p模拟物(miR-5089-5p组)及其阴性对照序列(阴性对照组);转染48 h后,qRT-PCR检测两组细胞miR-5089-5p表达水平;采用CCK-8法和划痕愈合实验分别检测两组细胞的增殖和迁移能力。用microRNA.org、Deepbase v2.0在线工具预测miR-5089-5p的靶基因,并采用双荧光素酶报告基因实验进行验证。qRT-PCR和蛋白质印迹法检测miR-5089-5p组、阴性对照组细胞靶基因的表达水平,同时采用蛋白质印迹法检测两组细胞增殖相关蛋白PCNA、Ki-67和迁移相关蛋白N-Cadherin、Twist表达。结果食管癌患者癌组织和癌旁组织中miR-5089-5p的相对表达量分别为1.54±0.53和7.07±1.25(t=24.06,P<0.01);各食管癌细胞株miR-5089-5p相对表达量均低于正常食管黏膜上皮HET-1A细胞(均P<0.05),相对表达量最低的是Eca109细胞(0.12±0.03)。与阴性对照组相比,随着转染时间延长,miR-5089-5p组Eca109细胞增殖能力逐渐降低,从48 h开始两组间差异均有统计学意义(均P<0.05);迁移能力亦降低[细胞划痕愈合率:(29±5)%比(64±8)%,t=3.91,P<0.01]。在线工具预测miR-5089-5p的靶基因可能是CTSB,双荧光素酶报告基因实验证实miR-5089-5p互补结合CTSB 3'UTR。qRT-PCR检测显示,与阴性对照组相比,miR-5089-5p组Eca109细胞CTSB mRNA相对表达量降低(0.23±0.04比1.01±0.09,t=8.27,P<0.01),蛋白质印迹法检测显示,CTSB蛋白表达水平亦降低,同时细胞增殖相关蛋白PCNA、Ki-67和细胞迁移相关蛋白N-Cadherin、Twist表达水平均降低。结论食管癌患者癌组织和细胞株中miR-5089-5p均低表达,miR-5089-5p能抑制食管癌Eca109细胞增殖和迁移能力,此作用可能是通过下调CTSB基因表达实现的。

Objective To investigate the effect of miRNA-5089-5p(miR-5089-5p)on proliferation and migration ability of esophageal cancer in vitro and its relationship with the expression of cathepsin B(CTSB)gene.Methods Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-5089-5p in 31 tissue samples from patients who underwent esophageal cancer resection and the corresponding pericarcinomatous tissues between in March 2017 and in December 2019 at Huangshi Central Hospital of Edong Healthcare Group,and TE-13,EC9706,Eca109,KYSE30 cell lines and normal esophageal mucosal epithelial HET-1A cells.The esophageal cancer cells with the lowest expression level of miR-5089-5p were divided into 2 groups:miR-5089-5p group transfected with miR-5089-5p mimics and the negative control group with negative control sequence.qRT-PCR was used to detect the expression level of miR-5089-5p after transfection for 48 h.CCK-8 method and scratch healing test were used to detect the proliferation and migration ability of cells in the two groups.The online tools microRNA.org and Deepbase v2.0 were used to predict the target genes of miR-5089-5p.The dual luciferase reporter gene assay was used to verify the target gene of miR-5089-5p.qRT-PCR and Western blot were used to detect the expression level of target genes in the two groups.The expressions of cell proliferation-related protein(PCNA and Ki-67)and migration-related protein(N-Cadherin and Twist)were detected by using Western blot.Results The relative expression level of miR-5089-5p in esophageal cancer and pericarcinomatous tissues was 1.54±0.53 and 7.07±1.25,respectively(t=24.06,P<0.01).The relative expression level of miR-5089-5p in the esophageal cancer cell lines was lower than that of normal esophageal mucosal epithelial HET-1A cells(all P<0.05),and the cell line with the lowest relative expression was Eca109 cells(0.12±0.03).Compared with the negative control group,the proliferation ability of Eca109 cells in miR-5089-5p group was gradually reduced with the transfection time extension,and the difference was statistically significant between the two groups since 48 h(all P<0.05),and the migration ability was also reduced[scratch healing rate:(29±5)%vs.(64±8)%,t=3.91,P<0.01].The online tool predicted that the target gene of miR-5089-5p might be CTSB,and the dual luciferase reporter gene assay confirmed that miR-5089-5p complemented CTSB 3'UTR.qRT-PCR results showed that compared with the negative control group,the relative expression level of CTSB mRNA in Eca109 cells of miR-5089-5p group was reduced(0.23±0.04 vs.1.01±0.09,t=8.27,P<0.01).Western blot results showed that the expression level of CTSB protein was reduced,and the expression levels of cell proliferation-related protein PCNA,Ki-67 and cell migration-related protein N-Cadherin,Twist were also reduced.Conclusions The expression level of miR-5089-5p in esophageal cancer tissues and cell lines is low.miR-5089-5p can inhibit proliferation and migration of esophageal cancer Eca109 cells.The mechanism may be achieved by down-regulating CTSB gene expression.