首页|微小RNA-93-5p/STEAP4分子轴调控肝癌细胞增殖转移的分子机制

微小RNA-93-5p/STEAP4分子轴调控肝癌细胞增殖转移的分子机制

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目的 探讨miR-93-5p靶向STEAP4调控肝细胞癌(HCC)细胞增殖转移的分子机制.方法 对Huh7细胞转染miR-93-5p mimic和阴性对照寡核苷酸(miR-NC),实时荧光定量聚合酶链反应(qPCR)检测miR-93-5p表达情况.通过噻唑蓝(MTT)、细胞克隆和Transwell实验检测miR-93-5p在HCC细胞增殖、迁移和侵袭中的作用.双荧光素酶报告基因确定miR-93-5p靶基因STEAP4.通过蛋白质印迹法(Western blot)、qPCR、MTT、细胞克隆和Transwell实验检测miR-93-5p/STEAP4轴对HCC细胞增殖、迁移和侵袭能力的影响.结果 对Huh7细胞转染miR-93-5p mimic和miR-NC后发现,miR-93-5p mimic 组 miR-93-5p 表达量(t=30.90,P<0.01)、细胞活力(t=4.93,P<0.01)、克隆形成(t=20.60,P<0.05)、迁移细胞数(t=56.93,P<0.01)和侵袭的细胞数(t=42.93,P<0.01)明显高于 miR-NC(20.93±0.63 比 1.20±0.05、1.33±0.01 比 1.02±0.06、163.30±2.40 比94.33±2.33、237.70±1.45 比 132.00±1.15、167.70±1.44 比 88.00±1.53),差异有统计学意义(P<0.05).通过生物信息学算法预测STEAP43'端非编码区(3'UTR)包含miR-93-5p的保守结合位点,是 miR-93-5p 靶基因.miR-93-5p mimic 与 pGL3-STEAP4-3'UTR-WT 共转染可显著抑制 Huh7细胞中的荧光素酶活性,miR-93-5p mimic+pGL3-STEAP4-3'UTR-WT组荧光素酶活性显著低于miR-NC+pGL3-STEAP4-3'UTR-WT 组(0.55±0.05 比 0.95±0.09),差异有统计学意义(t=48.99,P<0.01).通过 Western blot 和 qPCR 证实,在转染 miR-93-5p mimic 的 Huh7 细胞中,STEAP4 的蛋白(0.77±0.03 比 0.22±0.04)和 mRNA(1.005±0.007 比 0.400±0.002)表达显著降低,差异有统计学意义(t=39.38、24.37,P<0.05).miR-93-5p mimic 和 STEAP4 过表达质粒共同转染 Huh7 细胞,Western blot表明miR-93-5p mimic和STEAP4组中STEAP4蛋白表达显著低于STEAP4过表达组(0.86±0.04比1.25±0.03),差异有统计学意义(t=6.38,P<0.05).MTT、克隆形成、迁移细胞数和侵袭的细胞数 miR-93-5p mimic 和 STEAP4 组明显高于 STEAP4 组(0.86±0.02 比 0.71±0.01、95.26±1.22 比 63.67±1.20、125.67±1.20 比 85.33±1.76、75.47±1.32 比 55.67±1.85),差异有统计学意义(t=9.01、18.83、14.21、9.04,P<0.05).结论 miR-93-5p 通过靶向结合 STEAP4 显著调控HCC细胞的增殖、侵袭和迁移,为HCC患者提供了潜在的分子生物标志物.
Molecular mechanism of microRNA-93-5p regulating proliferation and metastasis of hepatocellular carcinoma cells by targeting STEAP4
Objective To study the molecular mechanism of microRNA(miR)-93-5p targeting STEAP4 regulating the invasion and metastasis of hepatocellular carcinoma(HCC)cells.Methods Huh7 cells were transfected with either miR-93-5p mimic or a negative control oligonucleotide(miR-NC),followed by quantification of miR-93-5p expression using real-time fluorescence quantitative polymerase chain reaction(qPCR).The influence of miR-93-5p on HCC cell proliferation,migration,and invasion was examined through MTT assay,colony formation assay,and Transwell assay.Subsequently,a dual-lu-ciferase reporter gene assay confirmed STEAP4 as a direct target of miR-93-5p.Finally,the impact of the miR-93-5p/STEAP4 axis on HCC cell proliferation,migration,and invasion capabilities was further eluci-dated using Western blotting,qPCR,MTT assay,colony formation assay,and Transwell assay.Results Following transfection of Huh7 cells with miR-93-5p mimic and miR-NC,the miR-93-5p mimic group exhibited significantly elevated levels of miR-93-5p expression(t=30.90,P<0.01),enhanced cell via-bility(t=4.93,P<0.01),increased colony formation(t=20.6,P<0.05),augmented migration(t=56.93,P<0.01),and elevated invasion(t=42.93,P<0.01)compared to miR-NC(20.93±0.63 vs.1.20±0.05,1.33±0.01 vs.1.02±0.06,163.30±2.40 vs.94.33±2.33,237.70±1.45 vs.132.00±1.15,167.70±1.44 vs.88.00±1.53),all of which were statistically significant(all P<0.05).Bioinformatics analysis identified a conserved miR-93-5p binding site within the STEAP4 3'untrans-lated regions(3'UTR),confirming STEAP4 as a direct target of miR-93-5p.Co-transfection of miR-93-5p mimic with pGL3-STEAP4-3'UTR-WT significantly suppressed luciferase activity in Huh7 cells,with lucif-erase activity in the miR-93-5p mimic+pGL3-STEAP4-3'UTR-WT group markedly lower than that in the miR-NC+pGL3-STEAP4-3'UTR-WT group(0.55±0.05 vs.0.95±0.09),demonstrating statistically significant difference(t=48.99,P<0.01).Western blotting and qPCR confirmed substantial reductions in STEAP4 protein(0.77±0.03 vs.0.22±0.04)and mRNA(1.005±0.007 vs.0.400±0.002)expression levels in Huh7 cells transfected with miR-93-5p mimic,with statistically significant differences(t=39.38,24.37,P<0.05).Western blotting confirmed substantial reductions in STEAP4 protein(0.86±0.04 vs.1.25±0.03)expression levels in Huh7 cells co-transfected with miR-93-5p mimic and STEAP4 overexpression plasmid,with statistically significant differences(t=6.38,P<0.05).Co-trans-fection of miR-93-5p mimic and STEAP4 overexpression plasmid notably enhanced MTT assay,colony for-mation,number of migrating cells,and invasive cells compared to the STEAP4 group(0.86±0.02 vs.0.71±0.01,95.26±1.22 vs.63.67±1.20,125.67±1.20 vs.85.33±1.76,75.47±1.32 vs.55.67±1.85),with statistically significant differences(t=9.01,18.83,14.21,9.04,P<0.05).Conclusion miR-93-5p can significantly regulate the proliferation,invasion,and migration of HCC cells by targeting and combining STEAP4.This study provides a potential molecular biomarker for HCC patients.

MicroRNAHepatocellular carcinomaProliferationMigrationInvasion

强勇、张小风、官兵

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荆门市人民医院荆楚理工学院附属中心医院普外科,荆门 448000

微小RNA 肝细胞癌 增殖 迁移 侵袭

2024

中华实验外科杂志
中华医学会

中华实验外科杂志

CSTPCD
影响因子:0.759
ISSN:1001-9030
年,卷(期):2024.41(9)