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.