RESUMO
OBJECTIVE@#To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect.@*METHODS@#The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments.@*RESULTS@#The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P < 0.05).@*CONCLUSION@#Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.
Assuntos
Humanos , Neoplasias Ósseas/genética , Proliferação de Células , MicroRNAs/genética , Osteossarcoma/genética , Proteínas rab3 de Ligação ao GTP/genética , RNA MensageiroRESUMO
AIM: To investigate the mechanism of baicalin-induced apoptosis in human laryngeal cancer cells. METHODS: AMC-HN-8 cells were selected for the study, and baicalin was applied to the cells at different concentrations (0, 10, 30, 100, and 300 μmol/L), and the half-inhibitory concentration (IC50) was measured by the CCK-8 method. Bax, cleaved-caspase-3, Cyto-c, IRF4 protein expression by protein blotting (Western blot); miR-125b-5p and IRF4 expression by RT-qPCR. Dual-luciferase reporter gene validation of Targetscan prediction (binding of miR-125b-5p to IRF4-3'UTR); apoptosis and necrosis inhibitors explore the way baicalein induces death in laryngeal cancer cells. AMC-HN-8 was then divided into blank group, baicalein (IC50), miR-125b-5p inhibitor group, baicalein + inhibitor NC group, baicalein+miR-125b-5p inhibitor group, and cell invasion and clone formation assays to detect cell invasion and proliferation ability, respectively. Apoptosis was detected by flow cytometry. RESULTS: Baicalein inhibited the proliferation of AMC-HN-8 cells in a dose-dependent manner with an IC50 value of 47.31 μmol/L. Compared with the blank group, 47.31 μmol/L baicalin induced apoptosis and inhibited cell invasion, while upregulating the expression of miR-125b-5p and suppressing the mRNA and protein levels of IRF4. The luciferase results showed that the miR-125b-5p mimic was able to inhibit the activity of the IRF4-3'UTR promoter relative to the NC mimic (mimic) group. Baicalein induces laryngeal cancer cell death in an apoptotic manner. In addition, the combination of 47.31 μmol/L baicalin and miR-125b-5p inhibitor affected the behavior of AMC-HN-8 cells, showing that compared with the blank group, the baicalin group showed a decrease in the number of cell clones, weakened invasion ability, and increased apoptosis; the miR - 125b-5p inhibitor group showed an increase in the number of cell clones, enhanced invasion ability and decreased apoptosis. The baicalin+ inhibitor NC group was consistent with baicalin, with no significant effect of inhibitor NC on cell behavior. The cloning, invasion, and apoptosis of cells in the baicalin+miR-125b-5p inhibitor group were intermediate between the baicalin and miR-125b-5p inhibitor groups. CONCLUSION: Baicalin inhibits the proliferation of AMC-HN-8 cells, and the mechanism may be related to miR-125b-5p targeting to inhibit the expression of IRF4, inducing the pro-apoptotic proteins Bax, cleaved-caspase3, and Cyto-c, and inhibiting the apoptosis suppressor protein Bcl-2 thereby inducing apoptosis.