Effect and mechanism of microRNA-125a-5p on proliferation of nasopharyngeal carcinoma cells
Objective To investigate the effect and mechanism of microRNA-125a-5p(miR-125a)on the prolifera-tion of nasopharyngeal carcinoma cell line HK-1.Methods Human nasopharyngeal carcinoma cells(HK-1 cells)and human embryonic kidney immortalized cells(293T cells)were cultured routinely.HK-1 cells were randomly divided into the control group 1,miR-125a overexpression group(transfected with miR-125a mimics),and miR-125a silencing group(transfected with miR-125a inhibitor).Some cells were further divided into the control group 2,miR-125a overexpression group(transfected with miR-125a mimics),and rescue group(transfected with miR-125a mimics and TAZ overexpression simultaneously).The transfection efficiency was verified by detecting miR-125a expression at 48 h after transfection.RT-qPCR was used to detect the expression levels of miR-125a-5p and TAZ mRNA,while Western blotting was used to detect TAZ protein expression.Cell proliferation capacity was assessed using the CCK-8 assay and colony formation assay.Tar-getScan and miRDB prediction websites were used to predict the binding site of miR-125a and TAZ.The 293T cells were divided into four groups:WT+miR-ctrl group(co-transfected with TAZ wild-type and miR-ctrl),Mut+miR-ctrl group(co-transfected with TAZ mutant and miR-ctrl),WT+miR-125a group(co-transfected with TAZ-WT and miR-125a mimic),and Mut+miR-125a group(co-transfected with TAZ-MUT and miR-125a).Dual-luciferase reporter gene experiments were conducted to further validate the downstream target of miR-125a.Results There were significant differences in the rela-tive expression levels of miR-125a among the control group 1,the miR-125a overexpression group,and the miR-125a si-lencing group(all P<0.05).Statistically significant differences were found in the proliferation activity(OD450 values)on the 2nd,3rd,4th,5th,and 6th days after transfection and colony formation numbers in the control group 1,miR-125a overexpression group,and miR-125a silencing group(all P<0.05).There was significant difference in the relative expres-sion level of TAZ protein among the control group 1,miR-125a overexpression group,and miR-125a silencing group(all P<0.05).Website prediction indicated the existence of binding sites between miR-125a and TAZ,and mutated sequences(TAZ-3'UTR MT)were designed.MiR-125a directly targeted TAZ in the non-coding region.The relative fluorescence in-tensity of the WT+miR-125a group was lower than those of the WT+miR-ctrl group and the Mut+miR-ctrl group(both P<0.05),while there was no significant difference in relative fluorescence intensity among the WT+miR-125a group,WT+ miR-ctrl group and the Mut+miR-ctrl group(all P>0.05).Statistically significant differences were found in proliferation activity on the 2nd,3rd,4th,5th,and 6th days after transfection and colony formation numbers between the control group 2,miR-125a mimics group,and miR-125a mimics+TAZ group(all P<0.05).Conclusion MiR-125a can inhibit the proliferation of nasopharyngeal carcinoma cells,and its mechanism of action is related to the inhibition of its downstream target TAZ expression.
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