Fig. 2.

The effect of transient transfection of miR-34a-5p mimic. a GAPDH siRNA was transiently transfected into KatoIII cells and used as positive control for transient transfection. The GAPDH mRNA was quantified using qRT-PCR analysis and normalized against β- actin internal control. The results displayed a significant reduction in GAPDH mRNA relative to the controls. b Different concentrations of miR-34a-5p mimic were transiently transfected into KatoIII cells. The qRT-PCR analysis verified that the miR-34a-5p was overexpressed in transfected cells relative to the control. c, d Transfection of double- or single- stranded miR-34a-5p mimic at 25 nM into KatoIII cells did not reduce the mRNA levels of β-catenin and Notch1. But, higher levels of target mRNAs were observed based on qRT-PCR analysis. e Western blot analysis showed that transient transfection with 25 nM miR-34a-5p mimic did not decrease the β-catenin protein level, but led to increase the protein level; thus, the data were consistent with qRT-PCR. f Different concentrations of miR-34a-5p mimic, including 15 nM, 10 nM, and 5 nM, were also transfected into KatoIII cells. Quantification of target mRNAs, β-catenin and Notch1, using qRT-PCR showed that miR-34a-5p mimic had inhibitory effect on these two target genes expression at lower concentrations, 10 nM and 5 nM, although the changes were not statistically significant. g, h The effect of transiently transfected miR-34a-5p mimic at 25 nM on the β-catenin expression was evaluated in BEAS-2B cell line. Using qRT-PCR analysis, a remarkably reduced level of β-catenin mRNA was detected in BEAS-2B cell line. i The effects of transient transfection of miR-34a-5p mimic on the apoptosis rate was assessed using flow cytometry analysis. Compared with the control cells, significant change was no observed in the percentage of apoptotic cells following transfection of miR-34a-5p mimic at 25 nM. *p < 0.05; **p < 0.01